In the current study, autologous ovarian transplantation by itself increased dysplastic changes in the ovarian surface epithelium in rats. It was observed that the use of Vit C significantly reduced these dysplastic changes. While serum and tissue MDA levels were significantly higher in the transplant-only group, a significant decrease was observed in the erythrocyte SOD level compared to the other groups. High SOD and low serum and tissue MDA levels in the Vit C group were both significantly different than those seen in the Vit E group. Therefore, it was determined that the use of Vit C was more effective than the use of Vit E for antioxidative protection during ovarian transplantation in rats.
According to the literature review conducted on Pub-Med (ovarian transplantation, ovarian surface epithelial dysplasia, ovarian follicle reserve, Vit E, Vit C), the current study is the first to be conducted on this specific subject.
Previous studies have been conducted on transplantation models using different substances, such as antioxidant agents or angiogenic factors. However, inflammation, oxidative stress, and apoptosis are all integral mechanisms that are involved in organ damage in organs susceptible to ischaemic reperfusion (I/R), such as transplanted ovaries. Therefore, this study hypothesised that the IP injection of either Vit E or Vit C prior to transplantation would protect the ovarian follicle reserve and surface epithelium from damage due to oxidative stress and consequently reduce the likelihood of postoperative complications.
Previous studies have shown that the transplanted ovaries suffer ischaemia and potential follicular atresia during and after tissue revascularisation, regardless of the transplant location (16). The mechanism of ischaemia causes energy depletion and oxidative stress, which in turn leads to the damage of lipids, DNA, enzymes, and structural proteins, which consequently results in cell death (17). In addition, transplanted tissues have been shown to have heightened sensitivity to hypoxia and undergo displacement of neutrophils and macrophages, causing tissue destruction and fibrosis (18).
Most ovarian cancers are thought to arise from surface epithelial cells that are disrupted during ovulation (19). During the follicular mechanics of ovulation, inflammatory mediators and reactive oxidant species are produced in the ovaries (20). In the current study, harmful cellular changes due to hypoxia in the ovarian surface epithelium and follicle reserve were histopathologically observed. The effect of this oxidative stress on normal cells was found to be consistent with the existing literature (21). Ovarian transplantation significantly increased ovarian surface epithelial stratification, tufting, chromatin irregularity, nucleus contour irregularity, nucleus size, nuclear-cytoplasmic ratio, pleomorphism, and hyperchromasia in rats. In rats that underwent ovarian transplantation without Vit E and Vit C treatment, a significant increase was seen in all parameters relating to the ovarian surface epithelium except in nuclear contour irregularity, nucleoli presence, nuclear-cytoplasmic ratio, mitosis, and chromatin irregularity. Vit C was found to be effective at protecting the ovarian surface epithelium from dysplastic changes. Therefore, using Vit C before ovarian transplantation can prevent the harmful effects of the inevitable I/R damage that occurs during ovarian transplantation. The insufficient protective effect of Vit E could be explained by the short exposure time or the use of a fatty form of Vit E, which may have resulted in inadequate exposure to the transplanted ovaries. Therefore, the differences in Vit E findings can be attributed to the methodological differences between studies, including the route of administration, the duration of exposure to Vit E after transplantation, or the transplantation site of ovarian tissue (22).
Ovarian follicle reserve was found to be significantly lower in the transplantation model group compared to the other groups, although a decrease was still seen in the groups that received Vit C and Vit E. One possible explanation for this finding is that the concentration of the single doses of Vit E and Vit C used in the current study may not have been sufficient to remove free radicals generated by prolonged ischaemia. Although the concentration of Vit C used appears to be effective at protecting follicle cultures, it is unknown whether this concentration is optimal in tissue (23). Therefore, future studies could investigate the clinical efficacy and relevance of using Vit C and Vit E to protect ovarian tissue from ischaemia after transplantation. Although it was observed that more follicles were lost during ischaemia when there was no antioxidant treatment, even with antioxidant treatments, over 50% of follicles were still lost due to ischemia. Therefore, the most critical issue for effective ovarian transplantation is to minimise ischaemia before and during angiogenesis. During the revascularisation of the transplanted tissue, hypoxia causes necrosis and subsequent follicular loss. Therefore, some degree of follicle loss during the procedure was inevitable in the current study.
In the existing literature, human vaccines have been transplanted into immunodeficient mice in order to elicit the effect of antioxidants. Abir observed a lack of antiapoptotic activity when grafts were incubated with Vit E before transplantation (24). In the current study, lower tissue and serum MDA levels in groups that received Vit E suggests that Vit E elicits more of an antioxidant effect on transplanted ovaries than Vit C. Tissue and serum MDA levels were higher in the transplantation model group. Vit C treatment resulted in lower serum and tissue MDA concentrations compared to Vit E treatment. This result is also correlated to the histopathological findings of Vit C, which showed that it prevented ischaemic damage during transplantation. According to Tavasoli, Vit C has physiological antioxidant properties to decrease I/R by reducing the expression of proinflammatory chemokines and lipid peroxidation. In addition, Tavasoli has shown that intravenous injection of Vit C has a protective effect on the intestinal mucosa (25). Another study showed that a single dose of Vit C immediately after bloodstream reperfusion significantly reduced I/R injuries in kidney tissue (26). According to Gunel, depending on the MDA levels, Vit C showed a more significant antioxidant effect in rabbits compared to Vit E regarding intestinal I/R injury (27). This finding is consistent with the current study.
In fresh and frozen/thawed cortical sections of bovine ovaries, stromal cells are more prone to ischaemia than primordial follicles, and the administration of Vit C reduces apoptosis in the ovarian cortex (28). The results of the current study showed that Vit C reduced tissue and serum MDA levels compared to the transplantation-only group, which is consistent with the existing literature. However, while Vit C and Vit E have significant beneficial effects on post-transplantation ovarian tissue, there are still marked differences compared to normal ovarian tissue. Furthermore, the control group had the lowest concentrations of both tissue and serum MDA, suggesting that further research is needed to determine how to control the oxidative stress mechanism.
SOD is an enzyme and antioxidant system marker that catalyses the superoxide (O2−) radical to ordinary molecular oxygen (O2) and hydrogen peroxide (H2O2). H2O2 is produced as a by-product of oxygen metabolism and, if not regulated, causes multiple types of cell damage (29). Therefore, it is an important target for antioxidant defence in all living cells. Although the use of Vit E and Vit C increased SOD levels when compared to the transplantation-only group, the highest erythrocyte SOD level was seen in the control group. This indicates that the concentrations used of Vit C and Vit E do not affect the oxidative stress mechanism as much as the normal tissue levels of both. Normal tissue level can be achieved by increasing the applied doses of Vit E or Vit C or by applying them together. Overall, it was found that antioxidant use is beneficial for reducing the damages caused during ovarian transplantation. These findings are consistent with the results of Tavasoli et al. (25). However, further research is needed to examine the molecular mechanism of administering a single Vit C and Vit E dose in autologous ovarian transplantation in rats.
Although the current study has promising results, it has some limitations. Since this is an animal study, the results may not be adopted directly and linearly to human beings. Additional studies should be conducted on this topic.
Using Vit E and Vit C in autologous ovarian transplantation in rats reduces the dysplastic changes of the ovarian surface epithelium and preserves the follicle reserve in the ovaries. While the use of Vit C and Vit E decreases tissue MDA and serum MDA levels, they increase erythrocyte SOD levels. Overall, Vit C was more effective in protecting against oxidative stress than Vit E.