补肾活血方中药对妊娠大鼠子宫内膜容受性的影响

目的:观察补肾活血方中药对妊娠大鼠子宫内膜容受性的影响.方法:选用妊娠SD大鼠随机分为空白组、模型组和中药组,于妊娠第l天模型组和中药组大鼠皮下注射米非司酮溶液1次(米非司酮溶液5 g/L,5 mg/kg)造成胚泡着床障碍模型,空白组皮下注射生理盐水1次(1 mL/kg).空白组和模型组从妊娠第l天起予生理盐水灌胃(6.7 mL/kg),2次,d,连续5 d.中药组从妊娠第1天起行补肾活血方中药灌胃(7.6g生药/kg),2次,d,连续5d.于妊娠第5天扫描电镜观察各组子宫内膜胞饮突发育情况,逆转录聚合酶链反应(RT-PCR)检测子宫内膜整合素αvβ3mRNA的表达,于妊娠第10天计数各组大鼠胚泡着床数.结果:模型组子宫内膜表面未见明显的胞饮突发育,表现为增生早期的子宫内膜变化.中药组可见发育中的胞饮突,发育形态明显超前于模型组,接近于空白组;模型组αvβ3mRNA表达水平(0.854 5±0.0747,0.3984±0.093 7)显著低于空白组(1.051 8±0.087 8,0.601 6±0.133 8)(P<0.01),中药组αvβ3mRNA表达水平(0.987 6±0.075 1,0.539 6±0.058 4)显著高于模型组(P<0.05);模型组胚泡着床数(4.63±3.66)显著低于空白组(12.63±2.07)(P<0.01),中药组胚泡着床数(10.75±2.44)显著高于模型组(P<0.01).结论:补肾活血方中药能明显改善胚泡着床障碍大鼠子宫内膜表面胞饮突的发育,并显著提高子宫内膜整合素αvβ3mRNA的表达,有助于子宫内膜容受性的建立,从而最终提高胚泡的着床率.     

对胚泡着床障碍小鼠子宫内膜容受性的研究

目的:建立胚泡着床障碍小鼠模型,并研究其子宫内膜容受性。方法:于妊娠d4上午利用米非司酮诱导昆明小鼠胚泡着床障碍,12h后处死小鼠,观察小鼠胚泡着床率及平均着床胚泡数,利用光镜观察小鼠子宫内膜形态结构,采用免疫组化SP法检测子宫雌、孕激素受体(ER、PR)表达,采用RT-PCR检测子宫ER、PR基因表达。结果:与对照组相比,模型组小鼠胚泡着床率及平均着床胚泡数明显降低,子宫内膜发育明显受抑制;子宫内膜腺体、间质ER、PR表达范围和强度均显著降低。结论:米非司酮能成功诱导建立胚泡着床障碍模型,其着床期子宫内膜发育受抑制,子宫内膜容受性降低,胚泡着床失败。     

醋炔诺酮丙酰肟对鼠的抗生育作用研究

醋炔诺酮丙酰肟在大鼠和小鼠妊娠1～5天口服的抗着床ED_(50)分别为0.068±0.02和0.31±0.1mg/kg,大鼠妊娠第2天单次肌注或口服的抗着床ED_(50)分别为0.53±0.1和3.5±0.7mg/kg。大鼠妊娠7～8天肌注5～10mg/kg醋炔诺酮丙酰肟有明显的终止早孕作用。醋炔诺酮丙酰肟在终止早孕剂量时,明显抑制假孕大鼠的蜕膜反应且使早孕大鼠的血清孕酮浓度明显下降。提示:醋炔诺酮丙酰肟的终止早孕作用可能与其抑制孕激素活性有关。     

Asoprisnil抗小鼠孕卵着床效果及对子宫内膜容受性的影响

目的:探讨Asoprisnil抗小鼠孕卵着床的效果及其对小鼠着床窗期子宫内膜容受性的影响。方法:将孕第1日小鼠随机分成4组,分别为Asoprisnil低(5μg/g)、中(10μg/g)、高(20μg/g)剂量组和对照组,每组22只,于孕第1~3日每日灌胃给药1次,对照组以体积分数1%羧甲基纤维素钠替代,第5日每组处死孕鼠5只取子宫组织,HE染色观察子宫内膜形态学改变,免疫组织化学S-P法检测子宫内膜PCNA表达。第8日处死余下的孕鼠,计数着床点数。结果:①Asoprisnil高、中和低剂量组的妊娠率分别为11.76%、35.29%和76.47%,与对照组(94.12%)比较差异有统计意义(P<0.001)。②Asoprisnil高、中、低剂量组着床胚泡数第50百分位数(P50)分别为13(10.5~14)、10(0.5~11)和0(0~12),与对照组0(0~0)比较差异均有统计学意义(P<0.001)。③对照组围着床期子宫内膜腺体弯曲折叠,为复层高柱状上皮细胞;基质细胞蜕膜变,细胞大且排列疏松,胞质丰富透亮;内膜中腺体和血管丰富。Asoprisnil组子宫内膜腺体为单层或者复层上皮细胞;内膜基质细胞蜕膜变不明显,基质细胞较小,排列致密;腺体和血管增生不明显。④围着床期小鼠子宫内膜腺体和基质中均有PCNA表达。内膜腺体中Asoprisnil高、中、低剂量组PCNA表达强度(AIOD)分别为0.15±0.01、0.16±0.03和0.14±0.02,与对照组(0.21±0.03)比较差异有统计学意义(P<0.001)。Asoprisnil高、中、低剂量组内膜基质细胞中PCNA表达强度(AIOD)分别为0.17±0.01、0.18±0.03和0.17±0.02,与对照组(0.15±0.02)比差异有统计学意义(P<0.001)。结论:Asoprisnil能显著抑制着床窗期子宫内膜腺体增殖,促进子宫内膜基质细胞增殖,但阻止基质细胞蜕膜化,降低子宫内膜容受性而发挥抗小鼠胚胎着床效应,显示出潜在的子宫内膜靶向避孕前景。     

