Abstract
Objectives
Patients scheduled to receive chemotherapy should be counselled on fertility preservation. Known gonadotoxic chemotherapies such as alkylating agents have a high risk of altering ovarian reserve. In some cases, the urgency of treatment requires the use of chemotherapy before fertility preservation, which will be carried out at a later stage. Usually the ovarian tissue is cryopreserved. The aim of our study is to investigate the impact of chemotherapies on follicular density and the apoptosis of reserve follicles.
Methods
We included 140 patients: 63 patients, mean age 18.8 years, were included in the group “no chemotherapy” (group A) and 77 patients, mean age 17.1 years, in the group “received chemotherapy before ovarian conservation” (group B). None of the patients had had pelvic radiotherapy prior to ovarian cryopreservation. The histological parameters studied were follicular density and the presence of malignant cells. We selected 12 patients from group A and 15 patients from group B, comparable in age and pathology, for whom we evaluated follicle apoptosis by immunostaining cleaved caspase‐3.
Results
We demonstrated an inverse relationship between follicular density and age (p < 0.0001), as well as a lack of effect of chemotherapy on follicular density (p = 0.87). We showed the impact of various chemotherapies, especially with alkylating agents, on the apoptosis of ovarian follicles (p < 0.0001). Three patients had ovarian tissue infiltration, two of which were malignant.
Conclusion
This work underlines the fact that conservation of ovarian tissue after chemotherapy remains possible.
Keywords: apoptosis, chemotherapy, quality of life, women's cancer
Ovarian follicle apoptosis by caspase‐3 cleavage staining: Section of ovarian cortex from a patient who received alkylating agents before preservation, with ovarian follicles labelled with an anti‐cleaved caspase‐3 antibody (×400 magnification).
1. INTRODUCTION
Patients scheduled to receive chemotherapy should be counselled on fertility preservation. Known gonadotoxic chemotherapies such as alkylating agents have a high risk of altering ovarian reserve. 1 In some cases, the urgency of treatment requires the use of chemotherapy before fertility preservation, which will be carried out at a later stage. The most common approach is ovarian tissue cryopreservation. To date, more than 200 live births have been published following the use of this procedure, which involves the slow freezing of ovarian tissue fragments, subsequent thawing and then orthotopic grafting, 2 , 3 , 4 , 5 and it is the reference method for ovarian tissue preservation. Two live births have been reported after the grafting of ovarian tissue that was cryopreserved by vitrification. 6 Since the technique of freezing fragments of human ovarian tissue has become more widely practiced in assistive reproductive technology (ART) laboratories, numerous studies have been carried out to investigate the effect of chemotherapy on follicular density in the tissue samples 7 , 8 , 9 , 10 , 11 and to search for tumour cells in these fragments, the presence of which is a contraindication to transplantation. 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 The first studies studied the effects of complete chemotherapy (ie induction, consolidation, and maintenance) on follicular reserve: Himelstein‐Braw et al and Marcello et al found a reduction in the total number of follicles after chemotherapy for acute leukaemia. 20 , 21 In addition, Marcello et al studied follicle quality and reported an absence of follicular abnormality. Finally, Familiari et al used electron microscopy to analyse the ovarian tissue of patients treated for Hodgkin's disease: abnormalities were found, with vacuolation of oocytes and granulosa cells, associated with thickening of the basement membrane. 22
When the procedure for freezing fragments of ovarian tissue was first developed, several articles were published on the population of patients who had received chemotherapy prior to ovarian cryopreservation. The aim of these studies was to highlight, or not, one or more differences (using optical and/or electron microscopy) between this population and the population of patients who did not receive chemotherapy prior to cryopreservation (ie the control group). For patients receiving chemotherapy prior to ovarian retrieval, the chemotherapy protocols vary greatly in intensity (from a single dose of induction chemotherapy to full chemotherapy, with the patient being referred by the onco‐haematologists when the disease progresses or recurs); some studies have found no histological difference between the chemotherapy group and the control group who had not received chemotherapy, prior to ovarian cryopreservation. 7 , 8 , 11 By contrast, other studies have found histological differences between these two groups. 11 Meirow et al studied ovarian tissue from patients who underwent surgery for ovarian cryopreservation before potentially sterilising chemotherapy. In the group of patients who had started chemotherapy, several abnormalities were present: blood vessel wall abnormalities, anarchic neovascularisation in the ovarian cortex, and focal cortical fibrosis. According to the authors of the study, all of these abnormalities are consequences of focal ischemia, which contributes to the decrease in ovarian reserve. However, the elderly patients in the control group exhibited these same anomalies. 9
The main objective of our study was the histological examination of ovarian fragments from 140 patients referred to the department of reproductive medicine of the Lyon hospitals for cryopreservation between August 1997 and March 2014. We compared the follicular density in women who had not received chemotherapy prior to cryopreservation with that of those women had received treatment. The secondary objective of the study was to compare follicle apoptosis within the ovarian cortex fragment in 12 treatment‐naïve women vs 15 women who received one or more courses of chemotherapy.
