Human ovarian tissue: vitrification versus conventional freezing.

Sir, In the challenging paper by Wang et al. (2008), the authors have reported about an effective method of vitrification of human ovarian tissue with direct contact of cells in liquid nitrogen, named needle immersed vitrification. Childbirth after cryopreservation of ovarian tissue is now a reality (Donnez et al., 2004; Demeestere et al., 2007; Meirow et al., 2007; Andersen et al., 2008) and to study the new modifications of cryopreservation protocols is very interesting. In fact, vitrification is technologically promising, it is simpler and one cryocycle is less time-consuming and cheaper than the conventional freezing method. However, the central goal of the cryotechnology is the preservation of intact follicles, and not the guarantee of simplicity and availability of technology for operator to the detriment of the post-warming quality of follicles. However, results of the above investigations (Wang et al., 2008, Fig. 2) have shown that proposed vitrification protocol cannot guarantee the storage of viable follicles after warming in contrast to conventional freezing. Analysis of histological preparation evidence that post-warming follicles are far from normality (Paynter et al., 1999): in both ‘postwarming’ follicles presented in Fig. 2B the vacuolization of cytoplasm, especially in the top follicle as well as the chromatin condensation in both follicles, can be noted; the right follicle in Fig. 2C, which is also denoted by authors as normal, has partly damaged cytoplasm. In our opinion, in the evaluation of normality of cryopreservation protocol it would be better (i) to demonstrate a bigger sector of tissue (see Isachenko et al., 2008a) and (ii) to evaluate the quality of follicles after long-term culture (see Fig. 1 and Isachenko et al., 2007, 2008a). Besides, in our opinion, the above-mentioned method of vitrification cannot be recommended for use in the medical practice because these protocols presuppose a direct contact with liquid nitrogen, which is a potential source of microbial contamination. In fact, any technology in reproductive biology and especially in a medical approach must ensure and guarantee the full protection of biological objects from micro-organisms. Liquid nitrogen, which is used for the storage of frozen material, can be a source of contamination by these micro-organisms (Tedder et al., 1995; Bielanski et al., 2003). Filtration or ultra-violet treatment of liquid nitrogen cannot guarantee the absence of contamination of biological material by viruses. Different types of viruses, which are simple and very cryostable structures, may increase their virulence after a direct plunging and storage in liquid nitrogen, such as hepatitis virus, papova virus, vesicular stomatitis virus and herpes virus. The above vitrification methodology is based on the direct cooling of cells in liquid nitrogen. In contrast, conventional freezing completely avoids the direct contact between the liquid nitrogen and the tissue.