Abstract
Transplantation of ovarian tissue (OT) is currently the only clinical option to restore fertility with cryopreserved OT. However, follicle loss caused by ischemia and slow revascularization occurs in transplanted OT. To shorten the ischemic period and promote angiogenesis, some angiogenic factors have been used. Angiopoietin-2 (Ang2) is one of the major angiogenic factors and has been reported to promote blood vessels and increase vascular permeability in ischemic and/or hypoxic environment. This study was performed to investigate the effects of Ang2 on follicle integrity and revascularization of transplanted mouse OT. Five-week-old B6D2F1 female mice were divided into a control group and two Ang2 groups, followed by ovary collection and vitrification. After warming, the ovaries were autotransplanted into kidney capsules with/without Ang2 injection (50 or 500 ng/kg), and then the mice were sacrificed at days 2, 7, 21, and 42 after transplantation. A total 2,437 follicles in OT grafts were assessed for follicular density, integrity, and classification by using hematoxylin and eosin staining. Apoptosis and revascularization were evaluated by using TUNEL assay and CD31 immunohistochemistry, respectively. Serum follicle-stimulating hormone (FSH) levels were measured by using enzyme-linked immunosorbent assay. Both Ang2 groups showed remarkable increase in morphologically intact follicle ratio across all grafting durations except D21. The numbers of CD31(+) vessels were significantly increased in both Ang2 groups compared with the control group at all durations, except in the 50 ng Ang2 group at D42. However, the mean numbers of follicles of the grafts, apoptosis ratios, and serum FSH levels showed no significant differences among the groups. Our results show that Ang2 treatment significantly increased the intact follicle ratios and the number of blood vessels of the mouse OT grafts. However, further studies performed with large animal or human OT are necessary before clinical application for fertility preservation in cancer patients, and the reliability of the systemic effects of Ang2 should be verified.
Highlights
Cancer treatment has had remarkable progress, which has enabled cancer patients to survive
The ovaries were vitrified by the two-step method described in our previous report [21] as follows: the ovarian tissue (OT) were placed in the equilibration solution consisting of Dulbecco’s phosphate buffered saline (D-PBS; Gibco; Paisley, UK) supplemented with 20% fetal bovine serum (FBS; Gibco), 7.5% ethylene glycol
Even though no significant difference was found between the 500 ng Ang2 and control groups in the gross examination of OT grafts on D7, we observed that the OT grafts treated with 500 ng Ang2 were better than the control OTs in color, follicle formation, and vessel numbers, which was proved by the histological examination and CD31 IHC results. These results suggest that the Ang2 treatment increased the angiogenesis of the OT grafts and facilitated the neovascularization, which led to the improvement of follicle survival and integrity
Summary
Cancer treatment has had remarkable progress, which has enabled cancer patients to survive. Some female survivors suffer from infertility caused by chemotherapy and/or radiotherapy, which lowers their quality of life [1,2]. To overcome these problems, several fertility preservation options have been introduced including oocyte, embryo, and ovarian tissue (OT) cryopreservation. OT cryopreservation and transplantation are regarded as an option that should be offered to female cancer patients. This procedure has many advantages for women who do not have a spouse and/or no time to delay chemotherapy. For prepubertal female patients, it could be the only option for fertility preservation [4,5]
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