2. MATERIAL AND METHODS
2.1. Population
Between August 1997 and March 2014, 220 patients benefited from ovarian cryopreservation in the context of fertility preservation at the Department of Reproductive Medicine of the Lyon Hospitals. Patients whose anatomopathological analysis was not performed at our centre were excluded. Likewise, patients for whom incomplete data were obtained were excluded. We therefore included 140 patients in our study. We defined two groups: group A was composed of patients who had not received any treatment prior to cryopreservation, and group B was composed of patients who had received one or more potentially gonadotoxic treatments prior to ovarian cryopreservation. Group A, consisting of 63 patients with a mean age of 18.8 ± 9.5 years (1‐34 years), was comparable to group B, which contained 77 patients with a mean age of 17.1 ± 9.41 years (1‐35 years). We retrieved the treatment protocols for the patients in group B. The chemotherapy molecules received were classified into five categories: alkylating agents (alkylating), topoisomerase inhibitors (TI), antimetabolites (AM), other inhibitors of DNA synthesis (AISA), and immunosuppressants (IS). None of the patients had had pelvic radiotherapy prior to ovarian cryopreservation.
2.2. Histological study
During the dissection of the ovary, which was performed in accordance with our laboratory freezing procedure, 23 a fragment of cortex and a fragment of medullary were systematically sent to the anatomy and pathology laboratory in order to count the ovarian follicles over the whole cortex and to look for tumour cell invasion. After fixation in 4% formaldehyde (VWR) at room temperature, the fragments were included in paraffin, and then cut into 4‐μm serial sections using a microtome. Six slices were obtained, with a spacing of 60 μm, which were stained with haematoxylin (Millipore), eosin (Sigma‐Aldrich), and saffron (RAL diagnostics) (HES) stain. The follicular density was calculated by relating the total number of resting follicles present on the histological slide to the total area of the biopsy. Double‐blind counting of all follicles was carried out by a pathologist and an embryologist (at the Anatomopathology Department of the Hospices Civils de Lyon) via a microscope follicle count and photography of the slides (surface measurements were obtained using the ImageJ software package) (Figure 1). The results were expressed in terms of the number of follicles per mm2. Where tumour cells were mentioned in the anatomopathological report, we examined the immunohistochemical results.
FIGURE 1.
Section of human ovarian tissue stained with haematoxylin, eosin and saffron (HES). Primordial and primary follicles are indicated by white arrows. (A) No chemotherapy before preservation; (B) chemotherapy before preservation
2.3. Study of apoptosis
Among the 140 patients included, we selected 12 patients from group A and 15 patients from group B, who were comparable in terms of age and pathology. Patients were classified into age groups: one group for patients under 10 years, and 5‐year age brackets thereafter. They were further classified according to pathology: haematological malignancy, solid tumour and benign haematological pathology. Among the 15 patients in group B, 6 had received alkylating agents outside of the doxorubicine, bleomycine, vinblastine, and dacarbazine (ABVD) protocol (the alkylating agents were cyclophosphamide and ifosfamide), 5 had been treated according to the ABVD protocol, and 4 had received chemotherapy containing no alkylating agent. We separated the patients treated with ABVD chemotherapy from those treated with alkylating agents because dacarbazine is a monofunctional alkylating agent, so the toxicity was low. This ABVD treatment is described by oncologists as low gonadotoxic. 24 For all patients, we obtained two slices (with a spacing of 60 μm), which we stained with the Cleaved Caspase‐3 Kit (Asp 175, ≠9661, Cell Signaling Technology) according to the manufacturer's protocol. This marking allowed us to evaluate the apoptosis of the follicles within the tissue. A confocal fluorescence microscope was used for double‐blind reading of the slides. On average, 18.3 follicles were counted per slide (range: 2‐43). We performed a positive control, which was a histological thymus slide (tissue with very high apoptosis) (Figure 2) and a negative control, which was a slide of ovarian tissue upon which we did not deposit the primary antibody (Figure 2). A follicle was considered positive when the whole follicle showed a fluorescence of more than 50% homogeneously (Figure 2) or when the oocyte was fluorescent.
