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While tissue engineering strategies for most organs stems primarily from a limited number of organ donors and side effects of immunosuppressive treatments, the demand for a transplantable artificial ovary (TAO) emerges as a procedure for fertility restoration in cancer survivors, who cannot benefit from available fertility restoration solutions. Indeed chemo- and radiotherapy can potentially be toxic for the ovaries, ultimately leading to sterility. This project represents the first attempt of ‘reverse engineering’ the human ovary. Reverse engineer biology means applying the engineering concept of taking apart a process or mechanism in order to understand it and re-engineer it (perhaps in a new way)—and applying it to the biological world. In this project, human ovarian cell’s microenvironment (matrisome) was deconstructed to better understand its proteomic, architectural and mechanical cues and their role in ovarian activity from prepuberty til menopause in order to reintegrate them into a biomimetic construct. Integrating these data will enable the TAO to be in the future a life changer for many female patients through making motherhood a possibility for them after cancer survival., (MED - Sciences médicales) -- UCL, 2021

An artificial ovary is a promising approach for preserving fertility in prepubertal girls and women who cannot undergo current cryopreservation strategies. However, this approach is in its infancy, due to the possible challenges of creating a suitable 3D matrix for encapsulating ovarian follicles and stromal cells. To maintain the ovarian stromal cell viability and proliferation, as a first step towards developing an artificial ovary, in this study, a double network hydrogel with a high water swelling capacity (swelling index 15–19) was developed, based on phenol conjugated chi-tosan (Cs-Ph) and silk fibroin (SF) through an enzymatic crosslinking method using horseradish peroxidase. The addition of SF (1%) to Cs (1%) decreased the storage modulus (G’) from 3500 Pa (Cs1) to 1600 Pa (Cs-SF1), and the hydrogels with a rapid gelation kinetic produced a spatially ho-mogeneous distribution of ovarian cells that demonstrated 167% proliferation after 7 days. This new Cs-SF hydrogel benefits from the toughness and flexibility of SF, and phenolic chemistry could pro-vide the potential microstructure for encapsulating human ovarian stromal cells., SCOPUS: ar.j, info:eu-repo/semantics/published

STUDY QUESTION: Can a reconstructed ovary using decellularized human ovarian tissue (DCT) support survival of pre-antral stage follicles? SUMMARY ANSWER: We have demonstrated an effective protocol for decellularization of human ovarian tissues and successful recellularization with isolated human ovarian cells and pre-antral follicles. WHAT IS KNOWN ALREADY: Survivors of leukemia or ovarian cancer run a risk of reintroducing malignancy when cryopreserved ovarian tissue is transplanted to restore fertility. A reconstructed ovary free of malignant cells could provide a safe alternative. Decellularization of ovarian tissue removes all cells from the extracellular matrix (ECM) including possible malignancies and leaves behind a physiological scaffold. The ECM offers the complex milieu that facilitates the necessary interaction between ovarian follicles and their surroundings to ensure their growth and development. Previous studies have shown that decellularized bovine ovarian scaffolds supported murine follicle growth and restoration of ovarian function in ovariectomized mice. STUDY DESIGN, SIZE, DURATION: Optimizing a decellularization protocol for human ovarian tissues and testing biofunctionality of the decellularized scaffolds in vitro and in vivo by reseeding with both murine and human pre-antral follicles and ovarian cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated human ovarian tissue and isolated pre-antral follicles were obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. Ovarian cortical and medullary tissues were decellularized using 0.1% sodium dodecyl sulfate (SDS) for 3, 6, 18 and 24 hours followed by 24 hours of 1 mg/mL DNase treatment and washing. Decellularization of ovarian tissues and preservation of ECM were characterized by morphological evaluation using Periodic Acid-Schiff (PAS) staining, DNA quantification, histochemical quantification of collagen content and immunofluorescence analysis for collage

PURPOSE: The aim of this study is to optimize fibrin matrix composition in order to mimic human ovarian tissue architecture for human ovarian follicle encapsulation and grafting. METHODS: Ultrastructure of fresh human ovarian cortex in age-related women (n = 3) and different fibrin formulations (F12.5/T1, F30/T50, F50/T50, F75/T75), rheology of fibrin matrices and histology of isolated and encapsulated human ovarian follicles in these matrices. RESULTS: Fresh human ovarian cortex showed a highly fibrous and structurally inhomogeneous architecture in three age-related patients, but the mean ± SD of fiber thickness (61.3 to 72.4 nm) was comparable between patients. When the fiber thickness of four different fibrin formulations was compared with human ovarian cortex, F50/T50 and F75/T75 showed similar fiber diameters to native tissue, while F12.5/T1 was significantly different (p value < 0.01). In addition, increased concentrations of fibrin exhibited enhanced storage modulus with F50/T50, resembling physiological ovarian rigidity. Excluding F12.5/T1 from further analysis, only three remaining fibrin matrices (F30/T50, F50/T50, F75/T75) were histologically investigated. For this, frozen-thawed fragments of human ovarian tissue collected from 22 patients were used to isolate ovarian follicles and encapsulate them in the three fibrin formulations. All three yielded similar follicle recovery and loss rates soon after encapsulation. Therefore, based on fiber thickness, porosity, and rigidity, we selected F50/T50 as the fibrin formulation that best mimics native tissue. CONCLUSIONS: Of all the different fibrin matrix concentrations tested, F50/T50 emerged as the combination of choice in terms of ultrastructure and rigidity, most closely resembling human ovarian cortex.

