Effects of Er:YAG laser treatment on re-vascularization and follicle survival in frozen/thawed human ovarian cortex transplanted to immunodeficient mice

Abstract

Purpose The huge loss of ovarian follicles after transplantation of frozen/thawed ovarian tissue is considered a major drawback on the efficacy of the procedure. Here we investigate whether Er:YAG laser treatment prior to xenotransplantation can improve re-vascularization and subsequently follicle survival in human ovarian tissue. Methods A total of 99 frozen/thawed human ovarian cortex pieces were included of which 72 pieces from 12 woman were transplanted to immunodeficient mice. Tissues from each woman were included in both an 8-day and an 8-week duration study and treated with either full-beam laser (L1) or fractionated laser (L2), or served as untreated controls. Vascularization of the ovarian xenografts were evaluated after 8 days by qPCR and murine Cd31 immunohistochemical analysis. Follicle densities were evaluated histologically 8 weeks after xenografting. Results Gene expression of Vegf/VEGF was upregulated after L1 treatment (p=0.002, p=0.07, respectively), whereas Angpt1, Angpt2, Tnf-α, and Il1-β were significantly downregulated. No change in gene expression was found in Cd31/CD31, ANGPT1, ANGPT2, ANGTPL4, XBP1, or LRG1 after any of the laser treatments. The fraction of Cd31 positive cells were significantly reduced after L1 and L2 treatment (p<0.0001; p=0.0003, respectively), compared to controls. An overall negative effect of laser treatment was detected on follicle density (p=0.03). Conclusions Er:YAG laser treatment did not improve re-vascularization or follicle survival in human ovarian xenografts after 8 days and 8 weeks grafting, respectively. However, further studies are needed to fully explore the potential angiogenic effects of controlled tissue damage using different intensities or lasers.

Effects of needle puncturing on re-vascularization and follicle survival in xenotransplanted human ovarian tissue

“…After 6 days we observed an increase in vessel formation compared to day 3 xenografts, however, the observed increase was not significantly higher. Between day 6 and 10 we observed a significant increase in vessel density in the human ovarian xenografts, reaching a vascularized area of approximately 3% of the grafts, which is comparable with our previous study [ 21 ]. These results show that the main vascularization of the grafted ovarian tissue occurs between day 6 and 10, confirming that areas of the graft are subjected to prolonged ischemia and hypoxia lasting up to at least 6 days.…”

“…These results show that the main vascularization of the grafted ovarian tissue occurs between day 6 and 10, confirming that areas of the graft are subjected to prolonged ischemia and hypoxia lasting up to at least 6 days. These results together with our previous findings [ 21 ] indicate that the burst in vessel formation occurs after 6 to 10 days, showing that the burst in vascularization happens during a short window of two to three days, giving us insight at which time point the ischemia/ reperfusion injury is the most intense. Furthermore, current results did not show any difference in vessel density between the needle group and control group, and we therefore conclude that needle puncturing did not increase revascularization of xenografted human ovarian tissue.…”

“…A murine liver section was included as a positive control, and non-grafted human ovarian tissue was used as a negative control. The IHC analysis was performed as described previously [ 21 , 26 ]. In brief, sections were de-paraffinized, rehydrated and antigen retrieval was performed using 10 mM sodium citrate (pH 6), following blocking using horse blocking serum (Vector Laboratories, Burlingame, CA, USA) and blocking of endogenous activity with 3% H 2 O 2 .…”

“…Scanned WSI files were analysed in the Visiopharm Integrator System (VIS) using the APP via the Visiopharm Author™ module. The APP used has been described earlier [ 21 , 45 ]. The APP automatically recognized the tissue as the ROI, and afterwards murine host tissue was manually excluded before running the APP.…”

Hormonal Characteristics of Women Receiving Ovarian Tissue Transplantation with or without Endogenous Ovarian Activity

“…Diffusion of oxygen and nutrients into the tissue requires some time to be established, during which reactive oxygen species and oxidative stress make the survival of follicles in the grafted tissue less favourable. Thus, the survival of nongrowing follicles at OTT is limited [6][7][8][9], and revascularisation of tissue in connection with OTT is an important step to improve and optimise the function of grafted tissue [10][11][12].…”