Study Of Serum Levels Of Inhinbin B In Women With Anovulatory Dysfunctional Uterine Bleeding

【Purpose】Diverse clinical manifestations and Diagnosis of complicated processes for anovulatory dysfunctional uterine bleeding disturb physical and mental health of perimenopausal women. It is a common but high incidence disease and the difficult problems of the gynecological clinic. The existence of the hidden dangers of iatrogenic menopause, lead to unnecessary medical disputes.It is particularly important to evalue ovarian reserve in patients with anovulatory abnormal uterine bleeding. Ovarian reserve is the memory left ovarian follicle quantity and quality. Reduced ovarian reserve have declined ovarian eggs and the quality of follicle cells, the number dropped to reduce sex hormone secretion and gonadotropin response to the decline. There are a series of evaluation indicators of ovarian reserve:basal FSH,basal E2,FSH/LH,Clomiphene stimulation test,Gonadotropin-releasing hormone stimulation test, exogenous FSH stimulation test and the basis of ovarian volume, ovarian volume of small follicles. Choice of clinically recognized indicators of ovarian reserve in this present study - the basal FSH, the basal E2 detect the ovarian reserve of anovulatory dysfunctional uterine bleeding . However, the basis of FSH in the menopausal transition period increased from time to time, but is not stable, and only continued to rise after menopause. Therefore, early detection of ovarian reserve can not function.Inbinhin B, secreted by the granulosa cells of preantral and small antral follicles, controls FSH secretion via a negative feedback mechanism and in the ovary through the side of local / autocrine regulation of E2 to increase production.Decreasing of ovarian function lead to progressive decrease in inhibin B.If only the level of inbihin B drop too much ,result in an early increase in follicle-stimulating hormone (FSH), which initially maintains folliculogenesis and estradiol secretion. Over time, follicle continue disappe- aring ,then estradiol and gonadotropin levels dramatic swings . Therefore, the serum inhibin B levels directly reflect decreased ovarian reserve.The aim of the present study is to evaluate whether the serum concentration of total inhibin B has the sensitivity/specificity characteristics ,based on analyses of FSH and E2, to become a diagnostic test for anovulatory dysfunctional uterine bleeding.【METHODS】Of 40 women, who exclude organic and internal and external genital diseases by systemic and gynecological examination, pelvic ultrasound, and other systemic examination and prove to be anovular menstruation cycle by examination of female sex hormones, referred for anovulatory dysfunctional uterine bleeding to the Second Clinical Hospital of Jilin University gynecology clinics in June 2008- August 2008. Patients are allocated into three groups on the basis of age. Group 1 (aged≤20 years) is adolescence,group 2 (aged 20-40 years) is reproductive ageing and group 3 (aged≥40) is the menopausal transition. 30 healthy female volunteers with regular menstrual cycles are recruited , at the same age allocated into adolescence,reproduction and menopausal transition.Blood specimens are collected from all people,study group in visiting day and control group in menstrual cycle day 3-5 . Use a serum separator tube (SST) and allow samples to clot for 2 hours before centrifugation for 20 minutes at approximately 1000×g. Remove serum and store samples at -80℃.Using a specific two-side enzyme-linked immunosorbent assay (ELISA), inhibin B levels are measured. Allow all reagents to reach room temperature. All the reagents should be mixed thoroughly by gently swirling before pipetting.Predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. Add 100μl of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37℃. Remove the liquid of each well, don't wash. Add 100μl of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37°C. Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400μl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.Add 100μl of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37℃.Repeat the aspiration/wash as in step 4.Add 90μl of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37°C. Protect from light. Add 50μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.Determine the optical density of each well at once, using a microplate reader set to 450 nm.In addition, serum FSH and estradiol are measured by chemiluminescent immunoassay in all serum samples. Add 50μl of Standard, or Sample per well,then add enzyme-conjugation 50 ul. Cover with the Plate sealer. Incubate for 1 hours at 37℃. Remove the liquid of each well, don't wash.Add 300ul eluant per well, waiting 10 seconds. Remove the liquid of each well, don't wash,repeating 5 time.Add detection Reagent A and B 50ul per well.,mixing light vibration, dark place at room temperature for 10 seconds. Determine the optical density of each well at once, using a Automatic analyzer Electrochemiluminescence microplate.【RESULTS】①Serum inhibin B levels, either expressed by study group and control group in adolescent group and reproductive group,do not show any significant variations,(p>0.05).Significant differences is revealed for study group and control group in menopause transition group,(p<0.05);②There are significant differences in control groups of adolescent group,reproductive group and menopause transition group for inhibin B,(p<0.05).The serum inhibin B in control group of menopause transition is the lowest;③Women in study group of menopause transition show serum FSH levels significantly higher than those in control group of menopause transition (P < 0.05).④There are no significant differences in adolescent group,reproductive group and menopause transition group for E2(p>0.05).【CONCLUSIONS】①Basal FSH in study group of menopause transition is higher than those in control group of it,so this note ovarian reserve in menopause transition of anovulatory dysfunctional uterine bleeding decreased.but basal FSH isn't sensitive indicator;②Inhibin B declining in control group of menopause transition and earlier changeing than basal FSH, is earlier and more sensitive indicators reflected ovarian reserve .