Expression Of FSHR,LHR MRNA Of The Ovarian Granulosa Cells From Patients With PCOS

Objective: Polycystic ovarian syndrome(PCOS) is a common reproductive syndrome of women characterized by ovarian hyper-androgenism, hyperinsulinemia , high LH level and LH/FSH. It is also one of the main causes of anovulatory infertility. It is characterized by a myriad of symptoms including oligomenorrhea or amenorrhea, anovulation or infertility, and so on. Drug-induced ovulation was effective in treatment of such infertility. Through controlled-ovarian hyper-stimulation process, ovarian response has obvious individual difference. Compared with normal ovary,patients with PCOS are more likely to OHSS. The mechanism still remains unknown. This study tested the levels of FSHR,LHR mRNA expression on ovarian granulosa cells after ovulatory induction ovarian by RT-PCR .The sexual hormone levels in serum at the day 3,the day 2 after pituitary down-regulation,the day of HCG administered and in follicular fluid were all measured by chemiluminescent method.The aim was to investigate the correlation between the levels of FSHR,LHR mRNA expression on ovarian granulosa cells after ovulatory induction ovarian and ovarian response to exogenous gonadotropins.Methods: 1 The study subject1.1 The PCOS group (The study group): To select 24 patients with polycystic ovarian syndrome who undergone IVF-ET because of infertility in the Second Hospital of HeBei Medical University from May 2007 to October 2007.Their mean ages were 28.83±3.16 years old.1.2 The control group: To random select 20 patients with eumenorrhea who undergone IVF-ET because of infertility, from May 2007 to October 2007.The patients were tubal infertility and their elementary hormone FSH,LH,E₂,PRL,T(MC:3day) were within normal limits. Their mean ages were 30.17±3.21 years old.The patients of PCOS group and control group should be the first time to receive controlled ovarian hyper- stimulation (COH). No woman had systemic diseases, galactorrhea, endometriomas and other endocrine diseases. They all have normal blood sugar and husbands'sperm condition in control group and PCOS group.2 Methods and Contents:2.1 Collecte the sample: All women receive OC long down-regulated protocols and controlled ovarian hypers- timulation (COH), GnRH-a / Gonal-F /HMG/HCG. Serial LH,E₂ levels and two-dimensional TVUS follicle measurements were performed until at least one dominant follicles reached at least 18mm or two dominant follicles reached at least 17mm or three dominant follicles reached at least 16mm in diameter. Then HCG 5000-10000iu was administered, followed by trans- vaginal oocyte retrieval 36h later. Follicular fluid uncontami- nated by blood was selected.They was centrifuged at 2000r for 10min (ordinary temperature), and the supernatant liquid were storaged at -20℃with refrigerator.At oocyte retrieval, all follicular fluid was collected. Granulosa cells were separated from follicular fluid by Ficoll density gradient centrifugation .They were stored in 1.5 ml Eppendorf tubes in Trizol (Invitrogen). Samples were snapping frozen in liquid nitrogen and were stored at -70℃for later RNA extraction.2.2 Mensurate the sample: FSHR and LHR mRNA were determined by RT-PCR .2.3 Hormone assays : Baseline serum FSH,LH,T,E₂,insulin as well as serum FSH,LH at pituitary down- regulation, LH,E₂ at the day of HCG administered , and follicular fluid FSH,LH,T,E₂ were all measured by chemiluminescent method.Results:1 The relative expression level of FSHRmRNA,LHRmRNA on ovarian granulosa cells from PCOS patients and control women1.1 The relative expression level of FSHRmRNA on ovarian granulosa cells from PCOS patients (0.675±0.106) was higher than that from control women (0.567±0.132). There were remarkably statistical significance (P<0.05).1.2 The relative expression level of LHRmRNA on ovarian granulosa cells from PCOS patients (0.647±0.123) was higher than that from control women (0.519±0.156). There were remarkably statistical significance (P<0.05).2 Multivariate linear regression analysis on data showed that there was a positive correlation between the number retrieved oocyte with the expression of FSHRmRNA on ovarian granulosa cells, but not with the expression of LHRmRNA. The regression equations in two groups were as followes: the PCOS group:Y=-22.18+50.51X, R²=0.66, F=20.36, P=0.0001;the control group:Y=-0.11+26.32X, R²=0.773, F=61.36, P=0.00013 To compare outcomes of serum sex hormone levels of the patients of PCOS group and control groupThe mean of baseline serum LH,T,insulin in PCOS group was higher than that in the control group,while FSH and T was similar. At day 2 after pituitary down-regulation, serum FSH,LH levels in two groups were similar. At the day of HCG administered, the mean of serum LH in PCOS group was higher than that in the control group, while E₂ levels in two groups were similar.4 To compare outcomes of follicular fluid sex hormone levels of the patients of PCOS group and control group:The mean of the FSH level in follicular fluid in PCOS group was lower than that in the control group (4.80±1.44 mIU/ml vs. 6.47±0.95 mIU/ml, P =0.003), while LH and T levels higher (P =0.004,P =0.005). There was remarkably statistical significance. The E₂ levels in follicular fluid in two groups were similar. 5 IVF cycle characteristics of control women and PCOS patients undergoing GnRH analog/rhFSHThe amount of rhFSH administered and the duration of rhFSH treatment were similar in control women and PCOS patients, causing comparable maximum serum E₂ levels on the day of hCG administration. Total number of oocytes retrieved was greater (21±8 vs. 15±4), whereas average follicle diameter was smaller, in PCOS patients vs. control women (18.02±1.80mm vs. 19.41±2.32mm). There were remarkably statistical significance (P<0.05).Conclusion: 1 The patients with PCOS existe more than one kinds of endocrine abnormality and abnormal growth of follicles.2 There was a positive correlation between the number retrieved oocyte with the expression of FSHRmRNA on ovarian granulosa cells in two groups,and the relative expression level of FSHRmRNA on ovarian granulosa cells from PCOS patients was higher than that from control women. The increase of the level of the expression FSHR may cause enhancement of ovarian response to exogenous gonado- tropins.This may be one of the cause that patient with PCOS are easy to occour OHSS.At the same time there was no correlation between ovarian response with the expression level of LHR.3 The relative expression level of LHRmRNA on ovarian granulosa cells from PCOS patients was higher than that from control women. To hint that PCOS follicles exposed to GnRH analog/FSH stimulation for IVF may exist abnormal luteinization.