Valérie Luyckx

Risk of transferring malignant cells with transplanted frozen-thawed ovarian tissue

Ovarian tissue cryopreservation and transplantation is a real option to preserve and restore fertility in young cancer patients. However, there is a concern regarding the possible presence of malignant cells in the ovarian tissue, which could lead to recurrence of the primary disease after reimplantation. A review of the existing literature was done to evaluate the risk of transplanting malignant cells in case of the main malignant indications for ovarian tissue cryopreservation. For ovarian tissue from patients with hematologic malignancies, it is of paramount importance to identify minimal residual disease before ovarian tissue transplantation. Indeed, these pathologies, reviewed here in detail, are considered to be most at risk of ovarian metastasis.

Transplantation of an alginate-matrigel matrix containing isolated ovarian cells: first step in developing a biodegradable scaffold to transplant isolated preantral follicles and ovarian cells.

For women diagnosed with leukemia, transplantation of cryopreserved ovarian tissue after disease remission is not advisable. Therefore, to restore fertility in these patients, we aim to develop a biodegradable artificial ovary that offers an environment where isolated follicles and ovarian cells (OCs) can survive and grow. Four NMRI mice were ovariectomized and their ovaries used to isolate OCs. Groups of 50,000 OCs were embedded in an alginate-matrigel matrix for further fixation (fresh controls), one week of in vitro culture (IVC) or heterotopic autografting. OC proliferation (Ki67), apoptosis (TUNEL), scaffold degradation, vessel formation (CD34) and inflammation (CD45) were analyzed. Ki67-positive OCs were found in 2.3%, 9.0% and 15.5% cells of cases in fresh, IVC and grafted beads respectively, while cells were TUNEL-positive in 0%, 1.5% and 6.9% of cases. After IVC or grafting, the beads degraded, losing their original round aspect, and infiltrating blood capillaries could be observed in the grafted beads. CD34-positive cells and 22% CD45-positive cells were found around and inside the matrix. In conclusion, our results demonstrate that an alginate-based matrix is a promising proposition to graft isolated OCs. After transplantation, this matrix was able to degrade, allowed vascularization and elicited a low inflammatory response.

A new step toward the artificial ovary: survival and proliferation of isolated murine follicles after autologous transplantation in a fibrin scaffold

OBJECTIVE To create an artificial ovary to provide an alternative way of restoring fertility in patients who cannot benefit from transplantation of cryopreserved ovarian tissue due to the threat of reintroducing malignant cells. DESIGN In vivo experimental study. SETTING Gynecology research unit in a university hospital. ANIMAL(S) Six-week-old female NMRI mice. INTERVENTION(S) Autografting of isolated preantral follicles and ovarian cells (OCs) encapsulated in two fibrin matrices containing low concentrations of fibrinogen (F; mg/mL) and thrombin (T; IU/mL): F12.5/T1 and F25/T4. MAIN OUTCOME MEASURE(S) Follicular density and development, OC survival and proliferation, inflammatory response, and vascularization. RESULT(S) After 1 week, the follicle recovery rate ranged from 30.8% (F25/T4) to 31.8% (F12.5/T1). With both fibrin formulations, all follicles were found to be alive or minimally damaged, as demonstrated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, and at the growing stage (primary, secondary, and antral follicles), confirmed by Ki67 immunostaining. Isolated OCs also survived and proliferated after grafting, as evidenced by <1% apoptotic cells and a high proportion of Ki67-positive cells. Vessels were found in both fibrin formulations, and the global vascular surface area varied from 1.35% (F25/T4) to 1.88% (F12.5/T1). Numerous CD45-positive cells were also observed in both F25/T4 and F12.5/T1 combinations. CONCLUSION(S) The present study is the first to show survival and growth of isolated murine ovarian follicles 1 week after autotransplantation of isolated OCs in a fibrin scaffold. The results indicate that fibrin is a promising candidate as a matrix for the construction of an artificial ovary. Xenotransplantation of isolated human follicles and OCs is the necessary next step to validate these findings.

