Novel needle immersed vitrification: a practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation

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Abstract
Ovarian tissue cryopreservation may be a potential method of preserving fertility in women who have experienced gonadotoxic treatments. To improve the efficiency of existing cryopreservation, we developed a practical and convenient vitrification method named needle immersed vitrification (NIV), which required a less concentrated and minimum volume of vitrification solution. Mouse ovaries and human ovarian cortex fragments were vitrified using the NIV method, the slow-freezing method or the dropping vitrification method. Their morphology, ultrastructure and viability were analyzed and compared with fresh group. Primordial follicles in human and mouse ovarian tissues vitrified by NIV were well preserved. In mice, the percentages of normal morphological primary and secondary follicles were greater in the NIV group than that in the slow-freezing group or dropping vitrification group (P < 0.001). Ultrastructure of the stromal cells was preserved better in the NIV group than the slow-freezing or the dropping vitrification group in both human (P = 0.039, P = 0.023, respectively) and mouse (both P < 0.001) models. The viability assessment on frozen–thawed human ovarian tissue strips revealed that the follicles and the stroma had a satisfactory viability in the NIV group. In mouse model, the ovarian functional restoration in the NIV group was the best among three freezing groups, which was demonstrated by follicle counting in grafts after transplantation (P = 0.009 and P = 0.010 versus slow freezing and dropping vitrification, respectively). The cleavage rate of oocytes from grafts of the NIV group was most similar to that observed in the fresh group. The NIV method could facilitate vitrification process, maximize the cooling rate and reduce the toxicity of the vitrification solution with a minimal volume of less concentrated cryoprotectants. NIV was practical and convenient for cryopreservation of ovarian tissues.

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