卵巢衰老体外细胞模型及抗苗勒管激素用于早期评估预警系统的建立
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摘要
本研究的目的是探讨小鼠窦前卵泡的最佳分离方法。取未成熟小鼠的卵巢，按照不同分离方法，单纯机械法或酶解法分离窦前卵泡，并用基础培养液进行培养，比较不同分离方法获得的卵泡数量、形态，及对其早期生长发育的影响。其中2mg/ml的胶原酶可获得大量基底膜完整的窦前卵泡及裸卵（P<0.01）；体外培养2天后，酶解组卵泡生长直径大(P<0.05)、GVBD率、放出第一极体率和7天生存率无明显差异（P>0.05）。由此可见胶原酶酶解法较单纯机械法更易获得大量高质量窦前卵泡，且对其早期发育无明显影响。
本研究的目的是探讨基础培养基、添加因子与血清相比，对乙烯雌酚（diethylstilbestrol, DES）处理的C57BL/6J未成年小鼠颗粒细胞(granulosa cells, GCs)增殖的影响，以建立一个无血清的体外原代培养体系，添加因子包括ITS（为5mg/ml胰岛素,5mg/ml转铁蛋白和5g/ml亚硒酸钠的混合物），牛血清白蛋白（bovineserum albumin，BSA），丙酮酸钠（sodium pyruvate，PNa）和表皮生长因子（epidermalgrowth factor，EGF）；此后，将此无血清培养体系用于评价这些添加因子对颗粒细胞甾体合成过程的影响以及相关的调节机制。DMEM/F12, McCoy’s5a, RPMI Medium1640, Medium199, a-MEM被选为基础培养基,其中McCoy’s5a能有效的促进颗粒细胞的增殖(P<0.05)并且颗粒细胞贴壁及形态均较好。颗粒细胞在有血清的培养基中较无血清的生长状态更佳； ITS，牛血清白蛋白，丙酮酸钠和表皮生长因子在无血清状态下对颗粒细胞增殖的促进能力逐渐增加，其中后三者在无血清的体系下拥有与血清相同的促颗粒细胞增殖能力。经过2天得培养，原代培养的细胞仍然为表达芳香化酶阳性的颗粒细胞，且维持正常的形态，当添加因子加入到无血清的体系中时，这些细胞亦无凋亡的表现。在所有的添加因子中仅有ITS能够诱导雌二醇的产生，同时ITS、牛血清白蛋白和丙酮酸钠能明显的增加孕酮的分泌提示其能促进细胞的分化。这些添加因子的作用通过部分增加类固醇生成性急性期调节蛋白（steroidogenic acuteregulatory protein，StAR）、3β羟类固醇脱氢酶（3beta-hydroxysteroid dehydrogenase，3β-HSD）、胆固醇侧链裂解酶（Cholesterol Side-Chain Cleavage Enzyme，P450scc）或芳香化酶（aromatase，P450arom）的转录水平达到。因此，McCoy’s5a培养基是一个较好的基础培养基，ITS和丙酮酸钠在无血清体系中同时促进原代颗粒细胞的增殖和分化，为颗粒细胞的生理学研究提供优良的培养体系。
目的：阐明生殖衰老分期系统（the Stages of Reproductive Aging Workshop stages，STRAW）中不同分期阶段及不同年龄阶段各种卵巢衰老相关指标的变化，确定最佳反映卵巢衰老的指标用于制定女性卵巢功能的参考值范围，建立卵巢衰老的评价标准；并通过区分生育年龄中期（mid reproductive age stage，MR）完善STRAW分期。
实验设计及方法：根据STRAW分期系统将18至74岁的健康女性进行分类，并检测月经第三天血清基础卵泡刺激素（follicle-stimulating hormone，FSH）、黄体生成素（luteinizing hormone，LH）、雌二醇（estradiol，E2）和抗苗勒管激素（anti-mullerianhormone，AMH）的水平以及卵泡期窦状卵泡数（antral follicle counts, AFCs）；记录另一组女性中行IVF治疗的获卵数及受孕结果。
结果：在生育年龄中期细分的情况下，随着整个STRAW分期系统的推进，血清FSH, LH水平及FSH/LH比值逐渐上升，血清E2, AMH水平和AFCs逐渐下降(P<0.001)。血清AMH(r=-0.679/-0.742)和AFCs(r=-0.620/-0.673)与STRAW分期紧密相关，且在25岁较早的时候就出现了明显的下降(P<0.01)，并和生育年龄中期阶段的年龄紧密相关(r=-0.451,-0.445,)。因此AMH和AFCs作为最佳卵巢衰老指标制定参考值范围；此外，0.982ng/ml AMH和3个窦状卵泡数(生育年龄中期25-30岁的较低水平)可作为临界值区分生育年龄中期为生育年龄早中期（early mid reproductiveage，EMR）和生育年龄晚中期（late mid reproductive age，LMR）。EMR较LMR女性可在IVF中获取更多的卵母细胞（P<0.05）；虽无统计学差异，由AMH水平区分的EMR相对于LMR受孕率稍高（57.9%）。
