小鼠卵巢不同发育时期SDF-1/CXCR4轴的动态变化

目的分析基质细胞衍生因子1(SDF-1)及其受体(CXCR4)在不同发育时期雌性ICR小鼠卵巢组织中的表达及意义。方法选择4周龄、7周龄和10周龄雌性ICR小鼠共30只,根据典型发育时期分为3组:性成熟前期(4周龄)组、性成熟期(7周龄)组及适龄繁殖期(10周龄)组,每组各10只。每组取5只收集卵巢组织,一侧卵巢提取RNA后利用qPCR检测SDF-1和CXCR4 mRNA表达水平;一侧卵巢固定包埋后切片,采用HE染色观察卵巢组织形态并进行各级卵泡计数,采用免疫组化方法观察SDF-1及CXCR4蛋白在小鼠卵巢组织的表达。余下每组5只促排卵后获取卵丘卵母细胞复合物(COCs),分离卵丘颗粒细胞与卵母细胞并提取RNA,利用qPCR分别检测两种细胞中SDF-1和CXCR4的mRNA表达水平。结果 3组小鼠中性成熟前期组卵巢SDF-1 mRNA表达量显著高于其他两组(P0.05),而CXCR4 mRNA表达量显著低于其他两组(P0.01)。HE染色显示3个发育阶段小鼠卵巢均可见发育正常的各级卵泡;适龄繁殖期组的卵巢总卵泡数目及初级卵泡数量显著高于其余两组(P0.01),次级卵泡数显著多于性成熟期组(P0.01)。免疫组化结果表明:SDF-1及CXCR4蛋白在卵巢各级卵泡的颗粒细胞和卵母细胞中都有表达,而在卵泡膜细胞中几乎不表达。COCs的qPCR结果显示:卵母细胞中,性成熟期组SDF-1 mRNA表达水平显著高于性成熟前期组(P0.05);卵丘颗粒细胞中,适龄繁殖期组SDF-1 mRNA表达水平显著高于其余两组(P0.01),性成熟前期组CXCR4 mRNA表达水平显著低于其余两组(P0.05)。结论 SDF-1及其受体CXCR4在不同发育阶段小鼠卵巢卵泡中均有表达且表达水平存在显著差异,提示其可能参与调控小鼠卵泡发育和性腺成熟的过程。     

大鼠脂肪间充质干细胞改善大鼠自体卵巢移植效果

目的观察大鼠脂肪间充质干细胞(ADMSCs)对大鼠卵巢自体移植的作用。方法8周龄SD大鼠分为4组:正常对照组、自体移植组、自体移植+ADMSCs组及去势组,在自体移植后28 d,行卵巢组织HE染色卵泡计数,用免疫组化观察血管生成情况,用TUNEL荧光染色技术进行细胞凋亡计数,ELISA测定血清FSH、AMH水平。结果自体移植+ADMSCs组总卵泡数、原始卵泡数均显著高于自体移植组(P<0.05)。自体移植+ADMSCs组细胞凋亡计数显著低于自体移植组(P<0.05)。与自体移植组相比,自体移植+ADMSCs组大鼠血清FSH水平更低,AMH水平更高(P<0.05)。自体移植+ADMSCs组CD31阳性细胞分布与正常对照组更接近。结论在大鼠卵巢自体移植过程中应用ADMSCs可改善大鼠卵巢自体移植效果,提高移植效率。     

FSH诱导转染细胞和颗粒细胞FSH受体内在化

目的 :探讨受体与激素螯合后 ,卵泡刺激素受体 (FSHR)的命运。　方法 :用一种能快速区分表面螯合和内在化激素的生化方法 ,在培养的猪颗粒细胞和表达重组猪FSHR的中国仓鼠卵巢细胞确定了卵泡刺激素 (FSH)诱导受体内在化的过程。　结果 :FSH内在化比较缓慢 ,内在化的放射活性在12 5I hFSH加入 10～ 12h后达到峰值。结论 :FSH可诱导FSHR缓慢内在化 ,可能是由于生理条件下FSHR蛋白未端缺乏某些特定氨基酸序列。     

