In this study, the effect of VEGF
165 on slow-frozen, cryopreserved ewe ovarian tissue that was grafted into a murine model was analysed three days and three weeks after transplantation. The results of this study suggest that VEGF
165 improves angiogenesis in ovarian grafts as soon as three days post-transplantation. In addition, this treatment enhanced vessel maturation because 50% of the ovarian transplants that were impregnated with VEGF
165 exhibited mature vessels that were surrounded by alpha-smooth muscle cells. This rapid reconstruction of blood vessels in ovarian grafts is important for limiting ischemic injury, which has been demonstrated to induce the depletion of 60-95% of the follicular reserve, including the loss of virtually the entire population of growing follicles, during autograft processes [
7,
37-
39]. This phenomenon has been associated with a dramatic reduction in graft size and significant fibrosis [
40]. In this study, three weeks after transplantation, the extent of fibrosis was similar between the control and VEGF
165-treated groups. We previously demonstrated that fibrosis that occurred three weeks after transplantation was partially due to the freeze-thawing procedure [
8]. Therefore, more rapid revascularisation of the transplant after grafting due to the pro-angiogenic effect of VEGF
165 was not likely to limit the development of fibrosis.
This study analysed ovarian fragments that were encapsulated in a collagen gel that contained VEGF
165. Indeed, given the short half-life time of VEGF and in order to have a longer local delivery, a matrix containing VEGF was necessary. In our laboratory, encapsulation of ovarian fragment in a collagen gel had already been developed and proved efficacious. However, other teams have also study the release of VEGF in different models like alginate scaffold [
41] or fibrin matrix [
42-
45]. In this study, the results regarding vascular improvement are in agreement with those from studies that used different systems, such as the encapsulation of ovarian fragments in a VEGF
168 fibrin matrix and autologous murine transplantation [
42-
45]. Additionally, the combined effect of VEGF and basic fibroblast growth factor (bFGF) was found to increase graft survival in an experimental rabbit model of xenografted human ovarian tissue by triggering angiogenesis and by reducing apoptosis and fibrosis [
45]. The present study confirmed that VEGF
165 triggers the angiogenic process; however, VEGF
165 did not reduce fibrosis or affect follicle survival.
The pre-existing vasculature can initiate the formation of new blood vessels either by sprouting, intussusception, or elongation via the incorporation of circulating endothelial cells [
46]. In the context of the physiological angiogenic process of folliculogenesis, most of these processes are effective [
47]. Ovarian tissue grafts are exposed to ischemic damage during the post-transplantation period until the vasculature develops. Vascular connections between the host and an ovarian strip that was grafted into a murine ovary were observed 5 days after transplantation [
13]. To determine whether the addition of VEGF
165 had a beneficial effect on vascular recruitment and limited the period of tissue hypoxia, transplanted fragments were analysed three days and three weeks after grafting. The analysis of vascularisation revealed that VEGF
165 effectively improved graft vascularisation as early as three days post-transplantation. Moreover, a higher proportion of functional blood vessels (dextran FITC-positive vessels) and mature vessels (α-SMA) was observed. Using species-specific CD31 immunohistochemistry and double staining for functional and mature blood vessels (Dextran-FITC and α-SMA), we demonstrated the dual origin of angiogenesis (from the host and from the grafted tissue), as previously described [
36]. Collectively, these data confirm the pro-angiogenic effect of VEGF
165 and the presence of murine endothelial cell migration towards the sheep transplant. However, this improved vascularisation did not modulate the follicular density and did not alter the follicular morphology. In this study, follicular morphology was evaluated in several sections using light microscopy scoring as previously described [
24]. VEGF
165 was used in this study, following our encouraging results with VEGF
111, because it offers the advantage, with respect to the 111 isoform, of existing both in free and matrix-bound forms, which could improve its release profile from the collagen matrix. However, in contrast to VEGF
111, which has been demonstrated to preserve primary ovarian follicles and improve angiogenesis [
23], VEGF
165 failed to significantly decrease fibrosis or increase oocyte survival and morphology. Various VEGF isoforms have exhibited differences in mitogenicity, chemotactic efficacy, receptor interaction, and tissue distribution [
48,
49]. However, a direct comparison of VEGF
111 and VEGF
165 in terms of the follicular pool, survival, and morphology preservation has yet to be performed. Another key factor that affects the efficacy of cytokine treatments is the route of administration of these molecules. Skaznik-Wikiel et al. previously reported that intraperitoneal injections of VEGF
164 for five days combined with subcutaneous injections of granulocyte colony-stimulating factor (G-CSF) maintained the primordial follicular pool [
43]. In addition, Friedman et al. found that graft incubation with hyaluronan-rich biological glue combined with VEGF-A and vitamin E decreased the number of atretic follicles and the levels of apoptosis two weeks after grafting in a xenograft model [
44]. Additionally, a recent study demonstrated that human ovarian graft incubation followed by hypodermic injections at the transplant site with a combination of VEGF and bFGF 7 days after transplantation improved angiogenesis and reduced apoptosis and fibrosis 6 weeks after transplantation [
45]. Although these studies have demonstrated the beneficial effects of VEGF, they often used a method of systemic delivery of VEGF, which is not reasonable in the context of clinical usage in patients with cancer. The major advantage of our model was limiting local exposure to VEGF to the site of transplantation. Other molecules used without VEGF such as S1P [
15], EPO [
16], or PRP [
17] have been shown to improve the quality of ovarian grafts and could therefore be interesting to study in combination with VEGF. Other molecules such as human menopausal gonadotropin (HMG) act indirectly, increasing endogenous VEGF secretion in a transplant after three hours of culture before grafting
. HMG treatment accelerated revascularisation of the transplant and increased the number of surviving follicles one month after transplantation [
50]. These data emphasise the potential benefits of combining different growth factors to prevent early ischemic damage, improve rapid revascularisation of the transplant, and maintain the follicular pool.