结论：血清AMH水平和AFCs早期就会出现显著的下降，可作为卵巢衰老较佳的指标评价卵巢功能；并进一步区分和定义生育年龄中期，显示了STRAW分期系统中年轻女性不同的卵巢衰老阶段，为IVF治疗及生育计划提供参考。
目的：绝经年龄个体差异性较大。本研究的目的是利用数学模型的方法比较不同年龄相关的卵巢内分泌和影像学指标在正常可孕女性中预测终末绝经时间的能力（final menopause period，FMP）并最终构建卵巢衰老预警系统。
方法：我们检测了358名正常健康可孕中国汉族女性的血清基础抗苗勒管激素（anti-müllerian hormone, AMH）,卵泡刺激素（follicular stimulating hormone，FSH）水平和窦状卵泡数量（Antral follicle counts, AFCs），利用这些指标与年龄构建模型并找出最佳拟合效果的方程式。继而通过最大似然估计的阈值方法计算出相应样本可能的绝经年龄，这些指标预测FMP的能力由它们预测的绝经年龄分布与实际的绝经年龄分布的匹配度确定。最终，根据每位女性的实际指标水平和年龄位于预测绝经年龄分布的百分比区段位置预测绝经年龄，达到预警目的。
结果： logAMH的二次回归拟合, FSH的三次回归拟合和AFCs的线性回归分别最佳的反映了相应指标与年龄的关系(R2=0.634,0.595和0.320)。血清AMH的下降比FSH的升高平均早4.5年，因此内分泌学指标中，AMH被选为较佳的指标与影像学指标-AFCs相比预测绝经年龄。由AMH指标模拟计算获得的预测绝经年龄分布与实际绝经年龄分布在41岁到53岁间有较好的一致性，比AFCs的拟合效果更好。最终，二次方程拟合的AMH联合实际年龄可以通过相应的百分比区段位置预测绝经年龄范围。
结论：血清AMH水平是一个较FSH和AFCs更佳的指标来预测女性个体的绝经年龄，与实际年龄合并可预测终末绝经时间，早期预示卵巢功能的下降。
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.
本研究的目的是探讨基础培养基、添加因子与血清相比，对乙烯雌酚（diethylstilbestrol, DES）处理的C57BL/6J未成年小鼠颗粒细胞(granulosa cells, GCs)增殖的影响，以建立一个无血清的体外原代培养体系，添加因子包括ITS（为5mg/ml胰岛素,5mg/ml转铁蛋白和5g/ml亚硒酸钠的混合物），牛血清白蛋白（bovineserum albumin，BSA），丙酮酸钠（sodium pyruvate，PNa）和表皮生长因子（epidermalgrowth factor，EGF）；此后，将此无血清培养体系用于评价这些添加因子对颗粒细胞甾体合成过程的影响以及相关的调节机制。DMEM/F12, McCoy’s5a, RPMI Medium1640, Medium199, a-MEM被选为基础培养基,其中McCoy’s5a能有效的促进颗粒细胞的增殖(P<0.05)并且颗粒细胞贴壁及形态均较好。颗粒细胞在有血清的培养基中较无血清的生长状态更佳； ITS，牛血清白蛋白，丙酮酸钠和表皮生长因子在无血清状态下对颗粒细胞增殖的促进能力逐渐增加，其中后三者在无血清的体系下拥有与血清相同的促颗粒细胞增殖能力。经过2天得培养，原代培养的细胞仍然为表达芳香化酶阳性的颗粒细胞，且维持正常的形态，当添加因子加入到无血清的体系中时，这些细胞亦无凋亡的表现。在所有的添加因子中仅有ITS能够诱导雌二醇的产生，同时ITS、牛血清白蛋白和丙酮酸钠能明显的增加孕酮的分泌提示其能促进细胞的分化。这些添加因子的作用通过部分增加类固醇生成性急性期调节蛋白（steroidogenic acuteregulatory protein，StAR）、3β羟类固醇脱氢酶（3beta-hydroxysteroid dehydrogenase，3β-HSD）、胆固醇侧链裂解酶（Cholesterol Side-Chain Cleavage Enzyme，P450scc）或芳香化酶（aromatase，P450arom）的转录水平达到。因此，McCoy’s5a培养基是一个较好的基础培养基，ITS和丙酮酸钠在无血清体系中同时促进原代颗粒细胞的增殖和分化，为颗粒细胞的生理学研究提供优良的培养体系。