抑制素B和抗苗勒管激素预测卵巢反应性

近年来应用抑制素B(inhibin B,INHB)、抗苗勒管激素(anti-mullerian hormone,AMH)水平等做为预测卵巢储备功能的指标。INHB是转化生长因子β超家族的成员,由卵巢中小窦卵泡颗粒细胞直接产生,主要生理作用是反馈性抑制垂体卵泡刺激素(FSH)分泌,其在年龄上的变化早于FSH水平,因而     

血清抗苗勒氏管激素在体外受精超排卵中预测卵巢反应的价值

目的血清抗苗勒管激素(AMH)由窦前和小窦卵泡分泌,是一个反映卵巢储备的指标。分析AMH在正常育龄人群、多囊卵巢综合征(PCOS)患者、卵巢储备功能减退(DOR)人群中的分布规律,探讨AMH预测卵巢反应的价值。方法回顾分析2008年6月至2012年6月在我中心接受诊治的患者,将其分为三组:(1)正常组:293例。(2)PCOS组:258例。(3)DOR组:59例。比较AMH水平在这三组间的差异,绘制ROC曲线选择判断PCOS及DOR的最佳临界值。进一步选取同时间段,在我中心初次行长方案体外受精/卵胞浆内单精于注射-胚胎移植(IVF/ICSI-ET)的患者,578例。按获卵数分为卵巢低反应组(0³个)、正常反应组(43个)、正常反应组(4¹9个)、高反应组(≥20个),评估年龄、基础窦卵泡计数(AFC)、基础卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E_2)水平,以及AMH、促性腺激素(Gn)用量与卵巢反应的关系。绘制相对工作特征(ROC)曲线,寻找AMH在IVF中判断卵巢反应的界点。结果 AMH水平随年龄增加而下降。AMH水平在PCOS组中较正常组显著升高,在DOR组中明显降低。2519个)、高反应组(≥20个),评估年龄、基础窦卵泡计数(AFC)、基础卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E_2)水平,以及AMH、促性腺激素(Gn)用量与卵巢反应的关系。绘制相对工作特征(ROC)曲线,寻找AMH在IVF中判断卵巢反应的界点。结果 AMH水平随年龄增加而下降。AMH水平在PCOS组中较正常组显著升高,在DOR组中明显降低。25⁴0岁人群中,当AMH界值取1.26ng/ml时,判断DOR的敏感性及特异性分别为88%及90%。当AMH界值取3.97ng/ml时,判断PCOS的敏感性及特异性分别为74%及63%。在IVF中,AFC和AMH水平在三组间均有显著差异(P<0.05)。当AMH界值为1.84ng/ml时,判断该患者本周期为低反应的敏感性及特异性分别为89%及75%,当AMH界值为3.95ng/ml时,判断为高反应的敏感性及特异性为79%及70%。结论 AMH反映卵巢储备状态。在IVF超排卵中,AMH能较好地预测卵巢反应性。     

CTRP6在小鼠卵巢中的定位、表达以及FSH对CTRP6的调控作用

目的探究C1q/肿瘤坏死因子相关蛋白6(CTRP6)在小鼠卵巢中的定位、表达以及FSH对其调控作用。方法免疫组化对56日龄小鼠卵巢中的CTRP6蛋白进行定位,实时定量PCR检测3 d、10 d、21 d和56 d小鼠、21日龄小鼠注射了孕马血清促性腺激素(PMSG)0 h、12 h、36 h、48 h及注射HCG 4 h、8 h、12 h卵巢中CTRP6 mRNA的表达水平。分别向人卵巢颗粒细胞(KGN)中加入0 ng/ml、1 ng/ml、10 ng/ml和100 ng/ml的FSH和LH,培养24 h后检测CTRP6 mRNA水平。结果小鼠卵巢免疫组化结果显示CTRP6可以在颗粒细胞和卵母细胞中表达,尤其在窦卵泡颗粒细胞中表达水平较高。另外,小鼠CTRP6 mRNA的表达水平随着年龄增加而升高(P0.05),且经PMSG处理不同时长后,CTRP6 mRNA相对表达量较PMSG处理前显著增加(P0.05),而与PMSG处理48 h比较,HCG处理后CTRP6 mRNA的相对表达水平显著降低(P0.05),且随着处理时间的延长呈降低趋势。不同浓度梯度(1、10、100 ng/ml)的FSH处理KGN细胞24 h后均使CTRP6 mRNA表达水平显著增高(P0.05),浓度为10 ng/ml时CTRP6 mRNA水平达峰值。选择10 ng/ml FSH于不同时间梯度(6、12和24 h)处理KGN细胞后,CTRP6 mRNA相对表达量均较未处理前显著增加(P0.05),且随着时间延长呈升高趋势。不同浓度梯度(1、10、100 ng/ml)的LH处理KGN细胞24 h后,CTRP6 mRNA的表达均较LH处理前无显著差异(P0.05)。结论 CTRP6可能参与了卵泡生长发育的调控,是卵泡形成的重要因子。     

