Isoform 165 of vascular endothelial growth factor in collagen matrix improves ovine cryopreserved ovarian tissue revascularisation after xenotransplantation in mice

Abstract

Background: Aggressive anti-cancer treatments can result in ovarian failure. Ovarian cryopreservation has been developed to preserve the fertility of young women, but early graft revascularisation still requires improvement.

Methods: Frozen/thawed sheep ovarian cortical biopsies were embedded in collagen matrix with or without isoform 165 of vascular endothelial growth factor (VEGF165) and transplanted into ovaries of immunodeficient mice. Ovaries were chosen as transplantation sites to more closely resemble clinical conditions in which orthotopic transplantation has previously allowed several spontaneous pregnancies.

Results: We found that VEGF165 significantly increased the number of Dextran-FITC positive functional vessels 3 days after grafting. Dextran- fluorescein isothiocyanate (FITC) positive vessels were detectable in 53% and 29% of the mice in the VEGF-treated and control groups, respectively. Among these positive fragments, 50% in the treated group displayed mature smooth-muscle-actin-alpha (alpha-SMA) positive functional vessels compared with 0% in the control group. CD31 positive murine blood vessels were observed in 40% of the VEGF165 transplants compared with 21% of the controls. After 3 weeks, the density of murine vessels was significantly higher in the VEGF165 group.

Conclusion: The encapsulation of ovarian tissue in collagen matrix in the presence of VEGF165 before grafting has a positive effect on functional blood vessel recruitment. It can be considered as a useful technique to be improved and further developed before human clinical applications in female cancer patients in the context of fertility preservation.

Figure 1

Encapsulation of ovarian graft in collagen matrix with and without VEGF ₁₆₅ . A. Type I collagen was extracted from rat tail tendons. B. Preparation of collagen matrix. C. Adjusting collagen mix to pH 7.4. D. Illustration of agarose rings. E. Filling of agarose ring with a first layer of the collagen matrix with or without recombinant murine VEGF_165. F. Frozen/thawed ovarian sheep explant deposit. G. Second layer of collagen matrix with or without recombinant murine VEGF_165. H. Agar ring removal before transplantation.

Figure 2

The analysis of functional blood vessels in ovarian grafts 3 days after transplantation. A. An illustration of functional blood vessels that were observed using dextran-FITC immunostaining. B. The quantification of FITC-positive vessels in ovarian transplants from the control and VEGF₁₆₅-treated groups. In the treated mice, 8 of 15 fragments contained functional vessels compared with only 4 of 14 fragments in the control mice. C. An illustration of double staining for functional and mature blood vessels in a transplant that was treated with VEGF₁₆₅. FITC was stained brown, and α-SMA was stained red. n corresponds to the number of transplanted ovarian fragments that contained functional blood vessels. Scale bar: 200 μm. *Corresponds to a p value < 0.05. Error bar are SEM (mean standard error).

Figure 3

- Murine blood vessels in sheep ovarian transplants after 3 days and 3 weeks reflect colonisation by host vessels into the graft. A. An illustration of murine blood vessels that were stained with a mouse-specific CD31 Ab in an ovarian fragment that was treated with VEGF₁₆₅ and removed 3 days after transplantation. The host tissue is identified by the dotted arrow, and the graft is identified by the plain arrow. B. The quantification of murine blood vessels in the grafted tissue 3 days after transplantation. In the treated mice, 6 of 15 fragments contained CD31-positive blood vessels compared with only 3 of 14 fragments in the control mice. C. The quantification of murine blood vessels in the grafted tissue 3 weeks after transplantation. All of the fragments in both the control and treated groups contained CD31-positive blood vessels. n corresponds to the number of transplanted ovarian fragments that contained murine blood vessels. Scale bar: 200 μm. **Corresponds to a p value < 0.01. Error bar are SEM.

Figure 4

The analysis of fibrosis and follicles 3 weeks after transplantation. A. The percentage of fibrotic tissue in the ovarian transplant. B. Primordial and primary follicle quantification in H&E-stained sections (mean number/mm²). C. An illustration of primordial follicles that were considered to be morphologically normal (the plain arrow) or abnormal (the dotted arrow) with disorganised granulosa cells. D. The proportion of morphologically normal and abnormal primordial and primary follicles in the control group and the VEGF₁₆₅-treated group. n corresponds to the number of ovarian fragments that were transplanted into mice. Scale bar: 200 μm. Error bar are SEM.