Human ovarian tissue cryopreservation: indications and feasibility

Catherine Poirot1,2,7, Marie-Cécile Vacher-Lavenu3, Pierre Helardot4, Juliette Guibert5, Laurence Brugières6 and Pierre Jouannet2

1 Laboratoire de Biologie de la Reproduction, Groupe Hospitalier Pitié-Salpêtrière, Université Paris VI, 75013 Paris, 2 Laboratoire de Biologie de la Reproduction, CECOS, Groupe Hospitalier Cochin-Saint Vincent de Paul, Université Paris V, 75014 Paris, 3 Service d'Anatomie et de Cytologie pathologiques, Groupe Hospitalier Cochin-Saint Vincent de Paul, Université Paris V, 75014 Paris, 4 Service de Chirurgie Pédiatrique, Hôpital Trousseau, Université Paris VI, 75012 Paris, 5 Service de Gynécologie Obstétrique, Groupe Hospitalier Cochin-Saint Vincent de Paul, 75014 Paris and 6 Service d'Oncologie Pédiatrique, Institut Gustave Roussy, 94800 Villejuif, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The cryopreservation of ovarian tissue may enable women exposed to gonadotoxic treatments to have children at a later date. METHODS: Between April 1998 and October 2000, we evaluated the feasibility of long-term ovarian tissue cryopreservation in 51 women who were all at risk of becoming sterile following treatment. RESULTS: Ovarian tissue was not cryopreserved in 20 cases because of the woman's age or premature ovarian failure. In 31 patients, ovarian tissue was frozen by a slow cooling technique using DMSO and sucrose as cryoprotectants. The patients were aged 2.7–34 years and 16 of them were <18 years old. Cryopreservation could be performed in all cases. Ovarian cortex histology was performed for all patients to evaluate the concentration of follicles. The mean number of primordial and primary follicles per mm2 was 20.36 ± 19.03 before 10 years of age, 4.13 ± 2.9 between 10 and 15 years of age and 1.63 ± 3.35 after 15 years of age. An average mean number of 26 ± 8.2 ovarian fragments (range 13–50) were cryopreserved per patient for future autografts or for in-vitro growth of follicles. CONCLUSION: Cryopreservation of ovarian tissue may be systematically proposed to young women and girls at risk of becoming sterile as a result of gonadotoxic treatment.

Key words: fertility preservation/gonadotoxic treatment/ovarian cryopreservation/ovarian follicle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The improvement in the survival of children and young women with cancer has led to consideration of the long-term side-effects of treatments. One of these side-effects is premature ovarian failure (Stillman et al., 1981Go; Teinturier et al., 1998Go; Byrne, 1999Go). Therefore, health professionals must consider the future fertility of cancer patients prior to chemotherapy and/or radiotherapy, which may be toxic for the gonads (Meirow, 2000Go). When the deleterious effects of the treatment are not reversible, mature oocytes, embryos and ovarian tissue may be cryopreserved to restore fertility following the completion of the treatment.

Mature oocytes cannot be cryopreserved easily. The cytoplasm volume and water content is much higher than in other cells but also the nucleus is blocked in metaphase II of meiosis which makes the spindle very fragile and sensitive to heat and osmotic stress (Glenister et al., 1987Go). Nevertheless, good oocyte survival rate (Fabbri et al., 2001Go) and successful pregnancies (Chen, 1986Go; Porcu et al., 1997Go) have been described after the cryopreservation of mature oocytes. However, the collection of mature oocytes requires ovarian stimulation, which is not always possible because the high levels of oestrogen induced may be deleterious in some cancers (Tavani and La Vecchia, 1999Go). Furthermore, this procedure should only be used in adult patients.

Embryo cryopreservation is an established clinical technique which results in acceptable pregnancy rates, depending on the age of the patient (Mandelbaum et al., 1998Go; Troup et al., 1990Go). However, the patient must have a partner or be in a stable relationship at the time of the treatment and again women with oestrogen-sensitive cancers cannot undergo the necessary ovarian stimulation before IVF.