FIGURE 2.
Ovarian follicle apoptosis by caspase‐3 cleavage staining: (A) Section of human thymus stained with an anti‐cleaved caspase‐3 antibody, positive control (×400) (99% of the cells were positive). (B) Section of human ovarian cortex without primary antibody, negative control (×400). (C) Section of human ovarian cortex from a patient who received alkylating agents before preservation, with ovarian follicles labelled with an anti‐cleaved caspase‐3 antibody (×400). (D) Section of human ovarian cortex from a patient who did not receive chemotherapy prior to preservation, with follicles no labelled (white arrow) (×400)
2.4. Statistical analysis
The statistical analyses were performed using the “R” software package (version 2.15.2). Comparisons between pairs of percentage values were carried out using a Chi square test, comparisons between pairs of average values were conducted using Student's t‐test, and comparisons between more than two average values were carried out using analysis of variance (ANOVA) testing. ANOVA was performed to investigate how follicular density varied according to the age of the patients. Test results were considered significant for p < 0.05.
3. RESULTS
3.1. Description of the population
Of the 140 patients included in the study, 63 had not received treatment prior to ovarian cryopreservation (group A) and 77 had received treatment prior to ovarian cryopreservation (group B). In Group A, 40 patients were referred prior to cancer treatment, 15 were referred for treatment of non‐malignant haematological diseases, 7 were referred for treatment of autoimmune diseases, and 1 had a genetic disease.
The 40 patients with cancer consisted of 14 cases of Hodgkin's disease, 6 cases of Ewing's sarcoma, 4 cases of sarcoma, 1 case of neuroblastoma, 2 cases of non‐Hodgkin's malignant lymphoma, 1 case of breast cancer, 3 cases of adenocarcinoma, 2 cases of myelodysplasia and 2 cases of mesotheliomas.
Non‐malignant hemopathies were represented by 6 cases of sickle cell disease, 2 cases of thalassemia, 3 cases of idiopathic bone marrow aplasia, 3 cases of Fanconi disease, and 1 case of agranulocytosis. The 7 cases of autoimmune disease comprised 2 cases of lupus, 1 case of vasculitis, 1 case of idiopathic thrombocytopenic purpura, 1 case of sarcoidosis, 1 case of scleroderma and 1 severe case of combined immune deficiency. Finally, the genetic disease was Turner syndrome, with a mosaicism of 8%; the patient had experienced spontaneous puberty associated with normal growth but showed a progressive decrease in anti mullerian hormon.
Group B, consisting of 77 patients, was composed of 75 cases of cancer and 2 of non‐malignant haematological disease. The 75 cases of cancers comprised 17 cases of Hodgkin's disease, 4 cases of Ewing's sarcoma, 17 cases of acute leukaemia, 11 cases of neuroblastoma, 9 cases of non‐Hodgkin's malignant lymphoma, 4 cases of renal cancer, 4 cases of medulloblastoma, 2 cases of chronic myeloid leukaemia, and 7 cases of other cancers.
Finally, the 2 non‐malignant hemopathies were major thalassemia (treated with hydroxycarbamide) and idiopathic bone marrow aplasia (treated with cyclophosphamide).
Group A contained more cases of non‐malignant hematopathies and autoimmune diseases than group B. By contrast, group B had more malignant haematological diseases, including acute leukaemia and neuroblastoma—pathologies that require emergency chemotherapy. None of the patients in group B received pelvic radiotherapy. 55 were treated with chemotherapy containing alkylating agents. Of these 55 patients, 9 had been treated according to the ABVD protocol.
3.2. Follicular density study
The mean follicular density was 8.8 follicles/mm2 (range: 0‐266.7). The number of ovarian biopsies without follicles on histological examination was comparable in both groups (9.7% in group A vs 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 3). This result was observed in all groups.
FIGURE 3.
