Fertility preservation and leukemia : cellular components of the artificial ovary and disease retransmission through the graft
(2015)
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- Ovarian tissue cryopreservation (OTC) prior to gonadotoxic therapy is currently the only means of preserving fertility in prepubertal girls and women requiring prompt treatment. Leukemia accounts for 11% of OTC procedures in our department. Despite growing success, with more than 60 live births to date, the safety of ovarian tissue transplantation remains of great concern in certain types of cancer, including leukemia, due to the possible risk of grafting cancer cells back to the patient. Previous studies appear to show low concentrations of malignant cells in ovarian tissue from leukemia patients, but there is no information on the number of leukemic cells capable of inducing disease. Our aim is to offer a realistic and effective fertility restoration strategy for leukemia patients and others undergoing OTC procedures, but in whom reimplanting ovarian tissue is not an option due to the risk of malignant cell involvement. In this context, our study has focused on the three following issues: 1. Minimal disseminated disease (MDD) in leukemia patients and risk of disease transmission through the graft: We confirmed the presence of malignant cells in cryopreserved ovarian tissue from leukemia patients by sensitive PCR techniques in 55% of cases. However, none of the grafted ovarian fragments from these patients resulted in leukemic masses in SCID mice after 6 months of grafting. We found that a high blood blast count (>200/µl) in patients at the time of OTC appears to be associated with positive PCR in ovarian tissue, but a low or negative blast count does not exclude ovarian involvement. We also showed that as many as 100 live leukemic BV-173 cells grafted inside an artificial ovary environment are insufficient to induce leukemia in SCID mice after 20 weeks of grafting. PCR proved more sensitive for leukemic cell detection than flow cytometry, at least for fibrous tissues like the ovary, where digestion and filtration cause a certain degree of malignant cell loss. 2. Safe follicle isolation for patients at risk of MDD: For patients running the risk of ovarian involvement, a safe alternative would be transplantation of ovarian follicles, enzymatically isolated from frozen-thawed ovarian tissue and embedded in 3D matrices. However, as follicles are recovered from potentially contaminated digested tissue suspensions, it is crucial to ensure that they are free of malignant cells before they can be safely grafted to patients. Using a model of healthy ovarian tissue artificially contaminated with leukemic cells, we showed that some malignant cells may possibly be retrieved along with isolated follicles during follicle pick-up. With this same model, we also demonstrated the usefulness of a simple additional procedure involving three washes to effectively eliminate leukemic cells present in follicle suspensions, while maintaining good follicle viability. A protocol for very sensitive detection of leukemic cells in isolated follicle suspensions, allowing detection of as few as 2 malignant cells by PCR, was subsequently set up. Using this protocol, our follicle isolation technique with three additional washes was tested on cryopreserved ovarian tissue from leukemia patients, and yielded recovery of a large number of highly viable preantral follicles. Most importantly, all isolated follicle suspensions were found to be completely negative for leukemic cell presence, even those recovered from contaminated digested cell suspensions, proving that these follicle suspensions can be safely grafted back to patients. 3. Origin of stromal cells for the artificial ovary: Addition of ovarian (stromal and endothelial) cells to the artificial ovary was found to be crucial to obtain a well vascularized ‘ovary-like’ structure after grafting, and is essential for follicle survival and growth. We studied the impact of freezing and thawing procedures, as well as the cortical or medullary origin of ovarian tissue, on isolated ovarian cell viability and in vivo proliferation and survival, with the aim of determining the best origin of stromal cells for the artificial ovary. We showed that cryopreservation procedures (slow-freezing) have a negative impact on isolated ovarian stromal cell number and viability. The medulla, on the other hand, was shown to yield more cells with greater viability and a higher percentage of endothelial cells than the cortex. This higher endothelial cell percentage was also correlated with increased graft vascularization. Our findings therefore point to fresh medullary tissue being the best source of stromal cells for the artificial ovary, as they can be isolated in larger numbers, show higher viability and are able to improve graft vascularization. Our experimental study has complemented previous understanding of the risk of ovarian involvement and disease transmission in leukemia patients, and examined key issues (including safe follicle isolation and origin of stromal cells) implicated in the creation of an artificial ovary as a fertility restoration option for these patients. We have highlighted certain areas that need to be addressed to continue making progress in this field. Finding the ideal scaffold for human follicle encapsulation, investigating and improving post-grafting isolated preantral follicle survival rates, and confirming the capability of isolated human ovarian stromal cells to differentiate into theca cells may help us turn our experimental model into a clinical reality.
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Soares, M. (2015). Fertility preservation and leukemia : cellular components of the artificial ovary and disease retransmission through the graft. https://hdl.handle.net/2078.5/26660