Effects And Mechanism Of Cyclophosphamide On The Ovarian Structure And Function Of Rats With Premature Ovarian Insufficiency

Premature ovarian insufficiency?POI?refers to the emergence of ovarian dysfunction in women before the age of 40,characterized with abnormal menstruation?amenorrhea or dilute menstruation?,elevated gonadotropin levels?FSH>25 U/L?,and the fluctuation of estrogen level.Genetic factors,iatrogenic factors,immune factors and other factors may lead to POI,and its pathogenesis is not clear.In recent years,the incidence of malignant tumor in the global range is increasing and the trend of youth is obvious.Although the improvement of modern medical technology is benefited more patients and prolongs their survival time,the incidence of iatrogenic POI is also increasing.POI caused the problems of menstrual disorder,amenorrhea,infertility,coronary heart disease and?or?cerebrovascular disease,osteoporosis and fracture,which seriously interfere with the life of the patients and affect the physical and mental health.Therefore,the treatment of premature ovarian insufficiency and improvement of ovarian function is a difficult problem to be solved in clinical work.Current clinical POI methods include hormone replacement therapy?HRT?,immunotherapy,phytoestrogens,traditional Chinese medicine,fertility-related treatments?such as embryo freezing,mature oocyte freezing,ovarian tissue freezing?,and other new therapeutic methods.However,these treatments have their limitations and it is difficult to fundamentally improve ovarian function.Therefore,it is necessary to actively explore new treatments.According to relevant studies,a certain number of follicles still remain in the ovaries of POI patients.In certain circumstances,the remaining follicles in the ovaries can continue to develop,mature,and even spontaneously ovulation,especially when a favorable external stimulus improves the local microenvironment.The study shows that low intensity pulsed ultrasound can improve the local microenvironment of inflammatory tissue,shorten the time of cells in the inflammatory stage,realize the anti-inflammatory effect and promote the repair of inflammatory tissue.Low-intensity Pulsed Ultrasound?LIPUS?is a pulse type ultrasonic wave.It has been widely used to promote bone and cartilage regeneration,soft tissue repair and other fields.Related studies have shown that LIPUS can inhibit the secretion of various inflammatory cytokines,promote the proliferation and migration of various cells,strengthen the osteogenesis,chondrogenic differentiation of stem cells and promote the development of ovarian follicles in the ovary.In this experiment,the effect of LIPUS on ovarian structure and function of POI rats and the mechanism of ovarian function of POI rats were observed by interfering with the model of chemotherapeutic premature ovarian failure,and the experimental basis for clinical treatment of POI was provided.Part 1 Construct premature ovarian insufficiency models in SD ratsObjective To establish the rat models with cyclophosphamide?CTX?-induced premature ovarian insufficiency?POI?.Methods Fifty female Sprague-Dawley?SD?rats were randomly divided into model group and control group.Rats in model group received intraperitoneal injection of loading dose CTX?50mg/kg?,and maintenance dose of 8mg/kg·d for 14 days;Rats in control group were given the same amount of saline during the same period.The modeling effects were judged by observation and comparison of estrous cycle,levels of sex hormone and morphological changes of organs between the two groups at different time.Results Compared with control group,the estrous cycle in model group was disordered;the levels of serum anti-mullerian hormone?AMH?and estrogen?E₂?decreased significantly and of follicle stimulating hormone?FSH?increased markedly;the quantity of follicles decreased obviously.Conclusion CTX may induce disturbance of estrous cycle,the decline of AMH and E2 levels,the increase of FSH levels,and the change of ovarian tissue structure in rats.The characteristics of the onset of premature ovarian insufficiency were simulated and the premature ovarian insufficiency models were successfully constructed.Part 2 The effects and mechanism of Low-intensity Pulsed Ultrasound on premature ovarian insufficiency modelsObjective To irradiate the POI rat models^'ovary by LIPUS,and investigate the effect and mechanism of it.Methods Thirty female SD rats were randomly divided into group A?low-intensitypulsedultrasoundearlyintervention group—CTX+LIPUS-A group?,group B?low-intensity pulsed ultrasound late intervention group—CTX+LIPUS-B group?and group C?CTX group?.After the modeling was completed,the three groups of rats were subjected to low-intensity pulsed ultrasound irradiation for 23 days according to different protocols,and the rats were sacrificed the day after the irradiation.The change of estrus cycle,the level of sex hormones,the histomorphological changes of ovarian tissue,the apoptosis of granulosa cells in the follicles,the inflammation-related factors and apoptosis-related proteins and other indicators were observed.Results Compared with group C,group A recovered the disordered estrus cycle of some rats;serum AMH,E2 levels increased,FSH levels decreased;the number of follicles at all levels increased;The level of inflammatory factors IL-1?,IL-6,TNF-?decreased,the level of VEGF-?increased in ovarian tissue homogenates.The apoptosis of granulosa cells decreased,the expression of Bax in the ovary of rats decreased,and the expression of Bcl-2 increased.The changes of each index in group B compared with group C showed no significant difference.Conclusion Early intervention with LIPUS can reduce the pathological damage of the ovary of rats with premature ovarian insufficiency and improve the reproductive and endocrine function of the ovary.The mechanism may be related to anti-inflammatory damage,improving the local microenvironment of the ovary;regulating Bax/Bcl-2expression and reducing granulosa cell apoptosis.Late intervention with LIPUS did not significantly improve ovarian function.