毓麟珠通过改善小鼠卵巢微环境防治早发性卵巢功能不全的作用机制研究

目的研究毓麟珠通过改善卵巢微环境防治早发性卵巢功能不全(POI)的作用机制。方法选取BALB/c雌性小鼠随机分为空白组、模型对照组、中药组和阳性药组,每组10只。除空白组外其余三组均皮下注射小鼠透明带3(ZP3)多肽建立免疫性POI模型,模型建立后分别给予生理盐水、戊酸雌二醇、毓麟珠溶液灌胃,每天一次,连续6周。高频超声系统动态监测卵巢面积、卵泡数、卵巢血流;取血检测血清激素水平。用BCA试剂盒检测卵巢组织超氧化物歧化酶(SOD)、活性氧(ROS)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)的水平。结果(1)模型对照组小鼠卵巢面积、卵泡数显著低于空白组(P<0.05);与模型对照组比较,阳性药组、中药组卵巢面积均显著增加(P<0.05),中药组卵泡数显著增加(P<0.05),且显著高于阳性药物组(P<0.05)。(2)模型对照组小鼠较空白组小鼠卵巢动脉脉压差增大,搏动指数、阻力指数增高(P<0.05);与模型对照组比较,阳性药组、中药组脉压差、搏动指数、阻力指数均显著降低(P<0.05)。(3)与空白组比较,模型对照组血清FSH、LH显著升高,而AMH显著降低(P<0.05);与模型对照组比较,阳性药组、中药组FSH、LH均显著降低,AMH均显著升高(P<0.05),且中药组较阳性药组FSH更低、AMH更高(P<0.05)。(4)与空白组比较,模型对照组卵巢组织SOD、GSH-Px显著下降,ROS、MDA显著上升(P<0.05);与模型对照组比较,阳性药、中药组卵巢组织SOD、GSH-Px均显著升高,ROS、MDA均显著下降(P<0.05),且中药组ROS显著低于阳性药组(P<0.05)。结论中药毓麟珠能通过上调抗氧化因子、下调缺氧相关因子改善卵巢微环境,促进卵泡发育,改善卵巢功能。     

卵巢巧克力囊肿术后血清AMH、FSH、LH和E_2检测意义

目的探讨卵巢巧克力囊肿腹腔镜术后血清抗缪勒氏管激素(AMH)、卵泡刺激素(FSH)、促黄体生成素(LH)和雌二醇(E_2)水平检测及临床意义。方法将本院2019年2月至2020年2月收治的66例卵巢巧克力囊肿患者随机分为缝合组和双极电凝组各33例,并选取同期在本院体检的33例健康女性作为对照组。两组患者均实施腹腔镜下巧克力囊肿剥除术。比较两组术后1、7个月的血清AMH、FSH、LH和E_2水平,分析FSH、LH水平与AMH、E_2水平的相关性。结果术后1、7个月,3组的AMH、FSH水平比较,差异显著(P0.05);双极电凝组的上述指标与对照组比较,差异显著(P0.05);缝合组的上述指标与双极电凝组、对照组比较,无显著差异(P0.05)。术后1、7个月,3组的LH水平比较,无显著差异(P0.05)。术后1个月,双极电凝组、缝合组的E_2水平与对照组比较,差异显著(P0.05);术后7个月,3组的E_2水平比较,无显著差异(P0.05)。FSH、LH水平与AMH、E_2水平呈负相关(r=-0.517、-0.334、-0.640,P0.05)。结论卵巢巧克力囊肿腹腔镜术后患者的AMH、FSH、LH、E_2可以作为评价其卵巢功能的有效指标,临床应加强对患者上述指标的检测。     

