Simvastatin and Fibrin Clots to Improve Human Ovarian Tissue Grafting

Introduction

Slow ovarian neovascularization after implantation leads to follicular loss. Simvastatin is an anti-ischemic and anti-inflammatory agent and increases vascularization. It was shown to protect grafted fresh and vitrified murine ovarian samples against ischemia and to enhance neovascularization. Moreover, fibrin clots were shown to enhance cellular vascularization.

Aim

To investigate if host treatment with simvastatin and/or transplantation of frozen-thawed human ovarian tissue embedded in fibrin clots will improve implantation in immunodeficient mice.

Material and Methods

Ovarian tissue was obtained from six patients (under IRB supervision), and frozen-thawed ovarian samples were transplanted into immunodeficient mice. Thawed tissue served as ungrafted controls. The ovarian tissue was transplanted into the following groups: 1) untreated (grafted control); 2) hosts treated with simvastatin; 3) tissue embedded in fibrin clots; 4) host treatment as in 2 and graft implantation as in 3. The mice were sacrificed three weeks after surgery, and the transplants were removed and fixed. Graft survival was assessed by follicle counts, apoptosis assay, immunohistochemical studies of Ki67 [follicular proliferation] and of platelet endothelial cell adhesion molecule 1 (PECAM 1) [neovascularization] expression.

Results

All the ovarian tissue grafts embedded in fibrin clots were recovered, maintained their original sizes and did not shrink, while ~75% were identified in groups 1 and 2 (NS). Fibrin clots embedding enabled easier implantation and the samples fused well with the host tissue. There was a significantly higher atretic follicle number in group 2 compared to all other groups (74% compared to 22-40%, P=0.03-P<0.0001). Two patterns of PECAM1 were identified: diffuse (scattered throughout the specimen) and peripheral. Both staining patterns were identified concomitantly only in groups 2 and 3 (NS), while in the ungrafted controls and groups 1 and 4 only diffuse staining was detected. There were significantly higher diffuse staining in group 2 than in group 1 (P<0.05). Apoptosis rates were lower in groups 3 and 4, but the differences did not reach significance. Ki67 staining was identified in all grafted/ungrafted follicles.

Conclusions

Fibrin clots seem to promote ovarian graft survival and neovascularization. Although simvastatin might also induce vascularization, group 2 samples had significantly more atretic follicles than all other groups. Further experiments with group 4 (simvastatin+fibrin clots) should be completed and studies with other-novel vasculrization markes are underway.