Development of a transplantable artificial ovary: influence of follicle stage on transplantation outcome

摘要

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 P 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.