血清抗苗勒管激素水平检测在预测卵巢低反应中的应用价值

目的 探讨血清抗苗勒管激素(AMH)水平对于卵巢储备功能低下患者在控制性超排卵(COH)中卵巢反应的应用价值. 方法 回顾性分析2010年1月至6月在本中心接受体外受精/卵胞浆内单精子注射-胚胎移植(IVF/ICSI-ET)治疗的308周期.患者分为卵巢低反应组(65例)和卵巢正常反应组(243例).清晨空腹静脉血测定AMH(无月经周期限制)和卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E2)水平(月经第2～5天),同时行阴道B超检查测定窦卵泡数(AFC),以获卵数为评价标准. 结果 与获卵数的相关性由强到弱是AMH水平、AFC、年龄、FSH/LH比值、基础FSH水平、基础LH水平;预测卵巢低反应ROC曲线下面积由大到小为AMH水平、AFC、基础FSH水平、FSH/LH比值、年龄、基础LH水平、基础E2水平;预测卵巢低反应的AMH水平界值≤2.21 μg/L(灵敏度77％,特异度80％). 结论 血清AMH水平是反映卵巢储备能力的理想指标,可预测COH中卵巢的反应性,指导临床选择合适的治疗方案.     

促排卵中卵泡刺激素的最大值

在促排卵过程中,卵泡的发育不仅取决于卵泡刺激素(FSH)的剂量,还取决于基础窦卵泡数,窦卵泡中颗粒细胞数量,颗粒细胞表面FSH受体的质量以及卵母细胞的质量.当FSH剂量达到阈值后,卵泡发育的关键就取决于窦卵泡数和卵泡发育的内在因素.窦卵泡在正常范围内,增加剂量可能增加获卵数,但并不增加妊娠率,因此促性腺激素(Gn)剂量只要超过需要卵泡数的阈值即可,一般建议为150～225 IU;而卵巢反应不良的患者,由于卵巢内能对FSH发生反应的小窭卵泡数减少,而且卵泡颗粒细胞和FSH受体数量下降,对FSH反应不敏感,因此增加剂量不能增加获卵数.系统综述、前瞻对照研究和回顾性研究结果均提示,FSH＞300 IU不能增加获卵数和妊娠率,因此对卵巢反应不良患者建议的最大FSH剂量为300 IU/d.     

Testosterone effects on luteinizing hormone and follicle-stimulating hormone responses to gonadotropin-releasing hormone in the mouse

Studies in the mouse have demonstrated for the first time in vivo regulation of gonadotropin-releasing hormone (GnRH) on the minute-to-minute dynamics of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release and the effects of testosterone on this regulation. Intact and castrated mice with different testosterone levels (3-9 ng/mL) were challenged with exogenous GnRH while under general anesthesia to block endogenous GnRH release. Plasma concentrations of LH and FSH were determined by radioimmunoassay from sequential blood samples collected from anesthetized mice with in-dwelling catheters. The release of LH was correlated with the infusion of different doses of GnRH (0.35, 3.5, and 35 ng) in both intact and castrated mice (r = 0.942, approximately 0.999). GnRH-stimulated LH release was significantly lower in intact mice and in castrated mice with high testosterone levels than in castrated mice with low testosterone levels (P < .05). However, GnRH did not induce FSH release except in castrated males with low testosterone levels and at the highest dose of GnRH. The profiles of FSH release in intact mice and castrated mice with the highest testosterone levels were significant lower than the other groups (P < .05). In conclusion, release of LH, but not FSH, was correlated with increasing dosages of GnRH (r = 0.970), and testosterone significantly suppressed GnRH-stimulated LH release in the mouse (P < .05).     

