Establish The In-vitro Cell Model And Early Estimated Warning System By Anti-mullerian Hormone Of Ovary Aging

The object of the study is to explore the optional isolation methods of preantral follicle.The ovary was obtained from premature mouse and dissected according to differentisolation methods, mechanical dissection or enzymatic digestion. Then they were culturedin the basal medium and the quantity, morphology and effects on early development wereassessed.2mg/ml collagenase was the optional isolation condition to obtain large amountof preantral and denuded follicles (P<0.01), and the follicles in enzymatic group grewfaster than mechanical group (P<0.05), but the GVBD rate, Polar body releasing rate andsurviving rate had no obvious difference(P>0.05). Collagenase isolation could obtain largeamount of high quality preantral follicles than mechanical methods, which also hadminimal effects on early development. The objectives of this study were to investigate the effects of basal media,supplements including mixture of5mg/ml insulin,5mg/ml transferrin and5g/mlselenium (ITS for short), bovine serum albumin (BSA), sodium pyruvate (PNa) andepidermal growth factor (EGF) compared with serum on diethylstilbestrol-treatedC57BL/6J mouse primary granulosa cells (GCs) proliferation to develop a serum-freeculture system and to use this system to evaluate the effects of these supplements on GCs’processes of steroidogenesis and related regulation mechanism. DMEM/F12, McCoy’s5a,RPMI Medium1640, Medium199, a-MEM were chosen as basal media, and McCoy’s5aproliferated the GCs greatly (P<0.05) with a better attachment and morphology. GCsshowed a better proliferation status in serum-containing media than serum-free media, BSA,ITS, PNa and EGF increased the proliferation of GCs in serum-free system gradually whilethe latter three had the same effect on supporting GCs proliferation with serum. After2-days culture, the cells cultured remained P450arom positive GCs that sustained a normalmorphology and there is no sign of apoptosis when supplements were added in theserum-free condition. Only ITS induced estradiol production while ITS, BSA, PNaenhanced progesterone secretion significantly indicating the promotion of differentiation.And these supplements functioned partly through increasing the transcription level of StAR,3β-HSD, P450scc or P450arom. In conclusion, McCoy’s5a medium was a better basalmedium, ITS, PNa promoted both the proliferation and differentiation of primary GCs inserum-free culture system, showing a satisfactory state for GCs’ physiological study. OBJECTIVE: To demonstrate the changes of ovarian aging markers across theStages of Reproductive Aging Workshop (STRAW) stages age, optimal markers weredecided to establish the reference value of ovary function and ovary aging criteria.Meanwhile，modify the STRAW stages with subclassification of mid reproductive agestage (MR).DESIGN: Healthy females were classified according to the STRAW system. Serumbasal FSH, LH, E2, and anti-Müllerian hormone (AMH) were detected, FSH/LH ratiocalculated, and antral follicle counts (AFCs) determined in follicular phase. Number ofretrieved oocytes and pregnancy outcome of women performing IVF were recorded inanother group of women in MR stages.RESULTS: Progression through the whole STRAW stages under MR stagesubdivided condition is associated with elevations in serum basal FSH, LH, and FSH/LHratio and decreases in E2, AMH and AFCs (P <0.001). Both serum AMH (r=-0.679/-0.742) and AFCs (r=-0.620/-0.673) were highly correlated with STRAW stages anddecreased early (after25years) and significantly (P<0.01; r=-0.451,-0.445, respectively)with chronological age in MR stage. Therefore, AMH and AFCs were selected as optimalmakers to give the reference value. Furthermore,0.982ng/ml AMH and3antral follicles(low level of MR25-30years) were set as cutoffs to distinguish MR stage into early midreproductive age (EMR) and late mid reproductive age (LMR) stages. The women in EMRstage compared with LMR could retrieve more oocytes in IVF treatment whether MR stagewas classidicated by AMH or AFCs. Though there is no significant difference of pregnancyrate between EMR and LMR stages, EMR classified by AMH has a higher pregnancychance (57.9%) CONCLUSION(S): The early and marked fall in serum AMH levels and AFCssuggest fine markers to evaluate the ovary function and further categorize and define theMR stage, demostrating disparate reproductive aging peroid in young age across theSTRAW stages and may help for IVF treatment and fertility plan. Objective: Age at menopause show considerable individual variation. Theobjective of the study was to compare the age related ovarian endocrine andultrasound markers in their ability to predict the final menopause period (FMP) innormal fertile women with mathematic model, and establish the early warning systemof ovary aging.Methods: We measured serum basal anti-müllerian hormone (AMH), follicularstimulating hormone (FSH) level and number of antral follicles in358normal healthyfertile volunteers from1,426Chinese Han women and modeled them withchronological age to find the equation with optimal goodness of fit. Their ability ofpredicting the FMP was decided by the degree of similarity between the predictivedistribution from their thresholds done by maximum likelihood and the actualmenopause condition. Finally, predictions of FMP follow from the level of anindividual woman’s marker and age relative to the corresponding percentiles of thepredictive distribution of FMP.Results: Quadratic regression of logAMH, cubic regression of FSH and linearregression of antral follicle counts (AFCs) proximally reflected their relationship withage respectively (R2=0.634,0.595and0.320). AMH declined mean4.5years earlierthan FSH elevated which was selected as endocrine marker and compared with AFCsto predict FMP. However, predictive distribution from declining AMH levels was ingood concordance with the actual menopause data distribution from41year to53year,which is better than AFCs. Finally, AMH in quadratic equation combined with agepredict the FMP in the corresponding percentiles.Conclusion: Serum AMH seems to be a favorable marker of ovarian function betterthan FSH and AFCs that predict the age at FMP for individual woman, warning anearly decline of ovary function.