| Background:Diminished Ovarian Reserve refers to a kind of disease with clinical manifestations such as decreased number of follicles in the ovary and/or decreased quality of oocytes,sexual hormone disorder and fertility decline.Age is the main risk factor for DOR and follicles continue to decrease at the rate of 1000 per month during the reproductive age.In recent years,the incidence rate of DOR has been rising,which has seriously affected women’s reproductive health and quality of life,and caused serious mental and economic pressure to many families.At present,drug induced ovulation therapy and assisted reproductive technology are mainly used to treat DOR.The effective rate is not high and the cost is expensive.Excessive stimulation of the ovary may further damage the ovarian function.Acupuncture and moxibustion therapy can produce certain therapeutic effect on the disease and has the advantages of high safety,no side effects,and low treatment cost.Specifically,acupuncture and moxibustion treatment can improve the serum FSH and E2 levels of patients with diminished ovarian reserve,increase the Antral Follicle Count,alleviate the symptoms of hot flashes,sweating,insomnia and dreaminess,and alleviate the patients’ anxiety,irritability and other negative emotions.Many studies have shown that acupuncture and moxibustion can also produce positive therapeutic effects in the process of assisted reproductive therapy.PI3K/AKT/FOXO3a intracellular signaling pathway is closely related to ovarian reserve,and is involved in regulating the proliferation and differentiation of granulosa cells,the growth of oocytes,and the dormancy,activation and atresia of primordial follicles.The previous study of our research group found that the fertility of 8-9 month old female SD rats has decreased significantly.Objective:(1)Explore the effect of electroacupuncture intervention on ovarian reserve function in DOR rats.(2)Explore whether the expression of FSHR,AMHR and their downstream PI3K/AKT/FOXO3a cell signaling pathways in ovarian tissue is involved in the mechanism of EA intervention to improve ovarian reserve function in DOR rats.(3)Exploring the effects of electroacupuncture intervention on pregnancy and offspring growth and development in DOR ratsMethods:(1)The vaginal exfoliated cell smears of rats were prepared by "vaginal washing method" every day,and the estrous cycle stage of rats was determined according to the morphological characteristics of cells and the number of different types of cells.The 8-month-old female SD rats with obvious disorder of estrous cycle were selected as the DOR model rats of this study,and the 3-month-old female SD rats with regular estrous cycle were selected as the normal control rats of this study.In order to ensure that the ovarian reserve function of DOR rats in each group is basically the same before the intervention,blood from the posterior orbital vein of the rats was collected at the interval of estrus.According to their serum FSH level,DOR rats were divided into model group and EA group by block random method.(2)The rats were bound with rat clothing,and the acupoints of Shenshu and Zhongliao were punctured on both sides.The current parameters in EA group were 2Hz and 3-5mA.The intervention frequency was once a day,30 minutes each time,for 14 consecutive days.The model group and control group were bound with the same rat clothing and pasted with adhesive tape.(3)Ovarian tissue sections were prepared by paraffin section-HE staining technique.The histological characteristics of ovaries of rats in each group were observed and the antral follicle was counted;Enzyme-linked immunosorbent assay was used to detect the levels of reproductive endocrine hormones in peripheral serum of rats in each group,including FSH,E2,LH and AMH.(4)The protein expression level of FSHR and AMHR,the phosphorylation level of PI3K/AKT/FOXO3a signal pathway related protein and the mRNA expression level were detected by western blot,real-time quantitative PCR and other experimental techniques.(5)The experiment design of 3:1 competitive cage mating was used to detect the pregnancy rate,live birth rate and the number of births of rats in each group;The physical development indicators of the early growth of the offsprings were measured,including the weight gain within 21 days after birth,the time of Pinna detachment,Incisor eruption and Eye opening;The learning and memory ability of the offsprings in adolescence was tested through the Novel object recognition test.Results:(1)The estrous cycle of 8-month-old female SD rats was almost disordered(p<0.001),the serum FSH level was significantly increased(p<0.05),E2 was significantly decreased(p<0.05),and the Antral Follicle Count was significantly decreased(p<0.001).(2)EA intervention increased the Antral Follicle Count in DOR rats to a certain extent(p<0.05).(3)EA intervention decreased the level of FSH in the peripheral serum of DOR rats during the diestrum period(p<0.05)and increased the level of E2(p<0.05).(4)There is a trend of recovery in the estrous cycle of DOR rats after EA intervention,.(5)EA intervention improved the ovarian quality index(p<0.05)and ovarian tissue morphology of DOR rats to some extent.(6)EA intervention increased the protein expression level of FSHR(p<0.01)and AMHR(p<0.05)in ovarian tissue of DOR rats.(7)EA intervention increased the protein phosphorylation level of PI3K,AKT and FOXO3a signaling molecules in the middle and downstream of PI3K/AKT signaling pathway in ovarian tissue of DOR rats(p<0.01),and was higher than that of normal control rats(p<0.05);EA intervention increased the mRNA level of AKT signaling molecule in PI3K/AKT signaling pathway in ovarian tissue of DOR rats(p<0.05).(8)EA intervention improved the delayed weight growth(p<0.05),the delayed Incisor eruption(p<0.001)and the delayed Eye opening development(p<0.01),and the impaired learning and memory ability of the offsprings of DOR rats to some extent.Conclusion:(1)EA intervention significantly improved ovarian reserve function in DOR rats,mainly manifested by an increase in the Antral Follicle Count,an improvement in peripheral serum reproductive endocrine hormone levels,and an improvement in ovarian quality index.(2)The increased expression of FSHR,AMHR and their downstream PI3K/AKT/FOXO3a cell signaling pathways in ovarian tissue may be one of the mechanisms by which EA intervention enhances ovarian reserve function in DOR rats.(3)EA intervention improved the growth and development delay of offspring of DOR rats to some extent. |
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