Successful vitrification and autografting of baboon (Papio anubis) ovarian tissue

STUDY QUESTION Can a vitrification protocol using an ethylene glycol/dimethyl sulphoxide-based solution and a cryopin successfully cryopreserve baboon ovarian tissue? SUMMARY ANSWER Our results show that baboon ovarian tissue can be successfully cryopreserved with our vitrification protocol. WHAT IS KNOWN ALREADY Non-human primates have already been used as an animal model to test vitrification protocols for human ovarian tissue cryopreservation. STUDY DESIGN, SIZE, DURATION Ovarian biopsies from five adult baboons were vitrified, warmed and autografted for 5 months. PARTICIPANTS/MATERIALS, SETTING, METHODS After grafting, follicle survival, growth and function and also the quality of stromal tissue were assessed histologically and by immunohistochemistry. The influence of the vitrification procedure on the cooling rate was evaluated by a computer model. MAIN RESULTS After vitrification, warming and long-term grafting, follicles were able to grow and maintain their function, as illustrated by Ki67, anti-Müllerian hormone (AMH) and growth differentiation factor-9 (GDF-9) immunostaining. Corpora lutea were also observed, evidencing successful ovulation in all the animals. Stromal tissue quality did not appear to be negatively affected by our cryopreservation procedure, as demonstrated by vascularization and proportions of fibrotic areas, which were similar to those found in fresh ungrafted ovarian tissue. LIMITATIONS, REASONS FOR CAUTION Despite our promising findings, before applying this technique in a clinical setting, we need to validate it by achieving pregnancies. WIDER IMPLICATIONS OF THE FINDINGS In addition to encouraging results obtained with our vitrification procedure for non-human ovarian tissue, this study also showed, for the first time, expression of AMH and GDF-9 in ovarian follicles. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie No. 7.4507.10, grant 3.4.590.08 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, and Department of Mechanical Engineering at Louisiana State University (support to Ram Devireddy), and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors has any competing interests to declare.

Endothelial cells are essential for ovarian stromal tissue restructuring after xenotransplantation of isolated ovarian stromal cells

BACKGROUND Grafting of isolated follicles represents an approach to prevent the risk of reimplanting malignant cells with cryopreserved ovarian fragments. Optimal conditions and cell types required to sustain human follicular growth need to be identified. To help improve the grafting technique, we investigated whether short-term xenografting of a suspension containing ovarian stromal and endothelial cells without follicles could enhance graft survival and revascularization. METHODS In human ovary, CD34 selectively labels endothelial cells of blood vessels. A CD34-replete ovarian stromal cell group, including stromal and endothelial cells, was obtained after enzymatic digestion of fresh human ovarian cortex. Magnetic-activated cell sorting was used to establish a CD34-depleted ovarian stromal cell group. Proportions of CD34-positive cells were evaluated by flow cytometry and immunocytochemistry. Cell suspensions were embedded in human plasma clots and grafted (n = 10 for each group, 7 days) to the ovarian bursa of nude mice. Angiogenesis was quantified after human/mouse CD34 immunostaining. RESULTS CD34-replete grafts had a well-organized and vascularized stromal structure, containing tubular components staining for human CD34 and corresponding to functional vessels, as evidenced by intraluminal red blood cells. CD34-depleted grafts tended to be smaller than CD34-replete grafts and poorly vascularized with central necrosis. Global microvessel density was higher in the CD34-replete than depleted group (337.9 versus 187.3 vessels/mm(2), P < 0.05), with a greater proportion of human vessels (68.02 versus 6.95%, respectively, P < 0.05). CONCLUSIONS We demonstrated the importance of co-transplanting ovarian endothelial and stromal cells to ensure the formation of a well-vascularized and structured ovarian-like stroma after short-term xenografting, for future application in the transplantation of isolated follicles.

Evaluation of cryopreserved ovarian tissue from prepubertal patients after long-term xenografting and exogenous stimulation.

OBJECTIVE To assess follicular development after long-term xenotransplantation and exogenous stimulation of cryopreserved prepubertal ovarian tissue. DESIGN Pilot study. SETTING Gynecology research unit in a university hospital. PATIENT(S) Cryopreserved ovarian fragments were obtained from five prepubertal patients aged 7.2-12.2 years. INTERVENTION(S) Xenografting of frozen-thawed prepubertal ovarian fragments to SCID mice for 5 months and exogenous stimulation. MAIN OUTCOME MEASURE(S) Follicular density, morphology, proliferation, development. RESULT(S) Follicular density varied between 1.05 and 47.89 follicles/mm(2) in frozen-thawed ovarian tissue and 0.48 and 32.74 follicles/mm(2) in grafted prepubertal ovarian tissue. Growing follicles at the last stage of follicular development were observed at significantly higher proportions after grafting. No statistical difference was evidenced in the number of primordial follicles, representing the largest proportion of the follicle pool (99.51% before grafting and 92.46% after grafting). Ki67 and antimüllerian hormone expression were observed in these growing follicles. CONCLUSION(S) This is the first description of transplantation of human cryopreserved prepubertal ovarian tissue to mice, demonstrating that a very high number of follicles survive after transplantation and a large pool of primordial follicles remains dormant. Growing follicles were observed, proving the responsiveness of prepubertal ovarian tissue to gonadotropins.