Metabolic and secretory recovery of slow frozen–thawed human ovarian tissue in vitro

Abstract

Within the options available for fertility preservation, cryopreservation of ovarian cortical tissue has become an important technique. Freezing and thawing procedures have been optimized to preserve tissue integrity and viability. However, the improvement of the tissue retransplantation is currently of great interest. Rapid angiogenesis is needed at the retransplantation site to accomplish sufficient blood supply to provide oxygen and nutrients. Many studies address this issue. However, we need to understand the physiology of the thawed tissue to gain further understanding of the complexities of the procedure. As freezing and thawing generally impairs cellular metabolism, we aimed to characterize the changes in metabolic activity and secretion of the angiogenic factor vascular endothelial growth factor-A (VEGF-A) of frozen-thawed ovarian cortical tissue over time. Biopsy punches of ovarian cortical tissue from patients undergoing fertility preservation were maintained in culture without freezing or after a slow-freezing and thawing procedure. VEGF-A secretion was measured after 48 h by ELISA. To examine temporary changes, metabolic activity was assessed for both fresh and frozen-thawed tissue of the same patient. Metabolic activity and VEGF-A secretion were measured at 0, 24 and 48 h in culture. Thawed ovarian cortical tissue secreted significantly less VEGF-A compared to fresh ovarian cortical tissue within 48 h of culture. After thawing, metabolic activity was significantly reduced compared to fresh ovarian cortex but over the course of 48 h, the metabolic activity recovered. Similarly, VEGF-A secretion of thawed tissue increased significantly over 48 h. Here, we have shown that it takes 48 h for ovarian cortical tissue to recover metabolically after thawing, including VEGF-A secretion.

Comparison of angiogenic potential in vitrified vs. slow frozen human ovarian tissue

“…This is in line with the observation that after cryopreservation the metabolic activity is reduced, accompanied by a vastly decreased secretion of VEGF. This effect recovers over the course of 48 h 28 . Only few factors were increased after OTC compared to the fresh tissue: bFGF, PDGF-BB (after vitrification/rapid warming), HGF and PlGF (after slow freezing/thawing).…”

“…Primordial follicles characterized by flat granulosa cells, primary follicles with cuboidal granulosa cells and early secondary follicles with more than a monolayer of cuboidal granulosa cells were observed. Moreover, follicles were viable as shown by the positive staining for PCNA 27 , 28 . Preserved viability and integrity of the tissue was observed, as shown in representative images in Fig.…”

“…EGF, HB-EGF and Leptin were barely above the detection limit. The key challenge regarding retransplantation is to counter ischemic deviations 28 , minimized by a prestaggered, optimized cryopreservation/thawing protocol that ensures substantial expression of cytokines involved in angiogenesis as well as a low apoptotic profile of the tissue prior to grafting.…”

Human ovarian tissue xenotransplantation in 3D-printed tissue trapper basket integrated with human umbilical cord mesenchymal stem cells

“…The increase in blood vessel number and diameter, along with higher VEGF gene expression, signifies enhanced tissue perfusion and angiogenic potential. Successful angiogenesis supports tissue integration and functional recovery (57).…”

Impacts of vitrification on the transcriptome of human ovarian tissue in patients with gynecological cancer

“…Based on live birth outcomes after OTC and transplantation in humans, slow freezing is still considered the gold standard technique for OTC. Because of ice crystal formation (Donnez et al, 2013), many studies have evaluated the negative effects on ovarian tissues of the slow-freezing method, including higher levels of DNA fragmentation (Xiao et al, 2013), gene under-expression (Labrune et al, 2020), and metabolic and secretory issues (Einenkel et al, 2022). Recently, Kim et al (2021) suggested that human OTC using a slow-freezing technique is associated with significant telomere shortening and altered senescence markers and mitochondrial structures (Boram et al, 2021).…”