Cryopreservation of prophase I oocytes contained in primordial follicles could be an attractive alternative for many reasons. Cytoplasmic differentiation is not complete in immature oocytes; nuclei are blocked in the germinal vesicle stage. There is a large number of immature oocytes in the ovarian cortex, so the method can be used for prepubertal girls and some promising uses of frozen ovarian cortex have been shown (Oktay and Karlikaya, 2000Go; Oktay, 2001Go; Radford et al., 2001Go). Thus, the cryopreservation of ovarian tissue is a potential alternative or addition to the cryopreservation of embryos or mature oocytes for women at risk of premature ovarian failure (Oktay et al., 1998Go; Pfeifer and Coutifaris, 1999Go).

We evaluated the feasibility of long-term cryopreservation of ovarian tissue in women and young girls admitted to undergo treatments that would probably render them sterile.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
From April 1998 to October 2000, 51 adult women, adolescents and prepubertal girls were referred by oncologists for ovarian tissue cryopreservation. Girls aged <18 years old were seen with at least one parent. During the first consultation, the patients and/or their parents were informed of the risks of the foreseen treatments on future fertility. Depending on the age and marital status of each patient, information was given on the various available methods of embryo, mature oocyte or ovarian tissue cryopreservations and on the foreseeable pregnancy rate of each technique. It was explained to the patients that there have been no studies showing that the transplantation of frozen-banked tissue would result in restoration of fertility. The risks of the procedure by which ovarian tissue is collected were also discussed. If patients agreed, informed consent was signed by the patients or by their parents if the patient was under 18 years old. The ethical aspects of the procedure have been extensively discussed in a multidisciplinary committee of the `Espace Ethique de l'Assistance Publique-Hôpitaux de Paris'.

Collection of ovarian tissue
Ovarian tissue was collected from 31 patients under general anaesthesia by laparoscopy or by laparotomy. In eight cases, the collection was associated with residual tumour removal, bone marrow collection or other surgical interventions needed by the treatment. At the beginning of the study, approximately half an ovary was collected so that the maximum volume of ovarian tissue was left in case ovarian function returned after treatment (n = 7). However, the electrocoagulation of the remaining part of the ovary was judged to be too deleterious. A thermal tissue injury, 3–5 mm from the cut edge, was noted by surgeons, therefore in the subsequent 24 cases one complete ovary was collected.

Cryopreservation of ovarian tissue
The protocol used was as previously described (Gosden et al., 1994Go). Briefly, immediately after collection ovarian tissue was transferred into Leibovitz 15® (Life Technologies, Cergy Pontoise, France) and transferred to the laboratory on ice. The ovarian cortex was isolated and tissue slices (1 mm thick and 1 mm2 to 1 cm2 in area) were prepared. The same thickness of all samples allowed similar penetration of cryoprotective agents (Newton et al., 1998Go) regardless of the surface of the slices. After rinsing in Leibovitz 15, each ovarian fragment was transferred into a cryovial (Nunc, PolyLabo, Strasbourg, France) containing 1 ml cryoprotectant [1.5 mol/l dimethylsulphoxide (Sigma, St Quentin Favalier, France) and 0.1 mol/l sucrose (Sigma) made up with Leibovitz 15 and 10% patient serum]. The thin slices of ovarian cortex were equilibrated for 30 min in the cryoprotective solution at 4°C. The cryovials were then loaded into an automated freezer (40 PC; Air Liquide Santé, Trappes, France) and the temperature was lowered from 4°C to –9°C at the rate of 2°C/min. After manual seeding, the vials were cooled to –40°C at the rate of 0.3°C/min. Finally, the vials were cooled to –140°C at 10°C/min before being transferred to liquid nitrogen for storage.

Histological analysis
For each patient, one sample of the ovarian cortex, selected before cryopreservation at random from the fragments prepared for freezing, was fixed in formaldehyde and embedded in paraffin. Sections (5 µm) were cut perpendicularly to the ovarian surface and stained with haematoxylin–eosin–saffron. At least 10 serial sections per patient (range 10–15) were analysed, the section area measured with a precalibrated ocular micrometer from 1 to 10 mm2. All follicles were systematically counted only once. Serial sections were useful for follicle classification.

The follicles were classified according to the modified Oktay system (Oktay et al., 1995Go) (Figure 1AGo): `primordial follicle' = an oocyte that was encapsulated by flattened pre-granulosa cells; `primary follicle' = when at least one of the pre-granulosa cells has become columnar or cubic until they form a single layer of cubic granulosa cells; `secondary follicle' = when the oocyte is encapsulated by two or more layers of granulosa cells without antrum formation; and `antral follicle' = when the oocyte is encapsulated by more than two layers of granulosa cells and an antrum has formed. Special care was taken when looking for tumour cells depending on the context. For each patient, the number of primordial and primary follicles per mm2 was measured within the ovarian cortex. The number of follicles surviving after thawing (Hovatta et al., 1996Go) has not been analysed in this study. It was decided to cryopreserve the maximum amount of ovarian tissue for future potential use, and it is planned to make this analysis when the tissue will be used.



