Fertility preservation in cancer patients : development of a transplantable artificial ovary prototype

Ovarian tissue cryopreservation before gonadotoxic treatment is the only means of preserving fertility in prepubertal girls and women requiring prompt therapy. Despite growing success, with over 100 births to date, the safety of ovarian tissue grafting is of great concern in certain cancers, like leukemia, due to the potential risk of transferring malignant cells back to patients. Our laboratory is working on transplantation of isolated follicles and creation of an artificial ovary to offer these patients a safer alternative. In order to construct an artificial ovary prototype, a modified follicle isolation procedure was first developed to maximize the number and quality of isolated follicles for future clinical use. Human ovarian tissue was then analyzed to characterize its physical properties and duly recreate an artificial bio-environment in which follicles can be temporarily encapsulated before grafting.(SP - Sciences de la santé publique) -- UCL, 201

Artificial ovary

Since the first publications on the artificial ovary, many advances have been made. At that time, no studies had yet proved the feasibility of the concept, so it might have appeared provocative to unable to propose this option as a future fertility restoration approach for women unable to undergo ovarian tissue transplantation. Today, different artificial ovary prototypes demonstrate that this technique is able to restore both endocrine and reproductive functions in a murine model, producing healthy pups. However, with a view to future clinical application, further studies with human ovarian tissue and human follicles are essential. The aim of this chapter is to highlight the latest developments and advances in the field, as well as future directions for prompt translation to a clinical setting

Allografting of murine ovarian follicles in a fibrin matrix: influence of follicle stage on graft outcome.

Introduction: For women with cancer at high risk of developing ovarian metastasis, transplantation of cryopreserved ovarian tissue is not recommended after disease remission. To restore fertility in these patients, a biodegradable artificial ovary containing isolated follicles and ovarian stromal cells (SCs) could be a safer option. We evaluated the influence of the developmental stage of isolated murine follicles embedded in a fibrin matrix and grafted to immunodeficient (SCID) mice. Materials and methods: Seventeen NMRI mice were ovariectomized to isolate murine ovarian follicles and SCs. Groups of around 50 primordial-primary (PP) and 50 secondary (S) follicles were embedded in a fibrin matrix with 50,000 SCs and grafted to SCID mice for 2 and 7 days. Shortly after isolation, follicle diameter and follicle viability (live/dead assay) were performed. The follicle recovery rate, follicle survival (TUNEL), development (Ki67), graft vascularization (CD34) and inflammation (CD45) were analyzed after grafting. Follicle ultrastructure was assessed by transmission electron microscopy. Results: On day 0, mean (±SD) follicle diameter was 40.6 ±4.4 µm in the PP group and 90.9 ±9.5 µm in the S group. The total percentage of viable follicles was 72%. All 34 fibrin clots were recovered after grafting. The mean (±SD) follicle recovery rate was 16% ±9% for PP follicles and 40% ±11% for S follicles on day 2, and 4% ±4% for PP follicles and 28% ±20% for S follicles on day 7. The S group showed a significantly higher recovery rate (p <0.001) than the PP group after both grafting periods. On day 2, mean (±SD) of growth rates attested by (Ki67) were 78% ± 18% and 100% ±0%, and on day 7, 100% ±0% and 98% ±48%, in the PP and S groups respectively. On day 2, no capillaries were found in clots. On day 7, vessel area corresponded to 0.8% of graft surface area in the PP group and 3.6% in the S group (p<0.001), where larger and functional vessels were evidenced by the presence of erythrocytes. TEM showed that both PP and S follicles had a normal aspect after grafting: granulosa cells were in close contact with the oocyte, which exhibited an enveloped nucleus and visible organelles in its cytoplasm. Discussion: Despite the healthy status of remaining follicles in both groups, our results suggest that S follicles are more likely to survive and develop after isolation, encapsulation and grafting than PP follicles. The higher recovery rate of larger follicles may be correlated with the lack of rigidity of our fibrin matrix. Indeed, in the mouse ovaries and those of other animal species, the PP follicles are located in ovarian cortex, whose composition and stiffness differ from the medullary region, where growing follicles tend to develop. Conclusion: In our fibrin matrix, S follicles were able to survive and grow up to the antral stage after isolation and transplantation, while PP follicles appear to be more sensitive. Further studies are needed to determine whether such findings are due to the follicles themselves, and/or the matrix used to encapsulate them