Reimplantation of cryopreserved ovarian tissue (OT) can successfully restore ovarian function in young cancer patients after gonadotoxic treatment. However, for patients with leukaemia, there is a risk of malignant cell transmission. Our objective was to evaluate minimal disseminated disease in OT from leukaemia patients and test a follicle isolation technique to obtain disease-free follicle suspensions. Cryopreserved OT from 12 leukaemia patients was thawed and analysed by histology and long-term xenografting in immunosuppressed mice. In 10 patients, follicles were isolated from OT, and polymerase chain reaction (PCR) was performed on tissue, digested ovarian suspensions and isolated follicle suspensions to investigate leukaemic cell presence. Mean patient age was 17·1 years. An average of 3·2 follicles were isolated per mm² of cortex. Xenografting of OT induced leukaemic masses in 2/12 mice. PCR identified leukaemic cell presence in 66% of OT. Malignant cells were also detected in digested ovarian suspensions. However, none of the follicle samples (>2300 follicles tested) showed any malignant cell presence after washing. This study demonstrates that it is possible to recover large numbers of viable follicles from cryopreserved OT of leukaemia patients. All isolated and washed follicle suspensions tested negative for leukaemic cells, giving leukaemia patients genuine hope of fertility restoration.

BACKGROUND: Ovarian tissue cryopreservation followed by transplantation after cancer remission is the most commonly applied fertility restoration approach in very young girls and women who require immediate cancer therapy. However, clinicians strongly advise against reimplantation of one's own ovarian tissue when there is a high risk of recurrence after grafting. For these patients, development of an alternative strategy, namely a transplantable artificial ovary, offers future hope of conceiving. The first essential requirement for an artificial ovary is the set-up of a safe and effective follicle isolation procedure. Despite encouraging results with different variants of this technique, none of them take into the account the physiology and great variability in follicular density inside individual tissue fragments and between different patients. The goal of this study was to improve our previously applied follicle isolation procedure in order to develop a tailored isolation procedure for human follicles according to individual tissue properties. To this end, enzymatic digestion was divided into three time intervals in order to initially recover the first follicles to be isolated, and then further dissociate undigested fragments of tissue containing entrapped follicles. RESULTS: After thawing frozen human ovarian tissue using a modified and tailored follicle isolation method, already 35% of follicles were fully isolated and recovered after 30 min of enzymatic digestion. Indeed, this protocol resulted in a higher follicle yield (p < 0.01) and greater numbers of primordial and primary follicles (p < 0.05) than the previous approach. However, no significant difference was found in caspase-3-positive and Ki67-positive staining between the two isolation protocols. In addition, greater follicle quality was demonstrated. When human follicles isolated using the modified protocol were encapsulated in a fibrin matrix with high concentrations of fibrinogen and thrombin and xeno

STUDY QUESTION: Do primordial-primary versus secondary follicles embedded inside a fibrin matrix have different capabilities to survive and grow after isolation and transplantation? SUMMARY ANSWER: Mouse primordial-primary follicles showed a lower recovery rate than secondary follicles, but both were able to grow. WHAT IS KNOWN ALREADY: Fresh isolated mouse follicles and ovarian stromal cells embedded in a fibrin matrix are capable of surviving and developing after short-term autografting. STUDY DESIGN, SIZE, DURATION: In vivo experimental model using 11 donor Naval Medical Research Institute (NMRI) mice and 11 recipient severe combined immunodeficiency (SCID) mice. Both ovaries from all NMRI mice were mechanically disrupted and primordial-primary and secondary follicles were isolated with ovarian stromal cells. They were then encapsulated in a fibrin matrix composed of 12.5 mg/ml of fibrinogen (F12.5) and 1 IU/ml of thrombin (T1) (F12.5/T1), and grafted to the inner part of the peritoneum of SCID mice for 2 and 7 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to conduct histological (H-E staining) and immunohistochemical (Ki67, TUNEL) analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Although all grafted fibrin clots were recovered, the follicle recovery rate on day 2 was 16 and 40% for primordial-primary and secondary follicles respectively, while on day 7, it was 6 and 28%. The secondary group showed a significantly higher recovery rate than the primordial-primary group (23%, P-value <0.001). Follicles found in both groups were viable, as demonstrated by live/dead assays, and no difference was observed in the apoptosis rate between groups, as evidenced by TUNEL. Their growth to further stages was confirmed by Ki67 immunostaining. LIMITATIONS, REASONS FOR CAUTION: As demonstrated by our results, secondary follicles appear to be more likely to survive a

