Follicle activation and 'burn-out' contribute to post-transplantation follicle loss in ovarian tissue grafts: the effect of graft thickness.

STUDY QUESTION: What are the effects of thin ovarian grafts compared with grafts of the standard thickness on follicle loss post-transplantation? SUMMARY ANSWER: Transplantation of reduced-thickness ovarian grafts led to intense activation and 'burn-out' a short time after transplantation resulting in significant folllicle loss. WHAT IS KNOWN ALREADY: Transplantation of fresh and frozen-thawed ovarian tissue has been proved successful, but techniques vary and are not optimised, often resulting in significant follicular loss. Follicle loss is mostly related to the freezing-thawing process and to post-transplantation hypoxia. STUDY DESIGN, SIZE, DURATION: Bovine ovarian tissue strips (n = 55) were prepared in two groups of conventional-thickness strips (1-2 mm) or thin strips (0.5-0.9 mm). Fresh or frozen-thawed samples were xenotransplanted into sterilized immune-deficient mice (n = 49). Non-transplanted conventional size fresh samples were used as controls (n = 6). Grafts from all study groups were recovered after 7 days for analysis. PARTICIPANTS/MATERIALS, SETTING,
METHODS: Morphometric differential counting of follicle classes was performed by two observers. Immunohistochemistry was conducted for proliferation (Ki67), cortical fibrosis (Masson tri-chrome) and blood-vessel density (CD31). Results were expressed as the mean number of dormant or growing follicle (GF) type per section or total follicle counts per graft. Blood-vessel density was calculated per mm(2). P-values <0.05 were considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: The loss of all follicle types, and most noteably of primordial follicles (PMFs), was observed 7 days post-transplantation (P < 0.05). The relatively high number of GFs and the positive Ki67 staining in all recovered grafts indicated that follicle activation was depleting the resting follicle pool. The reduced graft thickness had an adverse effect on the number of recovered follicles, especially on the resting non-GFs in the fresh, and more so in the frozen-thawed, samples (P < 0.05). Extensive stromal fibrosis and high blood-vessel density were observed in all grafts with no advantage in the thin prepared grafts. LIMITATIONS, REASONS FOR CAUTION: This study used only one species of ovaries (bovine) for xenotransplantation. The immediate post-transplantation events were not visualized directly nor were the molecules involved in follicle activation studied. WIDER IMPLICATIONS OF THE
FINDINGS: Follicle activation and 'burn-out' appear to be important in follicle loss after transplantation. Reducing graft thickness in an attempt to improve freezing conditions and reduce post-transplantation ischemia has adverse effects on the graft follicle pool due to increased activation and loss. Agents which prevent 'burn-out' will potentially improve follicle pool survival. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by research grants from the Israeli Science Foundation (No. 1675/10), the Israeli Jack Craps foundation and the Israel Cancer Research Fund (ICRF No. 12-3081). The authors have no competing interest to declare.