Distribution of follicular density (follicles/mm2) according to patient age (years)
The mean ± standard deviation of follicular density was 9.1 follicles/mm2 ± 33.8 (range: 0‐266) for group A, and 9.9 follicles/mm2 ± 18.6 (range: 0‐75) for group B (Figure 4). 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, 9 of whom had received ABVD. The mean age of this subgroup was 17.0 ± 9.6 years. The mean ± standard deviation of follicular density for the patients who received alkylating agents was 8.8 follicles/mm2 ± 14.8, which 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.
FIGURE 4.
Follicular density (follicles/mm2) according to groups: group A (without chemotherapy), group B (with chemotherapy before cryopreservation of ovarian cortex)
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), and age is known to impact follicular density (Figure 5).
FIGURE 5.
Follicular density (follicles/mm2) by treatment group (group A: without chemotherapy, group B: chemotherapy before cryopreservation of ovarian cortex) in different age subgroups
3.3. 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 in terms of age, which is an important factor in follicular reserve, and type of pathology. The patient groups were comparable (Table 1). The mean age in the “no chemotherapy” group was 19.5 ± 8.7 years (range: 3‐31) and the mean age in the “received chemotherapy” group was 19.9 ± 7.5 years (range: 6‐31) (p = 0.904). The “no chemotherapy” group consisted of 7 haematological malignancies and 4 solid tumours. The “received chemotherapy” group consisted of 9 haematological malignancies and 5 solid tumours. Each group contained one benign haematological pathology.
TABLE 1.
Comparison of the populations of the two groups
| Group A (without chemotherapy) | Group B (chemotherapy) | |
|---|---|---|
| Patients (n) | 12 | 15 |
| Age (years) mean ± SD | 19.5 ± 8.7 | 19.9 ± 7.5 (p = 0.904) |
| Range (years) | 3‐31 | 6‐31 |
| Haematological malignancies (n) | 7 | 9 |
| Hodgkin lymphoma | 5 | 6 |
| B cell lymphoma | 1 | 1 |
| Myelodysplasia | 1 | 0 |
| Acute leukaemia | 0 | 2 |
| Solid tumours (n) | 4 | 5 |
| Ewing sarcoma | 2 | 2 |
| Neuroblastoma | 1 | 1 |
| Rectal adenocarcinoma | 1 | 1 |
| Nephroblastoma | 0 | 1 |
| Benign haematological diseases (n) | 1 | 1 |
| Idiopathic thrombocytopenic purpura | 1 | 0 |
| Idiopathic medullar aplasia | 0 | 1 |
We analysed 202 follicles from patients in the “no chemotherapy” group and 291 ovarian follicles in the “received 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 “received chemotherapy” group (p = 0.741). The percentage of positive follicles in the “no chemotherapy” group was 5.9% vs 15.1% in the “received chemotherapy” group (p < 0.01). The extent of apoptosis was significantly higher in the group receiving chemotherapy prior to ovarian cryopreservation.
In the “received 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 vs 5.9% in the no chemotherapy group (p < 0.0001). The presence of alkylating agents outside of the ABVD protocol resulted in a significant increase in apoptosis in ovarian follicles.
A total of 83 follicles from the group that received ABVD prior to ovarian cryopreservation were analysed. Two follicles were positive, representing 2.4% of the total. 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 examined (ie 10.6% of the follicles, or twice the percentage of follicular apoptosis observed in group A). The lack of significance may be related to the low number of follicles analysed.
It is interesting to note that follicular apoptosis was always present in the granulosa cells. We did not observe any positive oocyte alone.
3.4. Histological study of the infiltration of the ovarian tissue
Three patients had medullar infiltration: in 2 of them (1.5% of patients), the infiltration was found to be malignant (lymphocytic infiltration in non‐Hodgkin lymphoma, and lymphoblast infiltration in chronic myeloid leukaemia). Only one infiltration was benign (lymphoplasmocyte infiltration without significance in lupus).
Of the 2 patients for whom histology tests found malignant cells, one was 25 years old at the time of cryopreservation and had acute chronic myeloid leukaemia in acute lymphoblastic leukaemia. They had been treated with imatinib and vincristine (AISA: other inhibitors of DNA synthesis) prior to ovarian cryopreservation. The second patient was 20 years old at the time of cryopreservation and had diffuse CD20+ large B‐cell lymphoma with osteomedullary localisation. The patient did not receive chemotherapy prior to ovarian cryopreservation.