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Figure 1. (A) Ovarian cortex section showing: `primordial follicle' (PF), in which the oocyte is encapsulated by flattened pre-granulosa cells; `primary follicle' (F1), in which at least one of the pre-granulosa cells becomes columnar or cubic until to form a single layer of cubic granulosa cells; `secondary follicle' (F2), in which the oocyte is encapsulated by two or more layers of granulosa cells without antrum formation. (B) Aspect of the ovarian cortex of a girl of 12.4 years old (patient no. 11), who had no previous chemotherapy and for whom ovarian tissue was cryopreserved before bone marrow graft for sickle cell disease. Number of primordial and primary follicles/mm2 is 8.5. Scale bar = 75 µm.

 
Statistical analysis
Differences in the mean concentration of primordial and primary follicles with or without previous chemotherapy were compared with the Mann–Whitney test. Statistical significance was assumed at P < 0.01.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovarian cryopreservation was carried out for 31 patients
Twenty-eight of the patients had a malignant disease: Hodgkin's disease (n = 8), neuroblastoma (n = 4), leukaemia (n = 5), Ewing's sarcoma (n = 4), lymphoma (n = 2), borderline tumour (n = 1), medulloblastoma (n = 2), breast cancer (n = 1) or myelodysplasia (n = 1). The three remaining women had sickle cell disease (Table IGo).


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Table I. Characteristics and follow-up of the 31 patients who had an ovarian tissue cryopreservation
 
Nineteen patients have already had between three and six courses of chemotherapy. The most frequent regimen combined cyclophosphamide, doxorubicin and vincristine with, in some cases, etoposide and cisplatin. In other cases, etoposide and cisplatin were given alone. The delay between the completion of chemotherapy and the ovarian tissue collection ranged from a few days to 6 months. In the three remaining cases, drugs such as methotrexate or hydroxyurea were given alone. In all cases included in this study, the drugs were not likely to cause sterility. In 13 cases, low doses of alkylating agents such as cyclophosphamide (1–12 g total dose) were used (Koyama et al., 1977Go).

The patient's mean age was 17.9 ± 9.3 years. The youngest patient was 2.7 years old and the oldest was 34 years old. The six patients under 10 years old had all received chemotherapy before.

Seven of 15 adult patients (44%) were married or were in a stable relationship, and one of them had a child.

The ovarian sample was collected by laparoscopy for 29 and by laparotomy for two of the 31 patients. In seven cases, another surgical procedure was carried out concomitantly to remove a residual abdominal tumour in two patients with neuroblastoma, and to collect bone marrow and/or to fit an intravenous catheter in four cases. In the final case, surgery was undertaken to remove a borderline peritoneal tumour.

An average of 26 ± 8.2 ovarian tissue fragments were cryopreserved per patient (range 13–50). The actual number depended on the age of the patient and the amount of cortical tissue that could be dissected from the collected ovarian piece.

The follow-up period was over 10 months for 23 patients. Seven of the patients did not survive, two relapsed and 12 were healthy. None of them have asked to use their ovarian tissue. The follow-up period was shorter than 10 months in eight cases because the delay after the end of treatment or after ovarian cryopreservation was too short, except if the patient died (n = 2).

Histological analysis of ovarian tissue
Follicles were not homogeneously distributed within the ovarian cortex and the number of follicles differed according to the patient's age (Figure 1BGo).

The total number of primordial and primary follicles per mm2 was dependent on the age of the patients and was independent of previous chemotherapy used for the patients included in this study, as shown in Figure 2Go.



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Figure 2. Mean number of primordial and primary follicles/mm2 in the ovarian cortex according to the age of patients and previous chemotherapy. {circ}, no previous chemotherapy; {blacklozenge}, previous chemotherapy.