目的：阐明生殖衰老分期系统（the Stages of Reproductive Aging Workshop stages，STRAW）中不同分期阶段及不同年龄阶段各种卵巢衰老相关指标的变化，确定最佳反映卵巢衰老的指标用于制定女性卵巢功能的参考值范围，建立卵巢衰老的评价标准；并通过区分生育年龄中期（mid reproductive age stage，MR）完善STRAW分期。
实验设计及方法：根据STRAW分期系统将18至74岁的健康女性进行分类，并检测月经第三天血清基础卵泡刺激素（follicle-stimulating hormone，FSH）、黄体生成素（luteinizing hormone，LH）、雌二醇（estradiol，E2）和抗苗勒管激素（anti-mullerianhormone，AMH）的水平以及卵泡期窦状卵泡数（antral follicle counts, AFCs）；记录另一组女性中行IVF治疗的获卵数及受孕结果。
结果：在生育年龄中期细分的情况下，随着整个STRAW分期系统的推进，血清FSH, LH水平及FSH/LH比值逐渐上升，血清E2, AMH水平和AFCs逐渐下降(P<0.001)。血清AMH(r=-0.679/-0.742)和AFCs(r=-0.620/-0.673)与STRAW分期紧密相关，且在25岁较早的时候就出现了明显的下降(P<0.01)，并和生育年龄中期阶段的年龄紧密相关(r=-0.451,-0.445,)。因此AMH和AFCs作为最佳卵巢衰老指标制定参考值范围；此外，0.982ng/ml AMH和3个窦状卵泡数(生育年龄中期25-30岁的较低水平)可作为临界值区分生育年龄中期为生育年龄早中期（early mid reproductiveage，EMR）和生育年龄晚中期（late mid reproductive age，LMR）。EMR较LMR女性可在IVF中获取更多的卵母细胞（P<0.05）；虽无统计学差异，由AMH水平区分的EMR相对于LMR受孕率稍高（57.9%）。
结论：血清AMH水平和AFCs早期就会出现显著的下降，可作为卵巢衰老较佳的指标评价卵巢功能；并进一步区分和定义生育年龄中期，显示了STRAW分期系统中年轻女性不同的卵巢衰老阶段，为IVF治疗及生育计划提供参考。
目的：绝经年龄个体差异性较大。本研究的目的是利用数学模型的方法比较不同年龄相关的卵巢内分泌和影像学指标在正常可孕女性中预测终末绝经时间的能力（final menopause period，FMP）并最终构建卵巢衰老预警系统。
方法：我们检测了358名正常健康可孕中国汉族女性的血清基础抗苗勒管激素（anti-müllerian hormone, AMH）,卵泡刺激素（follicular stimulating hormone，FSH）水平和窦状卵泡数量（Antral follicle counts, AFCs），利用这些指标与年龄构建模型并找出最佳拟合效果的方程式。继而通过最大似然估计的阈值方法计算出相应样本可能的绝经年龄，这些指标预测FMP的能力由它们预测的绝经年龄分布与实际的绝经年龄分布的匹配度确定。最终，根据每位女性的实际指标水平和年龄位于预测绝经年龄分布的百分比区段位置预测绝经年龄，达到预警目的。
结果： logAMH的二次回归拟合, FSH的三次回归拟合和AFCs的线性回归分别最佳的反映了相应指标与年龄的关系(R2=0.634,0.595和0.320)。血清AMH的下降比FSH的升高平均早4.5年，因此内分泌学指标中，AMH被选为较佳的指标与影像学指标-AFCs相比预测绝经年龄。由AMH指标模拟计算获得的预测绝经年龄分布与实际绝经年龄分布在41岁到53岁间有较好的一致性，比AFCs的拟合效果更好。最终，二次方程拟合的AMH联合实际年龄可以通过相应的百分比区段位置预测绝经年龄范围。
结论：血清AMH水平是一个较FSH和AFCs更佳的指标来预测女性个体的绝经年龄，与实际年龄合并可预测终末绝经时间，早期预示卵巢功能的下降。
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.
引文
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