4. DISCUSSION
The aim of our study was to evaluate the reserve follicles of ovarian tissues of patients before and after treatment with chemotherapies. To do this, we studied the follicular density and evaluated the apoptosis of ovarian follicles and the explored relationship of these characteristics to chemotherapy treatment. Our study is the largest cohort reported to date. We have demonstrated a link between patient age and follicular density. Herein, as age increases, follicular density also decreases. This correlation has been described in several other articles. 7 , 11 , 12
We have also shown the absence of an effect of chemotherapies, especially with alkylating agents, on follicular density. These results corroborate those found in other studies. 7 , 8 , 10 , 11 By contrast, Oktem and Oktay described a statistically significant decrease in ovarian reserve following chemotherapy with alkylating agents. However, the number of patients was low (n = 5) and the mean age of the group receiving alkylating agents was high (29.8 years old). 12 Meirow et al described an effect of chemotherapy on ovarian tissue. They reported neovascularisation and fibrosis in patients previously treated with chemotherapy. Neovascularisation was demonstrated using immunohistochemical markers (anti‐CD31 and anti‐CD34), which we did not use in our study. 9 The absence of any effect of the treatments on follicular density does not mean the absence of qualitative alterations in the ovarian follicles: follicular alterations were observed as intracytoplasmic oocyte vacuole, nuclear abnormalities of granulosa cells. 10 These anomalies can be explained by an initiation of apoptosis of the follicles. We evaluated the apoptosis of follicles according to the presence or absence of chemotherapy and found a significant increase in apoptosis of the follicles of women previously treated with chemotherapy and, more specifically, chemotherapies containing alkylating agents. These results corroborate those found by Soleimani et al and Li et al 25 , 26 Soleimani et al and then Li et al studied the effect of doxorubicin and cyclophosphamide in ovarian xenografts. 25 , 26 Recently a team has demonstrated another phenomenon of follicular loss; they showed follicular activation of reserve follicles after chemotherapy with alkylating agents. The number of growing follicles was increased a few days after treatment. 27 We did not observe this phenomenon, probably due to the time between treatment and preservation of ovarian tissue, which was longer in our cohort by several months. They did not observe an increase in follicular apoptosis immediately after chemotherapy. Another important point is the age of the patients: they were all pubescent, 27 unlike in our study, where the average age is lower. It would be interesting to evaluate this hypothesis for ovarian tissue from non‐pubertal girls.
We have demonstrated a lack of effect of chemotherapies on follicular density and an increase in follicular apoptosis. Chemotherapies did not quantitatively alter the ovarian follicles but probably did so qualitatively. Our results are in line with those of Luan's team; they showed that cyclophosphamide, which is an alkylating agent, did not decrease the number of primordial follicles but activated the signalling pathways of apoptosis. 28 The effect on these follicles could be modified after the transplant. It would be interesting to compare post‐graft apoptosis with that previously performed on fresh tissue to evaluate the repair mechanisms that may have been established. It is indirectly demonstrated by the live birth rate after ovarian tissue transplantation in patients who have received chemotherapy. This live birth rate was not affected by the history of chemotherapy. 5
5. CONCLUSION
In view of these results, it would be logical to carry out cryopreservation of ovarian tissue before treatment where possible. This work underlines the fact that patients treated with chemotherapy before ovarian cryopreservation can benefit from this fertility preservation technique. However, the use of alkylating agents prior to preservation surgery should be avoided. This should be discussed according to the clinical context because a balance must be found between the risk of invasion of ovarian tissue by malignant cells without the use of chemotherapies before ovarian conservation, and the use of alkylating agents that can prevent this invasion but alter the ovarian tissue. It is essential to remind oncology teams of the importance of referring patients promptly to reproductive medicine departments in order to preserve patients' fertility as optimally as possible.
AUTHOR CONTRIBUTIONS
Participation in the study design: EL, SB, BS, JL; data analysis: EL, SB, JL; execution and manuscript drafting: EL, SB, OL, GS; critical discussion: EL, SB, OL, GS, BS, JL.
FUNDING INFORMATION
This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.
CONFLICT OF INTEREST
The authors declare that they have no competing interests.
