Analysis Of The Correlation Of AMH And INS And Their Roles In Follicular Growth Of PCOS Rats

1. To compare progestrone plus hCG method with dehydroepiandrosterone (DHEA) method to induce polycystic ovary syndrome(PCOS) in young female Sprague-Dawley rats in order to provide an appropriate animal model for the follow-up study.2. To analyze the factors that may affect the abnormal follicular growth of PCOS and their possible mechanisms.3. To study the correlation between Anti-Mullerian hormone level and insulin resistance degree in serum.local ovary and single follicle induced by the preferred PCOS model method.4. To analyze the effect of insulin receptors on the interaction mechanism between the two we discussed and to understand the mechanism of the interaction.Methods:1. Construction of PCOS rat model:44 SD rats in Model groupâ… were induced by progestrone plus hCG method while other 44 SD rats in Model groupâ…¡by DHEA method. Using the following methods to compare and evaluate the two methods in order to provide an appropriate animal model for the follow-up study:vaginal epithelial cells in smears,detection of the serum gonadal hormone level and the ovarian pathohistological sturctuer of PCOS rats was observed by HE staining.2. Detection of serum related indicators:The level of serum luteinizing hormone(LH),follicle stimulating hormone(FSH), Estradiol(E2),Progesterone (P)and testoterone(T) was detected by radioimmuno-assay(RIA).The level of Anti-Mullerian hormone and insulin was measured by enzyme linked immunosorbent assay(ELISA).3. Histologic observation of ovarian tissue:The ovarian tissue of every group rats underwent paraffin imbedding, microtome section, HE staining and then observed their pathohistology by ordinary optical microscope.4. The correlation between the number of follicles and its possible impact factors:Slice all the rats'ovarian tissue, select five sections of each ovary,and randomly select five horizons of each section.Then count the number of follicles at all levels in every horizon. Analyse the correlations between the number of follicles and the level of serum E2,T,LH,FSH,the ratio of LH and FSH,AMH,FINS,FPG and HOMA-IR, and make regression analysis after screening variables.5. The correlation between the level of serum Anti-Mullerian hormone (AMH) and insulin resistance:HOMA-IR index of each rat was calculated,then the rats were divided into PCOS with IR group(n=10),PCOS without IR group(n=10) and the control group(n=10)according to HOMA-IR index. Compare the three groups and analyse the correlations among them.6. The correlation between the level of Anti-Mullerian hormone (AMH) and insulin resistanc in local ovary:A comparison and semi-quantitatively analysis was used to measure the compressions of ovarian local AMH and insulin receptor in different groups with immunohistochemical method. The positive unit(PU) was calculated to compare and analyse the correlations among them.Results:1. The rates of rats lost of menstrual cycle regularity were 86.4% and 77.3% in Model group I andâ…¡respectively; The body weight in DHEA induced PCOS group were lower than that in the control group (P<0.05);Serum E2 level in DHEA induced PCOS group was higher than that in the control group (P<0.05); The serum testosterone, fasting insulin, fasting blood glucose levels and the HOMA index were significantly higher in Model groups than those in control groups (P< 0.05). Serum E2 and P levels showed statistically significant difference between the two Model groups (P<0.05),while other indicators showed no difference.The ovaries in both experimental groups showed multiple follicular cysts, and the numberof theca cells and interstitial cells increased while the number of granulose cells decreased.2. In PCOS group, the number of primary follicles, preantral follicles and antral follicles significantly increased compared with the control group (P<0.05),while the number of primordial follicles had no significant difference with control group (P=0.343). The total number of follicles was two to three times compared with the normal control group. The number of follicles had a positive correlation with the level of serum AMH and T, and their Pearson correlation coefficients were 0.538 and 0.637 respectively (P<0.05). The equation was the number of follicles in PCOS group=107.182+33.067T +0.412AMH, but both influences were similar and small.3. The serum AMH level was obviously higher in experimental group (P<0.05) compared with control group.There was a signifieant difference (P<0.05) and a positive correlation (r=0.691,P=0.001) between serum AMH and HOMA-IR level. Multiple regression analysis showed there was a positive linear relationship between serum AMH and HOMA-IR,FINS (F=12.362,P=0.000),while T was removed.The equation was AMH=15.776HOMA-IR +4.751FINS-74.027,but both influences were similar and small.4. Immunohistochemistry of ovarian tissue showed AMH was mainly expressed in granulosa cells of preantral follicles and small antral follicles.Single follicle AMH levels showed no significant difference (x2=0.657,P=0.720),but varian local AMH levels and number of AMH-positive expression follicle were significantly different among the three groups(F=15.241, P=0.000).Local expression of AMH and number of AMH-positive expression follicle were positively correlated.5. Immunohistochemistry of ovarian tissue showed insulin receptors were widely expressed in the theca cells, granulosa cells and stromal cells, especially abundant in vascular endothelial cells. The expression of insulin receptor was not significantly different between model group and control group,among PCOS with IR group, PCOS without IR group and control group(P>0.05).There was no significant correlation beteen local ovarian AMH level and Insulin receptor expression(r=0.000, P>0.05).AMH and insulin receptor in single follicle had no significant difference(P>0.05) and correlation(r=0.011, P>0.05).Conclusions:1. The rat model induced by progestrone plus hCG was proved to be an ideal PCOS animal model with more similar morphological and hormonal changes to those of PCOS patients than those induced by DHEA.2. PCOS had an increase in the number of growing follicles, rather than an increase in primordial follicle recruitment. In PCOS,the excess follicle was mainly caused by the excess androgen. AMH may inhibit primordial follicle recruitment, and also was the main reason for follicle arrest.3. Under this modeling approach, the single follicle's AMH secretion of PCOS rats was still normal. In Model group, the elevated expression of AMH was caused by the increased number of AMH-positive expression follicles. The higher IR, and more small follicles led to higher AMH level.4. The number of insulin receptor had no obviously change compared with the control group, that meant:the reason of insulin resistence caused by PCOS generated in post-receptor level. Ir involved in PCOS ovarian interstitial fibrosis and the regulation of angiogenesis, participating the occurrence and development of PCOS.