Preservation of fertility in women : investigation of different approaches
(2007)
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- Progress in therapies for several chronic diseases has resulted in an increased number of long-term survivors. Unfortunately, ovaries are sensitive to chemo- and/or radio-therapy that severely affect the ovarian follicular store and subsequently lead to a loss of fertility and premature menopause. There are relatively few effective clinical options for preserving women fertility: embryo cryopreservation, oocyte cryopreservation and ovarian tissue cryopreservation. The choice depends on various parameters: the type and timing of therapy, the type of pathology, the patient's age and the partner status. Most female cancer patients of reproductive age do not have the option of utilizing established assisted reproductive technologies as embryo cryopreservation to safeguard their fertility. A promising alternative to prevent fertility loss in these patients is the cryopreservation and transplantation of ovarian tissue. Our study focused on cryopreservation and transplantation of ovarian tissue as cortical fragments, whole ovary with its vascular pedicle and isolated follicles, in order to propose the most suitable transplantation procedure in each clinical situation (type of cancer, patient's age, risk of cancer transmission). In view of their routine application in clinical practice, we analysed these three experimental options to preliminary establish whether these assisted reproductive technologies may compromise the oocyte and ovarian tissue morpho-physiology. Our objective was to evaluate the impact of cryopreservation, grafting or enzymatic isolation on the tissue and cell integrity of human ovarian tissue, using mainly TEM: 1) Cryopreservation and grafting of ovarian cortical fragments have already demonstrated to be clinically applicable, allowing restoring endocrine and gametogenic function in young patients with iatrogenic premature ovarian failure. However, transplantation procedure, as well as cryopreservation, may affect female gamete integrity. We analysed follicular integrity after cryopreservation and grafting of human ovarian fragments in both nude mouse model (xenotransplantation) and in a human autograft. We demonstrated that freeze-thawing and grafting do not appear to greatly affect human primordial/primary follicle ultrastructure. Interestingly, we observed an asynchrony between oocyte and granulosa cell development in well preserved secondary human ovarian follicles in short-term xenografts. This might result in functional uncoupling between oocytes and granulosa cells. Further studies investigating follicular ultrastructure after long-term grafting led us to better understand the developmental competence of these follicles. 2) Cryopreservation of whole ovary with its vascular pedicle allows minimizing post-transplantation ischemia improving the graft lifespan. We cryopreserved three intact human ovaries by perfusing the ovarian vessels with a passive cooling device. After thawing, we analyzed with particular attention the vascular compartment integrity that represents a critical factor for re-anastomosis between ovarian vascular pedicle and ovarian vessels. Our protocol ensured a well-preserved ultrastructure, as well as no induction of apoptosis, in follicular, vascular and stromal compartments after thawing. Our results are encouraging and human whole ovary transplantation may be a viable option in the future. However, further studies investigating vascular patency after whole ovary cryopreservation and transplantation are needed to assess the feasibility of this technique into clinical practice. 3) Isolation and transplantation of ovarian follicles may allow transplanting ovarian tissue in patient with cancer involving the ovary, avoiding transmission of malignant cells through the graft. The first step of this technique is to obtain well-preserved isolated ovarian follicles for their further successful processing, either for culture or transplantation. We set up a protocol to isolate primordial and primary follicles from ovarian cortical tissue using the Liberase enzyme blend. This protocol yielded good quality isolated follicles showing good morphology, well-preserved ultrastructure and high viability. We have also proved the ability of these isolated follicles to growth in a SCID mouse model, assessing their developmental ability after enzymatic isolation. Our studies show that TEM, combined to other in vivo and in vitro techniques, is a useful tool to set-up and to optimize experimental protocols to preserve female fertility.
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Camboni, A. (2007). Preservation of fertility in women : investigation of different approaches. https://hdl.handle.net/2078.5/112116