2.1.Description of the population
Of the 140 patients included in the study, 63 patients had not received treatment prior to ovarian cryopreservation (group A) and 77 patients had received treatment prior to ovarian self-conservation (group B). In Group A, 40 patients were referred prior to cancer treatment, 15 patients were referred for treatment of non-malignant hematological diseases, 7 patients were referred for treatment of autoimmune diseases, and 1 patient had a genetic disease.
The 40 cancers consisted of 14 Hodgkin's diseases, 6 Ewing's sarcomas, 4 sarcomas, 1 neuroblastoma, 2 non-Hodgkin's malignant lymphomas, 1 breast cancer, 3 adenocarcinomas, 2 myelodysplasias and 2 mesotheliomas.
Non-malignant hemopathies were represented by 6 sickle cell diseases, 2 thalassemias, 3 idiopathic bone marrow aplasias, 3 Fanconi diseases, and 1 agranulocytosis. The 7 autoimmune diseases were composed of 2 lupus, 1 vasculitis, 1 idiopathic thrombocytopenic purpura, 1 sarcoidosis, 1 scleroderma and 1 severe combined immune deficiency. Finally, the genetic disease was a Turner syndrome, with a mosaicism of 8%, which had had spontaneous puberty associated with normal growth but showed a progressive decrease in AMH.
Group B, consisting of 77 patients, was composed of 75 cancers and 2 non-malignant hematological diseases. The 75 cancers were 17 Hodgkin's diseases, 4 Ewing's sarcomas, 17 acute leukemias, 11 neuroblastomas, 9 non-Hodgkin's malignant lymphomas, 4 renal cancers, 4 medulloblastomas, 2 chronic myeloid leukemias, and 7 other cancers.
Finally, the 2 non-malignant hemopathies were major thalassemia and idiopathic bone marrow aplasia.
Group A is composed of more non-malignant hematopathies and autoimmune diseases than group B. On the other hand, group B had more malignant hematological diseases, including acute leukemia and neuroblastoma: pathologies that require emergency chemotherapy. As for the treatments received by the patients in group B, none of them included pelvic radiotherapy. 55 patients had been treated with chemotherapy containing alkylating agents. Of these 55 patients, 9 patients had been treated with the ABVD protocol.
2.2.Follicular density study
The mean follicular density was 8.8 follicles/mm2 (ranges: 0-266.7). The number of ovarian biopsies without follicles on histological examination was comparable in both groups (9.7% in group A versus 12.7% in group B, p=0.501). Follicular density was significantly inversely related to patient age (p<0.0001). The higher the age of the patient, the more the follicular density decreased (Figure 1). This result was observed in all groups.
The mean ± standard deviation of the follicular density of group A was 9.1 follicles/mm2 ± 33.8 (ranges: 0-266) and 9.9 follicles/mm2 ± 18.6 (ranges: 0-75) for group B (Figure 2). It is important to note that the average age of the two groups was comparable (18.8 ± 9.5 years for group A and 17.1 ± 9.4 years for group B). There was therefore no statistically significant difference in follicular density between the two groups (p=0.863). Chemotherapy treatments did not alter follicular density. The subgroup of patients who received alkylating agents consisted of 55 patients of whom 9 had received ABVD. The mean age of this subgroup was 17.0 ± 9.6 years. The mean ± standard deviation follicular density of the patients who received alkylating agents, which was 8.8 follicles/mm² ± 14.8, was comparable to that of group A (p=0.960). Seven patients had no follicles during histological analysis with a higher mean ± standard deviation age of 20 years ± 9.7.
The mean follicular density of group A was not statistically different from that of group B across age subgroups (p=0.638). There was no effect of chemotherapy; there was only an age effect (p=0.001), age being a known factor impacting follicular density (Figure 3).
2.3.Study of other histological parameters
Regarding the appearance of the vessels and the presence of cortical fibrosis of the ovarian tissue, the number of biopsies with these abnormalities was identical in both groups. In Group A, 8 biopsies had vessel abnormalities, which were statistically comparable to Group B, where 5 biopsies had the same abnormalities (12.7% vs. 6.5%). Similarly, cortical fibrosis was described in 4 patients in Group A versus 5 patients in Group B (6.3% vs. 6.5%).
Three patients had malignant medullar infiltration: in 2 of them (1.5% of patients), the infiltration was found to be malignant (lymphocyte infiltration in non-Hodgkin's malignant lymphoma, and lymphoblast infiltration in acute chronic myeloid leukemia). Only one infiltration was benign (lymphoplasmocyte infiltration without significance in lupus).
Of the 2 patients with histology finding malignant cells, one was 25 years old at the time of cryopreservation and had acute chronic myeloid leukemia in acute lymphoblastic leukemia. She had been treated with AISA (other inhibitors of DNA synthesis) prior to ovarian cryopreservation. The second patient was 20 years old at the time of cryopreservation, had diffuse CD20+ large B-cell lymphoma with osteomedullary localization. The patient did not receive chemotherapy prior to ovarian cryopreservation.
2.4.Study of ovarian follicle apoptosis by caspase 3 cleavage staining
In order to study the impact of chemotherapies on ovarian follicle apoptosis, we performed cleaved caspase 3 staining in 12 patients in group A and 15 patients in group B. We matched patients on age, which is an important factor in follicular reserve, and on the type of pathology. The patient groups were comparable (Table 1). The mean age in the "no chemotherapy" group was 19.5 ± 8.7 years (ranges: 3-31) and the mean age in the "presence of chemotherapy" group was 19.9 ± 7.5 years (ranges: 6-31) (p=0.904). The "no chemotherapy" group consisted of 7 hematological malignancies and 4 solid tumors. The "presence of chemotherapy" group consisted of 9 hematological malignancies and 5 solid tumors. Each group was composed of one benign hematological pathology.
We analyzed 202 follicles from patients in the "no chemotherapy" group and 291 ovarian follicles in the "presence of chemotherapy" group. The mean ± standard deviation number of follicles observed per patient was 16.8 follicles ± 9.2 in the “no chemotherapy” group and 18.2 ± 11.5 in the “presence of chemotherapy” group (p=0.741). The percentage of positive follicles in the "no chemotherapy" group was 5.9% vs. 15.1% in the "presence of chemotherapy" group (p<0.01). Apoptosis was significantly greater in the group receiving chemotherapy prior to ovarian cryopreservation.
In the "presence of chemotherapy" group, 161 follicles were derived from 6 patients who received non-ABVD alkylating agents before ovarian cryopreservation. The alkylating agents were cyclophosphamide, ifosfamide and/or oxaliplatin. The percentage of positive follicles was 22.9% in this group versus 5.9% in the no chemotherapy group (p<0.0001). The presence of alkylating agents, outside the ABVD protocol, resulted in a significant increase in apoptosis in ovarian follicles.
83 follicles from the group that received ABVD prior to ovarian cryopreservation were analyzed. 2 follicles were positive, representing 2.4% of the follicles. The use of ABVD did not result in more apoptosis than that observed in the group without chemotherapy.
Finally, the other chemotherapies did not seem to increase the apoptosis of the ovarian follicles. In fact, 5 follicles were positive out of the 47 follicles read, i.e. 10.6% of the follicles, or twice the percentage of follicular apoptosis of group A. The lack of significance may be related to the low number of follicles analyzed.
It is interesting to note that follicular apoptosis was always present in the granulosa cells. We did not observe any positive oocyte alone.