 
In patients aged <=7 years, the mean number of primordial and primary follicles was 20.36 ± 19.03/mm2 (n = 6). It was 4.13 ± 2.9/mm2 between 10 and 15 years old (n = 8) and 1.63 ± 3.35/mm2 in women aged over 15 years (n = 17). After 12 years old, only one patient had a mean concentration of >10 follicles/mm2 (Figure 2Go). No follicles were observed in one 32.3 year old patient with breast cancer who had not previously undergone chemotherapy and one 6.1 year old patient with leukaemia who had had previous chemotherapy. In two other patients who were 21 and 17.3 years old respectively, only a small number of secondary follicles could be seen. In 10 cases the mean number of primordial and primary follicles/mm2 was very low, between 0.02 and 1.0.

In patients over 10 years old, previous low-dose chemotherapy had no significant effect on mean follicle concentration (2.85 ± 3.7/mm2, n = 16 with prior chemotherapy versus 1.68 ± 2.80/mm2, n = 9 without). Of the four patients in whom no primordial or primary follicles were found, only two had previously undergone chemotherapy.

None of the cases had visible ovarian tumour components.

Ovarian cryopreservation was not carried out for 20 patients aged from 1 to 42 years old (Table IIGo). Their mean age was 25 ± 12.2 years, and five patients were under 18 years old. In five cases (A, B, C, G, T), the treatment was not judged to be a risk for future fertility, either because drugs such as bleomycin were to be used (A, B), because no alkylating agent had been administered (C, T) or the planned treatment had been changed because corticotherapy is better than cyclophosphamide for the treatment of a nephrotic syndrome (G). Three patients already had premature ovarian failure as shown by amenorrhoea and increased levels of FSH when ovarian cryopreservation was requested (D, F, I). They had all previously undergone chemotherapy, which was done in association with a bone marrow autograft in one case (D). Cryopreservation was not carried out in three women (E, S, R), aged 41.8, 42.1 and 38.5 years old, because of the very low probability of obtaining sufficient numbers of oocytes to preserve and the very low chance that efficient technologies could be used during the coming years to restore their fertility. Three patients did not consent to ovarian tissue cryopreservation, two (J, M) were adults and in one case the parents refused on behalf of their child (Q). They all said that they refused the technique because it was too new and uncertain. One woman (L) preferred to use an IVF treatment to cryopreserve embryos. In one case (H), the woman became pregnant and the treatment was delayed. Patient N was referred with a non-malignant ovarian tumour and no healthy ovarian tissue was surgically removed with the tumour. In patient O, who had a suspected borderline ovarian tumour, ovarian tissue collection for cryopreservation was cancelled because the diagnosis was not confirmed by tumour markers CA 125 and CA 19-9. In one case with sickle cell disease, the anaesthesiologist did not clear the patient for surgery because of the risks involved with giving general anaesthesia. Finally, in one patient with a borderline ovarian tumour (K), the histological analysis could not identify ovarian tissue and so the process was not continued.


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Table II. Details of the 20 cases in which no ovarian cryopreservation was done
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ability to detect various cancers at an early stage and the use of more efficient therapies have improved the long-term survival rate for many patients (Craft, 1998Go; Tamburini et al., 2000Go). However, the long-term side-effects of cytotoxic therapies, such as the loss of fertility, must be considered. The potential advantages of freezing and storing human oocytes and/or embryos successfully have already been discussed (Carroll, 1996Go) but the cryopreservation of ovarian tissue may be an alternative. Our study showed that this technique can be proposed to young women and prepubertal girls at risk of losing ovarian function in very different situations including bone marrow transplantation and treatments for cancer disease or non-malignant disease as sickle cell disease (Bernaudin et al., 1997Go). This is the first detailed report concerning a large number of patients requesting ovarian tissue cryostorage for fertility preservation.

The greatest advantage of ovarian tissue cryopreservation over oocyte or embryo cryopreservation is that it can be proposed to young girls. In our series, the youngest patient was 2.7 years old and 16 patients were under 18 years old. Furthermore, the chance of restoring fertility should be higher in younger girls as their ovarian cortex clearly contains more primordial and primary follicles.

The efficiency of ovarian tissue autografting and/or in-vitro maturation of follicles and oocytes has not yet been clearly demonstrated in humans. This should be taken into consideration and it should be seriously discussed when the procedure is proposed to older women. In our series it was decided not to cryopreserve ovarian tissue for women >35 years old. Young children are unlikely to ask to use their ovarian cortex; nevertheless they may have the chance of restoring fertility through the use of cryopreserved ovarian tissue when they reach reproductive age.