Transplantation of cryopreserved ovarian tissue is one of the alternatives to restore fertility in cancer survivors. However, this technique is not advisable for patients with certain types of cancer, since there is a risk of reintroducing malignant cells present in the cryopreserved tissue. For these patients, a safer alternative could be transplantation of isolated preantral follicles back to their natural environment. To encapsulate and protect isolated follicles, a matrix should be created. To this end, we have been testing different polymers to embed murine follicles. Our best results were obtained with a fibrin formulation containing low concentrations of fibrinogen and thrombin[1]. Despite these promising results with mouse follicles, our findings with human isolated preantral follicles were significantly inferior. The goal of this study was therefore to evaluate if a fibrin formulation with higher concentrations of fibrinogen and thrombin could improve survival of human isolated be suitable as a matrix to graft isolated human preantral follicles. Moreover, we would like to test if supplementation of the fibrin matrix with hyaluronan (HA) could also have a positive effect on follicle survival and growth.

STUDY QUESTION: What is the best source of ovarian cells for the artificial ovary: medulla or cortex, cryopreserved or fresh? SUMMARY ANSWER: Ovarian cells from fresh medullary tissue, which can be isolated in larger numbers, show higher viability and are able to improve graft vascularization. WHAT IS KNOWN ALREADY: In a previous study, addition of endothelial cells along with ovarian cells was found to be crucial for formation of a well-vascularized ovary-like structure. This study is the first to evaluate both the effect of cryopreservation and the source of ovarian tissue on isolated ovarian cells. STUDY DESIGN, SIZE, DURATION: Prospective experimental study in an academic research unit using ovarian tissue from seven patients undergoing surgery for benign gynecologic disease. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue was retrieved from seven patients, with one half processed as fresh (fresh group) and the other half frozen and thawed before processing (frozen group). In each group, ovarian cells from the cortex and medulla were isolated separately, and their viability was tested using a calcein AM/ethidium homodimer viability assay. Fifty thousand cells were then encapsulated in fibrin and grafted to peritoneal pockets in nude mice (14 in all). Grafts recovered after 7 days were analyzed by immunohistochemistry for the presence of ovarian cells (vimentin), proliferation (Ki67) and graft vascularization (double CD34). Cell apoptosis was analyzed by TUNEL assay. MAIN RESULTS AND THE ROLE OF CHANCE: Cryopreservation decreased ovarian cell yield (-2804 cells/mg, P = 0.015) and viability (-9.72%, P = 0.052) before grafting and had a considerable (5-fold, P = 0.2) but non-significant negative impact on ovarian cell presence in grafts. The medulla yielded many more cells (+3841 cells/mg, P < 0.001) with higher viability (+18.23%, P < 0.001) than did the cortex. Moreover, grafts with cells from the medulla exhibited a statistically significant 6.44- and 2

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. S, (MED - Sciences médicales) -- UCL, 2015

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.

BACKGROUND: Although transplantation of cryopreserved ovarian tissue is a promising approach to restore fertility in cancer patients, it is not advisable for women at risk of ovarian involvement due to the threat of reintroducing malignant cells. The aim of this study was therefore to find an alternative for these patients by development of an artificial ovary. METHODS: For construction of the artificial ovary matrix, we used a central composite design to investigate nine combinations of fibrinogen (mg/ml) and thrombin (IU/mL) (F/T): F1/T4, F12.5/T1, F12.5/T20, F25/T0.1, F25/T4, F25/T500, F50/T1, F50/T20 and F100/T4. From the first qualitative analyses (handling and matrix size), five combinations (F12.5/T1, F25/T4, F50/T20, F50/T1 and F100/T4) yielded positive results. They were further evaluated in order to assess fibrin matrix degradation and homogeneous cell encapsulation (density), survival and proliferation (Ki67), and atresia (TUNEL) before and after 7 days of in vitro culture. To determine the best compromise between maximizing the dynamic density (Y1) and minimizing the apoptosis rate (Y2), we used the desirability function approach. RESULTS: Two combinations (F12.5/T1 and F25/T4) showed greater distribution of cells before in vitro culture, reproducible degradation of the fibrin network and adequate support for isolated human ovarian stromal cells, with a high proportion of Ki67-positive cells. SEM analysis revealed a network of fibers with regular pores and healthy stromal cells after in vitro culture with both F/T combinations. CONCLUSION: This study reports two optimal F/T combinations that allow survival and proliferation of isolated human ovarian cells. Further studies are required to determine if such a scaffold will also be a suitable environment for isolated ovarian follicles.

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.