Growth hormone response to acute growth hormone releasing hormone administration in uraemic patients on haemodialysis.

To examine the response of growth hormone (GH) to growth hormone releasing factor (GHRF) in patients on haemodialysis, we performed the acute GHRF test (50 micrograms administered intravenously as a bolus) in 10 uraemic male patients on haemodialysis and eight normal controls. Each patient was tested before and after a haemodialysis session (at 08.30 and 12.30). Controls were tested on the same time schedule. At 08.30, patients had significantly greater basal and peak GH values (2.5 +/- 0.6 and 27.8 +/- 5.5 micrograms/l) than controls (0.68 +/- and 11.5 +/- 4 micrograms/l). After the haemodialysis session, basal and peak values declined significantly (P less than 0.01) in the uraemic group (0.5 +/- 0.03 and 3.1 +/- 1.1 micrograms/l), whereas the controls did not show such a change in the 12.30 test. Basal and intratest glycaemic values were comparable both before and after haemodialysis. After dialysis test results did not change either with the use of glucose-free dialysate or with bicarbonate buffer. Uraemic patients display a greater GH response to GHRF injection than normal subjects, and this response decreases after haemodialysis. The degree of reduction has no relationship with either glycaemia or the dialysate buffer. We suggest that other GH secretion regulating factors are altered by the haemodialysis procedure.     

Gonadal dysfunction after testicular torsion: luteinizing hormone and follicle-stimulating hormone response to gonadotropin releasing hormone

We studied 14 postpubertal patients at an average of 33 months after treatment for testicular torsion. Of these patients 11 had been treated by detorsion and 3 by orchiectomy. Five normal male volunteers of the approximate age of the study group served as controls. The patients treated by detorsion were subdivided into 3 groups based on the degree of atrophy of the detorsed testicle: group 1--no testicular atrophy (5), group 2--25 per cent testicular atrophy (2) and group 3--greater than 90 per cent testicular atrophy (4). Mean duration of torsion was greatest in the orchiectomy group (161 hours) compared to 6, 16 and 29 hours for groups 1, 2 and 3, respectively. The serum luteinizing hormone and follicle-stimulating hormone response to an intravenous bolus of 100 mcg. synthetic gonadotropin releasing hormone was measured in all patients. All groups had a greater mean follicle-stimulating hormone response to gonadotropin releasing hormone stimulation than controls (p less than 0.05). Patients who underwent orchiectomy had the greatest follicle-stimulating hormone response to gonadotropin releasing hormone stimulation. Mean luteinizing hormone response to gonadotropin releasing hormone stimulation was normal in patients without atrophy (group 1) but it was greater than controls in patients who had atrophy (groups 2 and 3) or who underwent orchiectomy (p less than 0.05). Several conclusions could be made from our study. All patient groups treated for torsion had evidence of testicular dysfunction. Patients who underwent orchiectomy displayed more testicular dysfunction than patients who had atrophy after detorsion. Testicular dysfunction after torsion is more likely to involve spermatogenic before Leydig cell function.     

Growth hormone responses to growth-hormone-releasing hormone and thyrotropin-releasing hormone in diabetic patients with and without retinopathy

Growth hormone (GH) responses to growth-hormone-releasing hormone (GRH) and thyrotropin-releasing hormone (TRH) were studied in 17 diabetic patients. Ten patients (group 1) had retinopathy corresponding to stage III-V (Scott's classification), and the remaining seven patients (group 2) had no retinopathy despite longer duration of diabetes in comparison with the patients in group 1. There were no differences in age, percent of ideal body weight, and serum HbA1 levels between the two groups. Basal serum GH levels were 1.9 +/- 0.4 ng/ml (mean +/- SEM) in group 1, and not different from the values in group 2 (1.6 +/- 0.7 ng/ml). However, GH responses to synthetic human GRH-44 (1 micrograms/kg body wt, i.v. bolus) were significantly greater in group 1, as judged by the maximal response or integrated GH secretion after the administration of GRH. There were no differences in serum insulin-like growth factor I (IGF-I) levels between group 1 (262 +/- 35 ng/ml) and group 2 (232 +/- 30 ng/ml), and no significant correlation was found between serum IGF-I levels and GH responses to GRH in either of the two groups. Paradoxical GH responses to TRH (500 micrograms, i.v. bolus) were found in only one patient in each group. We have thus demonstrated that GH responses to GRH are more pronounced in diabetic patients with retinopathy than in patients without this complication, although it remains to be determined whether or not greater GH responses to GRH are causally related to the development of diabetic retinopathy.     