Isolation and characterization of the human ovarian cell population for transplantation into an artificial ovary

To support survival and growth of follicles, the transplantable artificial ovary should mimic the original organ, offering a physical (3D matrix) and biological support (cells). In order to replicate the ovarian cell populations, the aim of this study is to assess the proportions of stromal and endothelial cells in the ovarian cortex. To this end, ovarian biopsies were obtained from six women (mean age: 49 years). The epithelial layer and medulla were carefully removed. The cortex was finely minced and enzymatically digested and the isolated cells were fixed. For cell characterization, immunostaining for CD31 (for endothelial cells) and inhibin-α (for granulosa cells) was performed. Positive cells in each staining were counted and the proportion of the different cell populations was estimated from the total number of isolated cells. Since there is no specific marker for ovarian stromal cells, we estimated the proportion of these cells by performing a vimentin immunostaining and subtracting the proportions of CD31- and inhibin-α-positive cells. Immunostaining showed that 84% of isolated cells were vimentin-positive. From this pool, 3% were endothelial cells and 1% granulosa cells. Consequently, the population of ovarian stromal cells was 80%. In conclusion, our findings show that stromal cells represent the larger population of cells in the human ovarian cortex. While this ensures follicle survival and development in a normal ovary, we believe that the low proportion of endothelial cells could have a negative impact on the angiogenesis in the artificial ovary after the first days of transplantation

A novel fibrin-based matrix for the creation of an artificial ovary prototype for human application.

Context: The development of an artificial ovary is actually an experimental fertility preservation strategy addressed to women affected by cancer with high risk of ovarian involvement who cannot benefit of the ovarian tissue cryopreservation and transplantation. Essential requirement for the creation of a bio-engineered ovary is the physical support needs to encapsulate isolated ovarian follicles. Ideally, this matrix should mimic the human ovarian tissue as far as possible in terms of microstructure and rigidity. Although fibrin-based matrices appear to be the best choice, identifying the appropriate combination of fibrinogen (F, mg/mL) and thrombin (T, IU/mL), the principal constituents of fibrin, for human ovarian follicle encapsulation and grafting remains a challenge. Objective: To improve the composition of the fibrin-based artificial ovary prototype by mimicking human ovarian tissue ultrastructure and rigidity. Methods: Fresh human ovarian cortex from different women (n=3) and four fibrin formulations using different concentrations of F and T (F12.5/T1, F30/T50, F50/T50, F75/T75) were investigated by scanning electron microscopy. Rheology was performed in fibrin matrices to evaluate their stiffness. Fibrin formulations most closely resembling human ovarian tissue were used to embed isolated human follicles, and compared in terms of follicle recovery rate after encapsulation. Results: Ovarian tissue cortex exhibited a similar ultrastructure, with a mean ± SD of fiber thickness of 66.4 ± 8.6 nm. Of the 4 fibrin formulations, only F12.5/T1 and F30/T50 showed significantly thicker fibers (137.5 ± 45.5 nm 100.8 ± 13.2 nm respectively), while F50/T50 and F75/T75 had comparable fiber thickness to ovarian tissue (64.5 ± 2.3 nm and 65.4 ± 18.4 nm respectively). Interestingly, fibrin stiffness was positively correlated to F and T concentrations, with F50/T50 rigidity similar to human ovarian tissue stiffness (Wood et al., 2015). F50/T50 and F75/T75 were therefore used to encapsulate isolated human preantral follicles, and recovery rates obtained (44% and 47% respectively) were not different between these two fibrin formulations. Conclusions: We showed, for the first time, that human ovarian cortex exhibits a similar ultrastructure in age-related women. This allowed us to standardize fibrin matrix architecture and select the fibrin formulation that best resembles native tissue in terms of ultrastructure and rigidity

A Tailored Protocol For Human Ovarian Follicle Isolation For Clinical Application

