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 I).
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
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 1B).
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 2.
In patients aged
7 years, the mean number of primordial and primary follicles was 20.36 ± 19.03/mm2
= 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
). 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 II). 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.