ACKNOWLEDGEMENTS
The authors would like to thank the Anatomopathology Department of the Hospices Civils de Lyon, particularly Dr Annie Buener.
Labrune E, Bianchetti S, Lepinasse O, Soignon G, Salle B, Lornage J. When to cryopreserve ovarian tissue: Determining the effects of chemotherapy on the ovarian reserve by studying follicular density and apoptosis. Cytopathology. 2023;34:146‐153. doi: 10.1111/cyt.13194
DATA AVAILABILITY STATEMENT
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
REFERENCES
- 1. Oktay K, Harvey BE, Loren AW. Fertility preservation in patients with cancer: ASCO clinical practice guideline update summary. J Oncol Pract. 2018;14(6):381‐385. doi: 10.1200/JOP.18.00160 [DOI] [PubMed] [Google Scholar]
- 2. Donnez J, Dolmans MM. Fertility preservation in women. N Engl J Med. 2018;378(4):400‐401. doi: 10.1056/NEJMc1715731 [DOI] [PubMed] [Google Scholar]
- 3. Dolmans MM, Falcone T, Patrizio P. Importance of patient selection to analyze in vitro fertilization outcome with transplanted cryopreserved ovarian tissue. Fertil Steril. 2020;114(2):279‐280. doi: 10.1016/j.fertnstert.2020.04.050 [DOI] [PubMed] [Google Scholar]
- 4. Marin L, Bedoschi G, Kawahara T, Oktay KH. History, evolution and current state of ovarian tissue auto‐transplantation with cryopreserved tissue: a successful translational research journey from 1999 to 2020. Reprod Sci. 2020;27(4):955‐962. doi: 10.1007/s43032-019-00066-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Shapira M, Dolmans MM, Silber S, Meirow D. Evaluation of ovarian tissue transplantation: results from three clinical centers. Fertil Steril. 2020;114(2):388‐397. doi: 10.1016/j.fertnstert.2020.03.037 [DOI] [PubMed] [Google Scholar]
- 6. Suzuki N, Yoshioka N, Takae S, et al. Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency. Hum Reprod. 2015;30(3):608‐615. doi: 10.1093/humrep/deu353 [DOI] [PubMed] [Google Scholar]
- 7. Poirot C, Vacher‐Lavenu MC, Helardot P, Guibert J, Brugières L, Jouannet P. Human ovarian tissue cryopreservation: indications and feasibility. Hum Reprod. 2002;17(6):1447‐1452. doi: 10.1093/humrep/17.6.1447 [DOI] [PubMed] [Google Scholar]
- 8. Seshadri T, Gook D, Lade S, et al. Lack of evidence of disease contamination in ovarian tissue harvested for cryopreservation from patients with Hodgkin lymphoma and analysis of factors predictive of oocyte yield. Br J Cancer. 2006;94(7):1007‐1010. doi: 10.1038/sj.bjc.6603050 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Meirow D, Dor J, Kaufman B, et al. Cortical fibrosis and blood‐vessels damage in human ovaries exposed to chemotherapy. Potential mechanisms of ovarian injury. Hum Reprod. 2007;22(6):1626‐1633. doi: 10.1093/humrep/dem027 [DOI] [PubMed] [Google Scholar]
- 10. Abir R, Ben‐Haroush A, Felz C, et al. Selection of patients before and after anticancer treatment for ovarian cryopreservation. Hum Reprod. 2008;23(4):869‐877. doi: 10.1093/humrep/dem413 [DOI] [PubMed] [Google Scholar]
- 11. Fabbri R, Vicenti R, Macciocca M, et al. Cryopreservation of ovarian tissue in pediatric patients. Obstet Gynecol Int. 2012;2012:910698. doi: 10.1155/2012/910698 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Oktem O, Oktay K. Quantitative assessment of the impact of chemotherapy on ovarian follicle reserve and stromal function. Cancer. 2007;110(10):2222‐2229. doi: 10.1002/cncr.23071 [DOI] [PubMed] [Google Scholar]
- 13. Abir R, Feinmesser M, Yaniv I, et al. Occasional involvement of the ovary in Ewing sarcoma. Hum Reprod. 2010;25(7):1708‐1712. doi: 10.1093/humrep/deq121 [DOI] [PubMed] [Google Scholar]
- 14. Dolmans MM, Marinescu C, Saussoy P, Van Langendonckt A, Amorim C, Donnez J. Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe. Blood. 2010;116(16):2908‐2914. doi: 10.1182/blood-2010-01-265751 [DOI] [PubMed] [Google Scholar]
