The Therapeutic Effects Of Low-intensity Pulsed Ultrasound On Premature Ovarian Failure Of SD Rats

Objective1.To explore the therapeutic effects of low-intensity pulsed ultrasound（LIPUS）on the rat model of premature ovarian failure induced by cyclophosphamide（CTX）.2.To explore the molecular mechanism of ovarian function recovery of POF rats after ultrasound treatment.MethodsThis study is divided into two parts.The first part was the therapeutic effects of LIPUS on POF rats.The second part was the mechanism of LIPUS to improve the ovarian function of POF rats.1.A total of 70 animals were randomly selected according to the random number table from 100 female SD rats to establish POF animal models.The rats were intraperitoneally injected with CTX 30mg/kg/d for 5days.At the end of the last injection,vaginal smear analysis was analysed at 8 a.m.for 14 days.The disturbance of the estrous cycle or no estrous cycle represented the success of the model.A total of 62 CTX-induced premature ovarian failure（POF）female SD rats were randomly divided into an ultrasound group（n=31）and a control group（n=31）according to the random number table.The 3rd group was a blank group which had 30healthy female SD rats.The ultrasound group was treated with LIPUS,30min/d,a total of 10 days.The blank and control groups were treated with the same method as the ultrasound group,but the transducer had no energy output.The estrous cycles and general situation of all rats were monitored in all rats.Seven days after treatment,21 rats（7 rats from each group according to the random number table）were selected to mate with 10 healthy and age-matched male rats respectively.The pregnant rate of female rats,number and weight of newborn rats of per nest were recorded.51 rats（17 rats from each group according to the random number table）were sacrificed,and the blood,uterus and ovary of these rats were collected.HE staining were used to observed the morphological changes of uterus and ovary,and then the number of follicles in different stages was recorded,ELISA method was used to test serum hormone levels.2.The remaining 12 rats were sacrificed.The ovary at right side was obtained.One part of it was placed in 4%paraformaldehyde,and was fixed for 24h at the normal temperature.The other part was put in EP tube,stored at-80℃.IHC and RT-qPCR techniques were used to evaluate the protein and the mRNA expression of growth differentiation factor-9（GDF-9）and bone morphogenetic protein-15（BMP-15）respectively.Results1.Compared to the blank group,the estrous cycles were disordered in the control group,with atretic follicles（P<0.01）and serum FSH（P<0.01）increased,and other levels of follicles（P<0.01）as well as serum levels of E₂ and AMH（P<0.01）decreased.After LIPUS treatment,compared with the control group,estrous cycles were returned to normal in the ultrasound group,with atretic follicles（P<0.01）and serum FSH（P<0.01）decreased,and other levels of follicles（P<0.01）as well as the serum levels of E₂（P<0.01）increased.There was no statistical difference in serum AMH level between the ultrasound and control groups.There were no significant differences of the pregnancy rate,the number and weight of newborns per net among three groups（P>0.05）.2.In the ultrasound group,the expressions of BMP-15 protein,both BMP-15 and GDF-9 mRNA were significantly increased（P<0.01）while compared with those in control group.Conclusions1.LIPUS could improve some ovarian functions of the POF rats.However,it could not improve the ovarian reserve function,and the results of our experiment had not found LIPUS had an effect on pregnancy rate and fertility of POF rats.2.LIPUS could promote the protein expression of BMP-15 and the mRNA expression of BMP-15 and GDF-9 in ovarian tissue.