We show here that vitrification of human ovarian tissue can be carried out in a clinical setting without any direct contact with liquid nitrogen. This enables us to easily comply with the quality requirements of the European tissue directive 2004/23, 2006/17/EG, and 2006/86/EG (www.EU).
Using our closed vitrification system, we can avoid the risk of contamination of the samples during the vitrification and storage procedures. This is a real advantage because ovarian samples for fertility preservation have to be cryo-stored for very long periods.
In a previous study, we compared slow freezing and vitrification of human ovarian tissue, and showed that the morphological integrity of particularly the stromal tissue after vitrification was better than that after slow freezing (Keros et al., 2009
). However, the procedure was not carried out in a clinical setting. In this study, we reported an extremely simple and feasible, closed vitrification system which avoids direct contact of ovarian tissue with liquid nitrogen.
The device that we used was a cryotube, which could be closed and then rapidly immersed into liquid nitrogen leaving the cap above the surface of the nitrogen to avoid leaking. It was a non-toxic, sterile cryotube containing internal thread with a silicone gasket to provide the best possible seal. Efficient vitrification requires extremely fast cooling, and apparently this very simple system enables such a rate preserving the morphology of both the pre-antral follicles and stromal tissue. This morphology was maintained following 24 h of culture. It is possible that lysed follicles would not have been identified both after warming and culture; however, this is an error in any assessment of human ovarian tissue following cryopreservation.
Further investigation such as xeno-transplantation may also give information about viability of the follicles within the tissue. Nevertheless, tissue culture is an excellent method to demonstrate the viability of the tissue (Li et al., 2007
; Keros et al., 2009
). Non-viable follicles do not have intact electron microscopic ultrastructure after 24 h culture (Keros et al., 2009
). We did not use any particular apoptosis assays because the most likely consequence of vitrification would be necrosis, which can better be demonstrated using careful morphological and ultrastructural analysis (Hovatta et al., 1996
; Keros et al., 2009
). An increased number of viable follicles may indicate better survival rate of the follicles during vitrification. Furthermore ultrastructural evaluation by transmission electron microscopy is the best known method to demonstrate the cryodamage (Gook et al., 1999
; Hreinsson et al., 2003
; Martinez-Madrid et al., 2007
The final proof of the viability of the tissue would still be a pregnancy and a healthy child, and this remains to be shown.
The data from comparative studies of vitrification and slow-rate freezing of human ovarian tissue are limited and opposing conclusions have been drawn by investigators. Some results have indicated that vitrification of human ovarian tissue is less efficient than slow-rate freezing (Gandolfi et al., 2006
; Isachenko et al., 2009
). Some authors concluded that modified vitrification is an effective technique for freezing of ovarian tissue as it showed presence of most normal follicles (80.3%) after warming (Li et al., 2007
). Our group showed that vitrification of human ovarian tissue was comparable with slow-rate freezing (Keros et al., 2009
). These different results may due to differences in the procedure, for example, cryoprotectant composition and concentrations, exposure times to cryoprotectants and speed of vitrification. The process of slow freezing or vitrification is not the issue. The issue is that the procedures have not been optimized for that particular biological material. The penetration rate of cryoprotectants and the temperature play an important role during the cryopreservations procedure (Newton et al., 1996
). For this reason, it is important to have a sufficient diffusion of cryoprotectant through the stroma and granulosa cells to the oocytes. In order to reach this aim, a compromise between the concentration of cryoprotectants, incubation time and temperature is required (Hovatta, 2005
The improved stromal morphology reported in the present study confirms our previous observations (Keros et al., 2009
) and is in contrast to the poor morphology of human stromal tissue previously reported using slow freezing (Gook et al., 1999
). It would appear that the use of the combination of DMSO, PrOH and EG together with the stepwise increase in their concentrations has achieved sufficient dehydration to successfully vitrify all components of human ovarian tissue. In this study, the long incubation time of the cryoprotectants was applied because in our earlier study we showed that good-quality stroma, composed of collagen fibres and stromal cells was observed in the vitrification group with 10 min incubation time (Keros et al., 2009
). Stromal cells probably play an important role in the proliferation and differentiation of granulosa cells (Hovatta et al., 1999
; Liu et al., 2000
). Co-operation between the granulosa cells, the stromal tissue and the oocytes is important for ovarian function (Hovatta, 2005
), and preservation of the integrity of these components is necessary (Gook et al., 2004
Our vitrification protocol can be performed in a clinical setting. We have shown excellent structural preservation of ovarian tissue. Our observations from LM showed similar morphology of the pre-antral follicles in both vitrified and non-vitrified tissue. This observation was confirmed by TEM assesses which revealed that the organelles of granulosa cells and oocytes were well preserved in vitrified tissues. The result of this study suggest that human ovarian tissue can be well preserved using simply a cryotube as a device in a closed system to avoid possible contamination via direct contact with liquid nitrogen.