Clinical value
The preservation effect for vitrification freezing of ovarian tissues was influenced by ovarian tissue size, refrigerant concentration, refrigerant balance time, the carrier system applied and other multiple factors. These induced the refrigerating fluid evenly penetrate into the ovarian tissue and ensure the consistency of the freezing rate of each tissue. Furthermore, the cryopreservation of ovarian tissues mainly adopts cryopreserved small tissues (area: < 1 cm
2, thickness: 1 mm) [
9]. However, the tissue slice volume was not too small, in order to avoid the missing or damaging of excessive follicles during cutting, which would thereby produce many unavailable tissue slices. Some studies have reported that the freezing and thawing process of ovarian tissues can cause a loss of 7% of follicles, and after transplantation and before blood supply reconstruction, a loss of more than 60% of follicles may be caused, reducing the practical significance for cryopreservation and transplantation of small ovarian tissues [
10]. Poirot et al. [
11] considered that the freezing of large ovarian tissues can acquire more follicles, protecting the preantral follicle with its large volume, and guaranteeing the structure in the tissue to better support follicle growth. Gook et al. [
12] considered that after cutting the ovarian tissue into slices with the size of 2.0–5.0 mm, the influence on freezing was not significant, and preantral follicles with a large volume were also protected. Anerdsen et al. [
13] transplanted a large ovarian tissue (close to 1/3 of the whole ovarian tissue) to one patient with the acute lymphoblastic leukemia, and made the ovarian tissue function continuously for seven years, and even longer. A certain amount of follicles is the basis to maintain ovarian function, and for patients with ovarian function failure, who underwent high-dose radiotherapy and chemotherapy. Theoretically speaking, it is very beneficial to transplant more ovarian cortexes with follicle growth activity [
13]. Hence, in order maintain ovarian function after transplantation for a long time, the cortex size should be increased as far as possible under the premise of considering the freezing effect and follicle survival rate.
Feasibility analysis
The key for vitrification freezing is to make the refrigerant penetrate into the ovarian tissue, and effective tissue penetration is the premise for tissues to smoothly enter into the vitrification state during cooling. In the present experiment, the tissue slice was trimmed to form an even hole clearance, and the physical diffusion principle was used to realize the effective penetration of the refrigerant and reduce balance time. In this vitrification freezing scheme, the cryotissue was used as a carrier to bear the large ovarian cortex slice. Meanwhile, in this scheme, the tissue was directly placed in a metal carrier that has been precooled in liquid nitrogen, in order to avoid heat conduction block due to the formation of bubbles generated from the evaporation of liquid nitrogen, and minimize the cryoprotective agent required for frozen tissue, thereby improving cooling speed and promoting the formation of a vitrification state [
14]. This experiment cryopreserved a large area of ovarian tissue slice, but the ice crystal was not obviously formed during vitrification freezing and after unfreezing. Approximately 79.6% of primordial follicles in the vitrification freezing group maintained a normal morphology, which was slightly lower than the normal morphology rate in fresh ovarian tissues (87.5%). However, such rate corresponded to the normal morphology rate of 70–90% of primordial follicles after freezing, as reported in other literatures [
15‐
17]. This proves that after the tissue slice was trimmed, the refrigerant was evenly penetrated to prevent the ovarian tissue from forming an obvious ice crystal during the transformation to the vitrification state, thereby leading to a good follicle preservation effect. At the same time, the main preservation object of ovarian tissue freezing includes the primary follicle, accounting for 20–30% of the total number of follicles, in addition to the primordial follicle. Through the morphological analysis of the follicle, by comparing with programmed freezing, vitrification freezing has a significant advantage on the cryopreservation of the primary follicle.
Due to the limitations of the morphological analysis in the evaluation of ovarian tissue development potentiality, the present experiment adopted the analysis of the apoptosis index to compare the effects of different freezing methods on the preservation of ovarian development potentiality. It was found that the follicle apoptosis rate was similar among freezing groups, while for the apoptosis rate of the primordial follicle, the difference was not significant. Meanwhile, the incidence of apoptosis of the interstitial cell in the vitrification freezing group was lower than that in the programmed freezing group, prompting that the vitrification freezing method can be used to better protect ovarian interstitial cells, providing better tissue samples for the clinical application of ovarian tissues after freezing and thawing. The apoptosis was closely correlated to the normal development of human ovarian tissues and its function. It is undeniable that vitrification freezing can cause a certain degree of damage on follicles, which may be due to the high concentration of the cryoprotective agent.
The present experiment selected subcutaneous tissues in the back of nude mice as the transplantation part. This was due to its loose nature and relatively rich blood supply, which is beneficial for the survival of the transplants. Denschlag et al. [
18] reported that a large ovarian tissue slice (diameter: approximately 2 cm, thickness: approximately 2–3 mm) can survive after autoplastic heterotopic transplantation without vascular anastomosis. However, the blood supply of ovarian tissues after transplantation completely relies on the growth of peripheral capillaries without the help of vascular anastomosis, and excessive long or thick tissues can make the blood vessels of an animal subject spend more time entering into the tissue by means of peripheral growth. The longer the new vessels completely cover the tissue, the larger the ischemic injury becomes, and will cause ischemic injury to the tissue at the early stage of transplantation, and influence the development of follicles. The present experiment revealed that the volume of partial transplants shrunk, and most follicles had locking, tissue fibrosis, obviously declined density, and other conditions. These were consistent with the phenomenon on the loss of many follicles due to ischemia reperfusion injury in the ovarian transplantation test on mice performed by Liu et al. [
19]. However, in the present experiment, there were many new vessels in the junction of the transplant and the host, and under an optical microscope, rich capillaries formed. Meanwhile, the follicle distribution was relatively rich, showing that after the ovarian tissue slice was trimmed through holes, the new vessels formed by the host could enter the tissue through these evenly distributed holes, reducing the ischemic injury of the tissue, and providing a possibility for survival and further development of follicles.
The proliferating cell nuclear antigen Ki-67 antigen expression was located in granule cells, and the oocyte cytoplasm and nuclei, and it is an index to evaluate the cell proliferation state. Its expression is positively correlated to the cell proliferation in the cell cycle of G1, S, G2 and M, other than G phase. Furthermore, it can be used as a specific marker of cell proliferation to evaluate follicle activity, and the proliferation of the granule cells is usually considered as an early marker of primordial follicle activation [
20]. The present experiment revealed that the expression of the Ki-67 antigen in the transplant was mainly embodied in the surviving follicle during the growth period, while the expression of primordial follicles was rare, which was mainly due to the primordial follicle in the dormancy period. In comparing the follicle positive rate of the Ki-67 antigen expression in the transplant among these three groups, the difference was not statistically significant. This might be associated to statistical error due to loss of follicles after the transplantation. However, in the vitrification freezing group, the positive staining of follicles during the growing period was identified, indicating that ovarian tissues after vitrification freezing continued to have a potential for proliferation in vitro. Furthermore, whether there is tissue and cell activity after human ovarian tissues are frozen and thawed, the activated cell mass, follicle growth, and its development ability are constrained. Other issues need to be further researched to carry out further confirmations and interpretations.