Altered responses of prolactin, luteinizing hormone and follicle stimulating hormone secretion to thyrotrophin releasing hormone/gonadotrophin releasing hormone stimulation in cyclical mastalgia

A generalized abnormality of hypothalamopituitary function was found in 17 patients with cyclical pronounced mastalgia compared with 11 controls by using a combined thyrotrophin releasing hormone and gonadotrophin releasing hormone test. The release of prolactin, luteinizing hormone and follicle stimulating hormone was significantly greater in cyclical mastalgia patients than in controls. Basal thyrotrophin, T3 and T4 levels were within the normal range in both groups indicating normal thyroid status in benign breast disease. The single measurement of oestrogen and progesterone in the luteal phase was not abnormal. These data demonstrate an alteration in lactotroph and gonadotroph function in patients with cyclical mastalgia. It is unknown at present whether this represents an appropriate cellular response to altered central or peripheral signals. There is no evidence to suggest, however, that the anterior pituitary cell types are abnormal per se.     

Relation between parathyroid hormone and adrenocorticotropic hormone in primary hyperparathyroidism

Parathyroid hormone is concerned with urolithiasis, and regulated by serum ionized calcium concentration. We thought that parathyroid hormone might also be regulated by a hormone. 1 mg of ACTH injection was given intramuscularly to 6 patients with primary hyperparathyroidism, 6 patients with urolithiasis, and 5 control subjects. Serum calcium significantly increased 2 h after ACTH injection in primary hyperparathyroidism. However in the other two groups, an increase of serum calcium was not observed. Parathyroid hormone increased after ACTH injection in most subjects of all three groups. Calcium concentration in a culture medium of parathyroidectomy increased in 4 cases, and the parathyroid hormone concentration in the culture medium increased in 3 cases after ACTH addition. From these data, we believe that ACTH directly influences the parathyroid glands, and that calcium is released from the parathyroid cells. Therefore, the decrease in calcium concentration in the parathyroid cells activates the excretion of parathyroid hormone. The fact that serum parathyroid hormone increases in most subjects in all groups supports our hypothesis, namely that ACTH acts directly on the parathyroid gland.     

Cervical sympathectomy affects adrenocorticotropic hormone and thyroid-stimulating hormone in rats

To examine the effects of bilateral cervical sympathectomy on the secretion of adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), growth hormone (GH), and prolactin (PRL), 18 male rats were divided into three groups: control (Cont), sham operation (Sham), and bilateral cervical sympathectomy (Symp). All rats were kept under a normal circadian rhythm for 2 weeks. Subsequently, blood was collected and plasma ACTH as well as serum TSH, GH, and PRL levels were measured. The difference in ACTH levels between the Cont and Sham groups was not significant, but ACTH levels in the Symp group were significantly higher than those in the other groups. The difference in TSH levels between the Cont and Sham groups was also not significant, but TSH levels in the Symp group were significantly lower than those in the Cont group. There were no statistically significant differences in GH and PRL levels among these groups. The present results suggest that cervical sympathectomy in the rat increases ACTH secretion and decreases TSH secretion in the pituitary. These effects seem to be due to a mildly increased secretion of melatonin in the pineal body that probably in turn increases corticotropin-releasing factor (CRF) secretion and decreases thyrotropin-releasing hormone (TRH) secretion in the hypothalamus. Extrapolation of these findings to humans suggests that longterm and repeated stellate ganglion block would affect the pituitary secretions of ACTH and TSH.