Mechanism Of Qigong Pill Regulating Granulosa Cell Autophagy In Patients With Phlegm-Dampness Syndrome Of Polycystic Ovary Syndrome Based On AMH/AMHRⅡ

Objective:To study the effect of Qigong Pill on the expression of anti-Mullerian hormone and AMH receptorⅡsignal pathway and autophagy-related genes in ovarian granulosa cells of polycystic ovary syndrome patients with phlegm-dampness syndrome,as well as the regulatory effect of AMH/AMHRⅡsignal pathway on autophagy,to explore the pathological mechanism of Qigong Pill in the treatment of PCOS phlegm-dampness syndrome.Methods:20 patients who met the criteria of PCOS phlegm-dampness syndrome were randomly divided into treatment group（Qigong Pill+IVF）and observation group（IVF）,with 10 cases in each group.In addition,10 patients with regular cycles and normal endocrine in the same period who underwent IVF simply because of unilateral or bilateral fallopian tube obstruction,adhesion,unilateral or bilateral salpingectomy,tubal ligation were selected as the control group;the treatment group was treated with Qigong Pill two months before controlled ovarian stimulation until the trigger day,while the observation group and control group received routine IVF,all three groups of patients with controlled ovarian hyperstimulation were treated with antagonist regimen.The general data of the three groups were recorded,and the contents of follicle stimulating hormone（FSH）,luteinizing hormone（LH）,testosterone（T）and AMH in serum on the third day of natural menstruation were recorded,and the scores of phlegm-dampness syndrome and body weight index（BMI）of the observation group and the treatment group before and after treatment were compared.The days of gonadotropin（Gn）use,the dosage of Gn,the level of LH,estradiol（E₂）and progesterone（P）,the number of eggs obtained,2PN rate and high quality embryo rate were compared among the three groups.The protein expression of AMH,AMHRⅡ,Beclinl,LC3-Ⅱ/LC3-Ⅰand SQSTM1/p62 in granulosa cells was analyzed by Western Blot.Results:1.TCM phlegm-dampness syndrome score:compared with the observation group,the score of phlegm-dampness syndrome after treatment in the treatment group was significantly lower than that before treatment（P<0.05）,but there was no significant difference in the observation group before and after treatment（P>0.05）.2.BMI:although there was no statistical difference before and after treatment in the treatment group（P>0.05）,it had a significant downward trend compared with that before treatment,while there was no significant change in the observation group before and after treatment.3.Gn days and Gn dosage:There was no significant difference among the three groups（P>0.05）.4.The levels of LH,E2and P on the trigger day:the E2 level on the trigger day in the experimental group was higher than that in the observation group and the control group（P<0.05）,but there was no significant difference in LH and P levels among the three groups on the trigger day（P>0.05）.5.The number of eggs obtained:The number of eggs in the treatment group was higher than that in the control group（P<0.05）,but there was no significant difference between the observation group and the control group（P>0.05）,but the number of eggs in the observation group was higher than that in the control group（P<0.05）.6.2PN rate,high quality embryo rate:There was no significant difference in 2PN rate among the three groups（P>0.05）,but the excellent embryo rate in the control group and the treatment group was significantly higher than that in the observation group（P<0.05）.7.Comparison of AMH、AMHRⅡ、Beclinl、LC3-Ⅱ/LC3-Ⅰand SQSTM1/p62 protein expression in GCs among the three groups:Compared with the control group,the expression of AMH、AMHRⅡ、Beclin1、LC3-Ⅱ/LC3-I in observation group was up-regulated（P<0.001）,and that of SQSTM1/p62 was down-regulated（P<0.001）;Compared with the observation group,the expression of treatment group AMH and AMHRⅡwas down-regulated（P<0.001）,Although the expression of Beclin1showed no obvious change（P>0.05）,it showed a downward trend.Down-regulation of LC3-Ⅱ/LC3-I expression（P<0.001）and Up-regulation of p62 expression（P<0.001）.Conclusions:1.In patients with PCOS phlegm-dampness syndrome,the up-regulation of AMH/AMHRⅡpathway in ovarian granulosa cells may activate autophagy and hinder follicular development and maturation;2.Qigong Pill can down-regulate AMH/AMHRⅡpathway,inhibit autophagy,improve the clinical symptoms of infertile patients with PCOS phlegm-dampness syndrome,and improve the quality of eggs and embryos.