Investigation of the effects of melatonin and vitamin D on the ovary in a model of premature ovarian failure induced by Cyclophosphamide in rats.

Chemotherapy can lead to premature ovarian failure (POF) and infertility by triggering primordial follicle apoptosis or excessive activation of primordial follicles in the ovary. However, an effective treatment method has not been found yet due to the mechanisms effective in the development of premature ovarian failure have not been fully elucidated. In the presented study, it was aimed to investigate the effects of the combined use of melatonin and vitamin D as a pharmacological approach to preserve fertility in an experimental premature ovarian failure model induced by cyclophosphamide, on ovarian follicle reserve and structure. In the study, 49 adult female rats were randomly divided into 7 groups. Group 1 was evaluated as intact control group, group 2 as melatonin control group, group 3 as vitamin d control group, group 4 as POF group, group 5 as cyclophosphamide and melatonin group, group 6 as cyclophosphamide and vitamin D group, and group 7 as combined treatment group. Intracardiac blood samples were taken from the rats under anesthesia on the 11th day, for serum FSH, LH, E2 and P level analyzes. Ovaries were prepared for examination by light microscopic, electron microscopic and molecular biological methods. During light microscopic examinations, primordial, developing, atretic follicles and corpus luteum number were counted in serial sections and statistical analyzes were performed. Ovarian fibrosis was evaluated by Masson trichrome staining method, PTEN, FOXO3a, AMH gene expression levels were evaluated by qRT-PCR technique, and PTEN, FOXO3a, AMH, TNF- α and caspase-3 expressions were evaluated by immunohistochemical method. It was observed that FSH and LH levels in cyclophosphamide-treated rats were increased significantly, E2 and P levels decreased significantly, and FSH and LH levels were decreased in the melatonin and vitamin D treatment groups, whereas there was a significant increase in E2 and P levels. It was determined that cyclophosphamide caused a decrease in the numbers of primordial and developing follicles and an increase in the number of the corpus luteum, atretic follicles and fibrosis, and the values in the treatment groups were close to the control groups. It was observed that PTEN, FOXO3 and AMH mRNA expression in the ovarian tissue were decreased with cyclophosphamide administration, while PTEN, FOXO3 and AMH mRNA expressions were increased in the treatment groups, and immunohistochemical examinations showed a similar correlation with these findings. While an increase in TNF- α and Caspase-3 expressions were detected in ovarian damage caused by cyclophosphamide, significant structural changes were also detected in primordial and developing follicles, surface epithelium and ovarian stroma. Significant decreases in TNF- α and caspase-3 expression were observed in the treatment groups, it was noted that structural changes in the follicles and ovarian stroma were milder. When all the findings are evaluated together, in ovarian damage caused by cyclophosphamide, as a result of the decrease in PTEN, FOXO3a and AMH gene expressions, which are known to have a negative regulatory effect on follicular growth, and the excessive activation of primordial follicles, many follicles enter the development process and eventually suffer atresia. It was determined that combined melatonin and vitamin D therapy protected ovarian reserve by changing PTEN, FOXO3 and AMH gene expression levels. It was also concluded that melatonin and vitamin D are effective in restoring ovarian function through their antifibrotic, antioxidant and anti-inflammatory effects and can be evaluated clinically as a therapeutic agent. Key words: Cyclophosphamide, Folliculogenesis, Premature ovarian failure, PTEN, FOXO3a, AMH