Optimal vitirification protocol and transplantation condition for mouse ovarian tissue

Abstract

Introduction: In clinical medicine, ovarian tissue (OT) cryopreservation together with transplantation has been used to restore their fertility for the women suffering from infertility caused by cancer treatment. Lots of studies on transplantation of cryopreserved-thawed OTs have been performed, and now this is the promising alternatives to preserve fertility in cancer patients. Due to the limited availability of human OT in experiments, the mice have been used as an effective model for such purposes in many studies. Cryopreservation and transplantation of murine ovaries is a useful tool to assess the risk of malignant recurrence after re-transplantation of OT, to analyze the recovery of ovarian function, and to improve the related protocols for future clinical application. Optimization of cryopreservation and transplantion procedures is the most important steps to improve OT survival. The aim of this study was to determine the optimal cryopreservation especially vitrification protocols for mouse OT survival, assess the impact of 4 different OT grafting sites and evaluated the effect of angiopoietin-2 (ANG-2) as an angiogenic factor on the follicular pool and OT integrity after grafting.

Methods: For the vitrification experiment, a total of 644 ovaries were collected from BDF1 mice. Of these, 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition, such as EDS, ES, ED, EPS, EF, EFS, E, and EP, respectively (E: EG, D: DMSO, P: PrOH, S: sucrose, and F: Ficoll). And the remained 73 ovaries were used for control group. After warming, each 8 group of ovaries was further randomly divided into 4 subgroups and in vitro cultured for 0, 0.5, 2, and 4 h, respectively. Ovaries of the best 2 groups among 8 groups were autotransplanted after IVC. To evaluate the quality of vitrified OTs, OT morphological histology, follicular apoptosis, proliferation and FSH level was observed. For the grafting site experiment, the B6D2F1 mice were randomly assigned to 4 groups. One group was used for the control (sharm). The ovaries from the other 4 groups were collected and autotranplanted directly to the different transplantation sites: back muscle (BM), fat pad (FP), kidney capsule (KC) and subcutaneous (SC). Assessment of the follicular density, integrity, classification, apoptosis, fibrosis, FSH level and the quantity and the quality of oocytes from the OT grafts was carried out on day (D) 2, 7, 21 and/or 42 after grafting. The Five-week-old B6D2F1 female mice were divided into 3 groups (a control and two ANG-2 groups) followed by ovary collection and vitrification. After warming, the ovaries were autotransplanted into kidney capsules with/without ANG-2 injection (50 or 500ng/kg), and then killed at day(D)2, 7, 21 and 42 after transplantation. Total 2,437 follicles in OT grafts were assessed for the follicular density, integrity and classification by hematoxylin and eosin staining. Apoptosis and revascularization were evaluated by Terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate Nick End Labeling assay and CD31 immunohistochemistry respectively. Serum FSH levels were measured by Enzyme-linked immunosorbent assay.

Results: Morphologically intact (G1) and apoptotic follicles were compared after vitrification. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8% and 46.6%, respectively, whereas the EP group showed the worst results at 42.2% and 12.8%, respectively. Apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All the 8 groups showed significant decreases in G1 follicle ratio and increases in apoptotic follicle ratio as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 ratio (84.9%). For the proliferation ratio, only the ES 4 h group (80.6%) showed significant decrease. There was no significant difference in apoptotic ratio and FSH level between all the groups. In the grafting site experiment, the graft recovery rate was lowest in the FP group (83.9%), and the mean number of follicles only in the KC group (D7: 14.7 and D21: 15.9) was comparable to that of the control group (D7: 17.0 and D21: 18.8). The antral follicles appeared in BM (0.9%) and KC (2.2%) groups on D2 while no antral follicles in FP and SC groups yet, and were increased as the grafting duration increased showing the lowest percentage in the SC group (7.8%) on D21. For the intact (G1) follicles, all the grafting groups showed low ratios (31.5%-37.7%) compared to the sharm group (61.3%) on D2 followed by dramatic increase in FP (74.0%) and KC (71.9%) groups on D21 (sharm: 63.0%, BM: 59.5%, and SC: 70.9%). The ratios of apoptotic follicles were lowest in the KC group (8.2%) and highest in the SC group (24.5%) on D7, but no significant difference was observed on D21. Marked increase in tissue fibrosis was observed in the FP (8.3%) and SC (15.0%) groups on D2, and it was continued in the FP group (D7: 5.9% and D21 7.5%). The FSH levels were significantly increased in all grafting groups on D2, but only the SC group showed the high FSH level (5.5ng/ml) compared to the others (1.6-2.5ng/ml) on D7 followed by no sign of significant difference among the groups on D21. When collecting the oocytes from the experimental 4 sites, the mean oocyte numbers of 4 sites ovarian grafts from 4 graft sites were significantly highest in KC group and lowest in SC group (BM

4.6±1.6, FP

3.5±1.3, KC

10.4±2.3 and SC

2.1±0.9 respectively). However the ratio of MII oocyte and normal spindle were not different among the 4 experimental groups. For the ANG-2 experiment, all the ANG-2 groups (50ng and 500ng) showed remarkable increase of morphologically intact follicle ratio across all the grafting duration except D21 (no statistical difference). The numbers of CD31 positive vessels (the sum of 3 fields at x400 magnification) were significantly increased in both ANG-2 groups compared with the control group at all the grafting duration. Especially at D42, the 500ng ANG-2 group showed significantly more vessels than the 50ng ANG-2 group as well as the control group. However the mean follicle numbers of grafts, apoptosis ratio and serum FSH levels showed no significant difference among the groups.

Conclusions: We compared 8 vitrification protocols according to CPA composition and IVC duration and found the EDS to be the optimal protocol among them. For the grafting site, we confirmed that the different grafting sites influenced the outcomes of the grafting. The KC site was the most optimal for OT transplantation in murine model. In contrast, the SC sites were not favorable to OT grafts. And we found that ANG-2 treatment to reduce the ischemic damage and improve angiogenic ability could preserve ovarian function via its beneficial effects on transplants.