Context: In recent years different research teams have been working in the development of a transplantable artificial ovary with a view to restoring fertility in cancer patients at high risk of ovarian involvement who cannot benefit of any of the currently fertility preservation strategies. First essential requirement for developing an artificial ovary is a safe and efficient follicle isolation procedure. Objective: The aim of this study was to improve our previously established human ovarian follicle isolation procedure and adapt it according to the unique tissue properties of each individual patient. Methods: Thawed ovarian tissue from 22 patients was equally distributed between the previous (Vanacker et al., Fertil Steril 2011) and newly modified protocol, which involved fractionating enzymatic digestion over 3 time intervals in order to recover the first fully isolated follicles. Follicle yield and viability were compared soon after isolation. Follicles isolated using both protocols were then encapsulated in fibrin clots and their survival (caspase-3 and Ki67) and developmental stage (hematoxylin-eosin) were assessed. The efficacy of the modified protocol was further investigated by encapsulating and xenografting 100 human follicles together with 100,000 ovarian stromal cells to a SCID mouse for 7 days. Results: More follicles (124 ± 122) were isolated using the modified protocol than the previous procedure (88 ± 85) (p<0.01). After follicle encapsulation, the modified protocol also yielded a higher percentage of primordial (15% vs 7%) (p<0.05) and primary (77% vs 66%) follicles (p<0.05). On the other hand, no difference was found in the percentage of caspase-3- and Ki67-positive follicles with either protocol. After one week of xenografting, a higher percentage of follicles (35%) was observed than in our previous study (Paulini et al., 2016 Reprod Biomed Online). They appeared to be healthy, with 72% and 28% at primordial and primary follicle stages respectively. Conclusions: The modified protocol was found to maximize the number of isolated primordial and primary follicles without affecting their survival or development after isolation and xenografting. Such a protocol can also be specifically tailored to the tissue properties of each individual patient

Allografting of murine ovarian follicles in a fibrin matrix: influence of follicle stage on graft outcome.

Introduction:For women diagnosed with a cancer at high risk of developing ovarian metastasis, transplantation of  cryopreserved ovarian tissue is not recommended after disease remission. To restore fertility in these  patients, a biodegradable artificial ovary containing isolated follicles and ovarian cells (OCs) could be a  safer option.  Materials and Methods:14 NMRI mice were ovariectomized in order to isolate murine ovarian follicles and OCs. Groups of 50  Primordial Primary (PP) and 50 Secondary (S) follicles were embedded in a fibrin matrix with 50,000  OCs and grafted to SCID mice for 2 and 7 days. For control purpose, follicles and cells were processed  for live/dead assays to evaluate their viability before grafting. The follicle recovery rate was evaluated  by histology. Follicle survival (TUNEL) and development (KI67) and graft vascularization (CD34) were  analysed by immunohistochemical analysis after grafting. Follicle ultrastructure was assessed by  transmission electron microscopy. Results: Before grafting the total percentage of viable follicles was 55.4% (<10% dead granulosa cells). All 28  fibrin clots were recovered after grafting. The follicle recovery rate was 16.1% for PP follicles and 39.2%  for S follicles on day 2, and 5.9% for PP follicles and 26.8% for S follicles on day 7. Compared to the PP  group, the S group showed a significantly higher recovery rate. On day 2, percentages of growing  follicles were 81.8% and 100%, and on day 7, 100% and 96.7%, in the PP and S groups respectively. On  day 2, new capillaries were found in the tissue surrounding the clot but not inside. On day 7, capillaries  were observed around and inside the fibrin clot. Vessel area corresponded to 0.8% of the graft surface  area in the PP group and 3.6% in the S group. Moreover, in the histological analysis, S follicles showed  larger and functional vessels, as demonstrated by the presence of erythrocytes, than PP follicles. The  preliminary results of transmission electron microscopy showed that follicles from PP and S groups had  a normal aspect.    Discussion  Despite the healthy status of remaining follicles in both groups, our results suggest that secondary  follicles are more likely to survive and develop after isolation, encapsulation in fibrin clots and grafting  than primordial and primary follicles. These results lead us to hypothesize that the higher recovery rate  of the larger follicles can be correlated with the mechanical properties of the fibrin matrix. Indeed in  the mouse ovary, as well as in other animal species, the primordial follicles are located in the ovarian  cortex, which composition and stiffness differ from the medullar region, where growing follicles tend  to develop.  Despite the preliminary results regarding the ultrastructure of the both populations of follicles further  insights are necessary to assert with certainty that fibrin matrix is an optimal candidate to develop an  artificial ovary.