Elettra | SYRMEP X-ray phase-contrast microtomography to monitor revascularization in ovarian tissue transplantation In recent years, fertility preservation has become a critical need for young women undergoing gonadotoxic cancer therapies. Among the options, Ovarian Tissue Cryopreservation (OTC) followed by transplantation (Ovarian Tissue Transplantation, OTT) remains the only viable strategy for prepubertal girls and women who cannot postpone oncological treatments. However, the procedure faces a significant challenge: the ischemic window posttransplantation, which can result in the loss of over 60% of ovarian follicles. For this reason, novel approaches to shorten this interval are urgently needed. Moreover, since ovarian angiogenesis is closely linked to follicular maturation, promoting vascularization is expected to enhance graft functionality and hormone production. This study proposes a novel approach to counteract this issue by leveraging tissue engineering strategies. A combined Advanced Therapeutic Medicinal Product (ATMP) was developed by coupling cryopreserved human ovarian tissue with a biocompatible scaffold pre-seeded with Endothelial Cells (ECs) derived from the same ovarian biopsies. This strategy aimed to accelerate graft revascularization and improve tissue survival. OVarian Endothelial Cells (OVECs) were isolated from healthy ovarian tissue obtained from patients enrolled at IRCCS Burlo Garofolo (Trieste, Italy). The cells were characterized by immunofluorescence and flow cytometry, confirming a purity of approximately 90% for endothelial markers such as CD31. OVECs were then cultured on four commercially available dermal scaffolds to evaluate their adhesion and proliferation capacity. Among the tested options, the bovine collagen matrix (Integra®) showed superior biocompatibility and promoted robust cell growth. Furthermore, OVECs cultured in 3D-conditions raised the levels of important genes that help blood vessel formation, including Vascular Endothelial Growth Factor-A (VEGF-A) and Placental Growth Factor (PGF), confirming the scaffold’s potential to support vascular regeneration. To validate these findings in vivo, cryopreserved ovarian tissue - with or without OVEC - was transplanted subcutaneously into immunodeficient female mice. After 14 days, grafts were excised and stained for human CD31 (hCD31) and OVEC-supplemented grafts showed markedly improved vascularization. Confocal imaging further revealed the formation of chimeric vessels, resulting from the integration between human OVECs and the host mouse vasculature. These newly formed vessels were supported by NG2-positive perivascular cells, which play a key role in vessel maturation and stability. Importantly, OVEC supplementation also led to a significant increase in tissue viability. Nuclear counting revealed that the supplemented grafts had more cells, while molecular analysis showed elevated expression of the anti-Müllerian hormone (AMH), which is a biomarker of ovarian reserve. These results suggest that OVECs not only enhance vascularization but also contribute to maintaining the structural and functional integrity of the transplanted ovarian tissue, possibly supporting follicular survival, endocrine function, and reproductive potential. The wellpreserved state of grafted ovarian tissue was also confirmed by x-ray phasecontrast microtomography (microCT) (Fig. 1 a) that allowed us to identify structures that are compatible with healthy primary or primordial follicles within the tissue (Fig. 1 b). X-ray phase-contrast microCT, also known as “virtual histology,” was used to assess the vascularization of the tissue, since it allows three-dimensional visualization of tissues without physical sectioning it. H&E staining (Fig. 1 c) and x-ray phasecontrast microCT (Fig. 1 d) revealed that OVECs, when grafted with ovarian tissue seeded onto a bovine collagen matrix, form vascular-like structures containing red blood cells—indicating functionality. In contrast, control grafts without OVECs (Fig. 1 e,f) lacked such vascularization. MicroCT further visualized these vessellike structures in 3D, confirming the ability of OVECs to create a vascular bed within the scaffold. Iodine staining was used to enhance contrast and allow deeper visualization of cellular and vascular compartments. Fig. 1 (g) shows a maximum-intensity projection of a stained OVECsupplemented graft, where both endogenous and newly formed vascular networks are visible. Large vessels can be seen at the center of the ovarian tissue, surrounded by denser regions consistent with endothelial cells accumulation and vascular infiltration into the scaffold. The combination of these imaging techniques provided a detailed and non-destructive view of the graft microarchitecture, supporting the interpretation of histological and immunofluorescence data. Overall, this work presents a promising and innovative strategy to enhance the clinical outcomes of ovarian tissue transplantation. By shortening the ischemic phase through the use of autologous pre-vascularized scaffolds, the approach significantly improves graft survival and function. In parallel, the integration of advanced synchrotron-based imaging tools such as x-ray phase-contrast microCT offers unprecedented insight into tissue morphology and vascular development, without the need for destructive processing. These results contribute to the emerging field of bioengineered reproductive tissues and pave the way for the development of an “artificial ovary”—a composite system to restore fertility and endocrine balance in cancer survivors and other patients facing reproductive challenges. The integration of cellular therapy, scaffold technology, and highresolution imaging holds great potential for personalized medicine in female reproductive health. Elettra Highlights 2024–2025 60
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