- 15. Dolmans MM, Jadoul P, Gilliaux S, et al. A review of 15 years of ovarian tissue bank activities. J Assist Reprod Genet. 2013;30(3):305‐314. doi: 10.1007/s10815-013-9952-x [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Dolmans MM, Luyckx V, Donnez J, Andersen CY, Greve T. Risk of transferring malignant cells with transplanted frozen‐thawed ovarian tissue. Fertil Steril. 2013;99(6):1514‐1522. doi: 10.1016/j.fertnstert.2013.03.027 [DOI] [PubMed] [Google Scholar]
- 17. Rosendahl M, Andersen MT, Ralfkiær E, Kjeldsen L, Andersen MK, Andersen CY. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia. Fertil Steril. 2010;94(6):2186‐2190. doi: 10.1016/j.fertnstert.2009.11.032 [DOI] [PubMed] [Google Scholar]
- 18. Bittinger SE, Nazaretian SP, Gook DA, Parmar C, Harrup RA, Stern CJ. Detection of Hodgkin lymphoma within ovarian tissue. Fertil Steril. 2011;95(2):803.e3‐803.e6. doi: 10.1016/j.fertnstert.2010.07.1068 [DOI] [PubMed] [Google Scholar]
- 19. Bastings L, Beerendonk CCM, Westphal JR, et al. Autotransplantation of cryopreserved ovarian tissue in cancer survivors and the risk of reintroducing malignancy: a systematic review. Hum Reprod Update. 2013;19(5):483‐506. doi: 10.1093/humupd/dmt020 [DOI] [PubMed] [Google Scholar]
- 20. Himelstein‐Braw R, Peters H, Faber M. Morphological study of the ovaries of leukaemic children. Br J Cancer. 1978;38(1):82‐87. doi: 10.1038/bjc.1978.166 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Marcello MF, Nuciforo G, Romeo R, et al. Structural and ultrastructural study of the ovary in childhood leukemia after successful treatment. Cancer. 1990;66(10):2099‐2104. doi: [DOI] [PubMed] [Google Scholar]
- 22. Familiari G, Caggiati A, Nottola SA, Ermini M, Di Benedetto MR, Motta PM. Ultrastructure of human ovarian primordial follicles after combination chemotherapy for Hodgkin's disease. Hum Reprod. 1993;8(12):2080‐2087. doi: 10.1093/oxfordjournals.humrep.a137985 [DOI] [PubMed] [Google Scholar]
- 23. Demirci B, Lornage J, Salle B, Frappart L, Franck M, Guerin JF. Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols. Fertil Steril. 2001;75(4):754‐762. [DOI] [PubMed] [Google Scholar]
- 24. Lee SJ, Schover LR, Partridge AH, et al. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. J Clin Oncol. 2006;24(18):2917‐2931. doi: 10.1200/JCO.2006.06.5888 [DOI] [PubMed] [Google Scholar]
- 25. Soleimani R, Heytens E, Darzynkiewicz Z, Oktay K. Mechanisms of chemotherapy‐induced human ovarian aging: double strand DNA breaks and microvascular compromise. Aging (Albany NY). 2011;3(8):782‐793. doi: 10.18632/aging.100363 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Li F, Turan V, Lierman S, Cuvelier C, De Sutter P, Oktay K. Sphingosine‐1‐phosphate prevents chemotherapy‐induced human primordial follicle death. Hum Reprod. 2014;29(1):107‐113. doi: 10.1093/humrep/det391 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Shai D, Aviel‐Ronen S, Spector I, et al. Ovaries of patients recently treated with alkylating agent chemotherapy indicate the presence of acute follicle activation, elucidating its role among other proposed mechanisms of follicle loss. Fertil Steril. 2021;115(5):1239‐1249. doi: 10.1016/j.fertnstert.2020.11.040 [DOI] [PubMed] [Google Scholar]
- 28. Luan Y, Edmonds ME, Woodruff TK, Kim SY. Inhibitors of apoptosis protect the ovarian reserve from cyclophosphamide. J Endocrinol. 2019;240(2):243‐256. doi: 10.1530/JOE-18-0370 [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
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Data Availability Statement
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.