Vitrification Of Large Pieces Of Human Ovarian Tissues And Xenotransplantation To Nude Mice After Cryopreservation

Background and purposeSurvival rates for cancers that occur in young girls and women of reproductive age have improved dramatically in recent years. However, anticancer treatments pose a high risk of ovarian failure in female survivors. Because a growing number of patients wish to preserve their fertility before receiving anticancer treatment, the preservation of fertility for female cancer survivors has become a major concern. Ovarian tissue cryopreservation has received growing attention as a promising technique following embryo freezing and oocyte cryopreservation. In most centers worldwide, ovarian samples from cancer patients are cryostored using slow freezing methods. However, a rapid, simple and inexpensive method termed vitrification has recently been applied to the preservation of ovarian tissue for tissue banking. Vitrification is a procedure in which the cells or tissue are plunged directly into liquid nitrogen, saving both time and cost. This ultra-rapid cooling process can produce a glass-like solidification of cells by increasing the solution's viscosity during cooling, thereby avoiding the formation of lethal intracellular ice crystals. Ovarian tissue preservation by vitrification has been reported in humans, and the results have shown that vitrification may represent an effective alternative to the slow freezing method in human ovarian tissue cryopreservation. However, vitrification is still considered experimental compared with the vitrification of embryos or oocytes and the slow freezing method. A live birth after transplantation of vitrified-warmed human ovarian tissue has not yet been reported, while pregnancies have been successfully achieved through the slow freezing procedure. At present, there is no standard vitrification procedure for human ovarian tissue. Most reports describe the ovarian tissue as being cut into small fragments(approximately 1 mm in thickness, area ? 1 cm2). However, the size of the ovarian tissue sample should not to be too small in order to prevent loss or damage to the follicles, resulting in useless tissue slices during the process of preparing the cortex into small pieces of tissue. Moreover, it is wildly known that the cryopreservation procedure leads to follicle loss compared with the isolation of fresh ovarian tissue. In addition, a large number of grafted follicles could be lost due to ischemia that might occur in post-grafted fragments. For those reasons, the desired effect may be lost when cryopreserving and transplanting small ovarian tissue fragments. In theory, cryopreserving and transplanting a certain number of follicles is beneficial to maintaining ovarian function and female fertility, especially in patients who have undergone a high dose of radiation or chemotherapy with ovarian function failure. However, few studies have been specifically designed to evaluate the vitrified and warmed effectiveness when tissues are cut into large fragments.In this study, we developed a procedure to cryopreserve large-sized samples of human ovarian tissue(approximately 10 × 18 mm2 in area) using a vitrification method. We compared the effects of this cryopreservation method to the slow freezing method. All of the tissues were transplanted into the backs of nude mice subcutaneously and recovered after 3 weeks. Histological analysis and apoptosis were investigated using light microscopy and the terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL) assay, respectively. Graft survival was assessed by follicle counts and proliferative activity was evidenced by immunostaining with anti-Ki-67 antibodies. This study was designed to assess the effects of vitrification on large pieces of human ovarian tissues with the use of a xenografting model.Methods: Ovarian tissues from 18 patients were collected. The size of each tissue piece was about 1mm(thickness)×10mm(width)×18mm(length).All the pieces were randomly divided into vitrification group(group A),slow freezing group(group B) or fresh group(group C). All the tissues were transplanted into the back of nude mice subcutaneously and recovered after 3 weeks. The effects were investigated by histology using light microscope and apoptosis assessed by TUNEL assay. Graft survival was assessed by follicle counts and proliferative activity was evidenced by immunostaining with anti-Ki-67 antibodies.Results:(1)7days after cryopreservation, the proportion of normal primordial follicle had no differences between group A and group B(P<0.05).(2)No significant difference of the proportion of apoptotic follicles was observed between frozen-thawed groups( P>0.05).The proportion of apoptotic stromal cells in group A was lower than those in group B(P<0.05).(3)Three weeks after transplantation, the folicular density of group A had no differences with that in group B(P>0.05),and the proportion of Ki-67-positive follicles in group A had no differences with that in group B(P>0.05).Conclusions(1) Compared with slow freezing method, vitrification freezing procedure m aybe has more advantages in the preservation of ovarian stromal cells.(2) Vitrification of large size pieces of human ovarian tissue could become a possible way to preserve ovarian tissue compared with slow freezing method. However, further studies are still necessary and essential to opti mize vitrification procedure.