Our histological analysis provided some interesting information on ovarian tissue cryopreservation. The concentration of primordial and primary follicles appeared not to be influenced by previous low-dose chemotherapy since the mean number of follicles was not significantly lower in patients, over 10 years old, previously treated with such a regimen. Cryopreservation of ovarian tissue could not be undertaken for three patients aged 24–33.5 years who had already received high-dose chemotherapy and who presented the clinical and biological symptoms of ovarian failure. Clearly more information is needed to evaluate the effect of drugs on ovarian function and to determine the indications for ovarian tissue cryopreservation.

From our results, it is possible to define the size of the ovarian tissue sample that should be preserved to provide a given number of follicles after thawing. We found that the mean concentration of primordial and primary follicles was ~350–400/mm3 before 10 years, 70–80/mm3 between 10 and 15 years and 30–35/mm3 from 15 to 34 years, thus the size of the ovarian sample should change with the patient's age. For example, if the thickness of the dissected ovarian tissue is ~1 mm, the surface area of each fragment should be ~3 mm2 before 10 years, 15 mm2 between 10 and 15 years and 50 mm2 from 15 to 34 years in order to cryopreserve >=1000 primordial or primary follicles. Indeed it is the number of functional follicles after freezing and thawing which should be estimated. It has been shown previously that the follicle survival rate was 74% after freezing, using the same method, thawing and xenografting (Newton et al., 1996Go). Therefore, >700 follicles are expected to be available from each fragment. If more follicles are needed, the size of the frozen fragment or the number of thawed samples could be adapted accordingly. In this study, it was chosen to collect half, then a whole, ovary and to freeze a large number of fragments. This strategy could be revised once clinical pregnancies have been established with oocytes from frozen–thawed ovarian grafts.

In our study, seven patients had ovarian tissue collected in association with another surgical procedure, so no more anaesthesia was necessary. In the 24 remaining cases an additional anaesthesia had to be performed. Usually the patients had to stay 2 days in the hospital. It was generally well accepted and no post surgical complications were reported. The cancer treatment could begin very quickly when the ovarian collection was made by laparoscopy. In most cases, commencing treatment was not delayed because of the procedure. This required close collaboration between the various partners involved in the programme.

The cost of the ovarian tissue cryopreservation process has not been evaluated in this study. It could be separated into three parts: the cost of the surgical act, which is similar to a standard laparoscopy (€1843, in France); ovarian tissue freezing, which might be similar to the cost of freezing of testicular sperm obtained after testicular dissection (€157, in France); and the annual cost of ovarian tissue storage, which might be similar to the cost of embryo cryopreservation (€32, in France).

Nine of the patients died during the months following the cryopreservation, seven of whom were under 18 years old. This questions the legitimacy of proposing the technique when the prognosis is very bad. This is a difficult question. In this feasibility study, we chose not to refuse the procedure only because of a bad prognosis and we left the women or the young girls and their parents to take the decision after supplying information and counselling. We observed in some cases that providing patients and/or their parents with the information on ovarian tissue cryopreservation was beneficial, since it offered an optimistic perspective for the future in an otherwise stressful context. Furthermore, there is no clear indicator of an individual's prognosis and children with ovarian failure could survive for long periods following treatment (Teinturier et al., 1998Go). Clearly, the place of the future health status of the women and the young girls among the criteria to decide the ovarian tissue cryopreservation needs to be discussed further in relation to ethical, legal and medical issues. Indeed, the patients and their parents should be offered careful psychological counselling and follow-up both when the cryopreservation is foreseen and if the storage of the ovarian tissue has to be stopped in the case of the child's death.

In conclusion, this first study shows that cryopreservation of ovarian tissue may be proposed for a large variety of diseases, that younger patients should be given priority and that it is well-accepted by motivated patients despite the current uncertainties of using thawed ovarian tissue to restore fertility. Further studies should be done to determine the incidence of patients who will need and accept ovarian tissue cryopreservation.


    Notes
 
7 To whom correspondence should be addressed at: Laboratoire de Biologie de la Reproduction, Pavillon Benjamin Delessert, Groupe Hospitalier Pitié-Salpêtrière, 83, bd. de l'Hôpital, 75013 Paris, France. E-mail: catherine.poirot{at}psl.ap-hop-paris.fr Back


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 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 10, 2001; resubmitted on November 2, 2001; accepted on January 23, 2001.