Mouse ovarian tissue vitrification on copper electron microscope grids versus slow freezing: a comparative ultrastructural study

Abstract

There are many reasons, including cancer therapy, for premature ovarian failure and infertility. Oocyte, embryo and ovarian cryopreservation are current options for fertility preservation. Ovarian tissue cryopreservation is essential in patients whose cancer therapy cannot be delayed, including prepubertal girls, and is mostly performed using slow freezing. In the present study, mouse ovarian tissues were vitrified on copper electron microscope grids (n=18) or conventionally slow frozen (n=18). Post-thaw tissues were examined histologically using light and electron microscopy and compared with the control group. According to light microscopy observations, antral follicles were found to be better preserved with the slow freezing technique rather than vitrification. Electron microscopy revealed swollen mitochondria in the oocyte cytoplasm, condensations in the zona pellucida, breakages in the junctions of granulosa cells and vacuolisation in the extracellular space in pathologic follicles, which were relatively more frequent, in the vitrification group after thawing. These results indicate that ovarian slow freezing is preferable than vitrification on copper electron microscope grids, especially for larger follicles. Conversely, vitrification of ovarian pieces using cooper grids is user-friendly and provided good protection for primordial follicles and stromal cells. There is a need for further studies into advanced tissue vitrification techniques and carriers.

Expression of inhibitor proteins that control primordial follicle reserve decreases in cryopreserved ovaries after autotransplantation

“…Slow-freezing of OTs was performed using a temperaturecontrolled freezer (IceCube; SyLab, Neupurkersdorf, Austria). The procedure was carried out according to the protocol described previously [20]. Briefly, OTs were taken into cryovials containing 1 mL of cryoprotective medium composed of 1.5 M dimethyl sulfoxide (DMSO), 20% fetal bovine serum (FBS), and 0.1 M sucrose and incubated at 4°C for 30 min prior to loading into the freezer.…”

Bio-inspired solute enables preservation of human oocytes using minimum volume vitrification

“…Various vitrification devices, including the conventional plastic straw, have been used for the vitrification of many cells, including oocytes (Asghar et al, 2014;Berthelot, Martinat-Botté, Locatelli, Perreau, & Terqui, 2000;Chung et al, 2000;Hyttel, Vajta, & Callesen, 2000;Nöthling & Shuttleworth, 2005;Topal-Celikkan, Ozkavukcu, Balci, Serin-Kilicoglu, & Atabenli-Erdemli, 2015). However, due to their low cooling rates (~1000°C/min), these methods require high CPA concentrations (~8 M), which further subject the cells to high levels of toxicity.…”

Cryopreservation of Endangered Ornamental Plants and Fruit Crops from Tropical and Subtropical Regions

“…Detailed procedures of cryo-mesh cryopreservation have been presented by Yamamoto et al [ 90 ], Funnekotter et al [ 100 ], and Wang et al [ 6 ]. Another possibility is the user-friendly vitrification of tissues on electron microscope grids (made of copper) for cryopreservation, which so far has been successfully used with animal specimens (oocytes) [ 101 ].…”