AP-1蛋白在胚胎着床前后小鼠子宫内膜的表达

目的 :研究子宫内膜原癌基因AP 1蛋白在小鼠胚胎着床前后的动态变化 ,了解AP 1在胚泡着床中的作用。方法 :采用Immuno blot和Western blot方法检测AP 1蛋白的表达。结果 :AP 1蛋白的表达高峰出现在小鼠早孕的第 4天、第 5天 ,一直维持到妊娠第 7天 ,每毫克组织的积分光密度值分别为 6 0 .2± 4 .2 1,5 2 .6± 7.0 9,5 0 .2± 5 .89和 5 0 .0±3.6 1。结论 :AP 1蛋白参与了胚胎着床过程中的信号传导并可能与胚胎的早期分化有关。     

胚泡和内膜发育状态对子宫内膜“着床窗口”形成的影响

本文应用胚泡移植和改进的内膜上皮细胞与胚泡共培养方法研究了胚胎和子宫内膜不同发育状态对子宫内膜对胚泡接受性的影响.实验证明正常胚泡移植到子宫内膜的“着床窗口”出现时间以妊娠d4天上午9时到下午18时.如果被移植的胚泡是取自延缓着床胚泡,则它们的“着床窗口”出现时间有明显缩短,只能到下午14时为止.另外也证明.子宫内膜本身发育状态对内膜的接受性也有影响.以妊娠d5天内膜的胚泡附着数最多.所以.可以认为胚胎和内膜的发育状态都是可以影响内膜“着床窗口”的出现与消失的.     

米非司酮和孕酮对子宫内膜异位症患者子宫内膜白细胞介素6分泌的影响

目的探讨米非司酮和孕酮对子宫内膜异位症(内异症)患者异位子宫内膜(异位内膜)与正常子宫内膜(在位内膜)白细胞介素6(IL-6)分泌的影响.方法采用酶联免疫吸附试验(ELISA)方法,检测米非司酮浓度为1×10-6mol/L(米非司酮1组)、1×10-4mol/L(米非司酮2组),和孕酮浓度为1×10-7mol/L(孕酮1组)、1×10-5mol/L(孕酮2组)与体外培养的异位内膜细胞和在位内膜细胞上清液作用后的IL-6水平.结果米非司酮可降低异位内膜细胞IL-6水平,米非司酮1组和米非司酮2组的IL-6分为(1914.33±799.28)μg/L和(990.25±58.40)μg/L,两组与空白组比较(下同),差异有极显著性(P＜0.01).孕酮也可降低异位内膜细胞IL-6水平,孕酮1组和孕酮2组的IL-6分为(2575.89±119.75)μg/L和(1736.25±750.89)μg/L(P＜0.01).米非司酮还可使在位内膜细胞IL-6水平降低,其中米非司酮1组和米非司酮2组分别为(346.96±24.32)μg/L和(270.22±36.15)μg/L(P＜0.01).孕酮对在位内膜细胞IL-6水平无明显影响(P＞0.05).结论米非司酮和孕酮可抑制异位内膜和在位内膜细胞IL-6的分泌.     

保胎液对胚泡着床障碍小鼠子宫内膜降钙素表达的影响

目的:观察保胎液对胚泡着床障碍模型小鼠子宫内膜降钙素水平的影响.方法:采用米非司酮小鼠受精卵着床障碍模型.将孕鼠随机分为正常组、模型组、西药组和保胎液(高、中、低3种剂量)治疗组,从妊娠第1天起分别给予0.9％氯化钠溶液、地屈孕酮和保胎液(高、中、低3种剂量)灌胃治疗.于妊娠第4天上午皮下注射米非司酮,每只0.08 mg.正常组不予任何处理.于妊娠第5天取小鼠的子宫组织,运用实时荧光定量PCR技术检测小鼠子宫内膜降钙素的含量.结果:模型组降钙素表达量与正常组、中药高剂量组、中剂量组及西药组比较,差异有统计学意义(P＜0.05).中药高、中剂量组分别与西药组比较,差异无统计学意义(P＞0.05);中药中剂量组与正常组比较,差异无统计学意义(P＞0.05);中药中剂量组降钙素水平明显高于高、低剂量组(P＜0.05).结论:保胎液可以调节小鼠子宫内膜降钙素的表达量,特别是中剂量效果最明显,从而改善受精卵着床障碍小鼠子宫内膜容受性,促进受精卵着床.     

AF-113的抗生育作用研究

大鼠,小鼠和仓鼠受孕后第1(d_1),第2(d_2)和第7(d_7)天灌胃(Po)AF-113 0.56—15mg/kg 有明显的抗生育作用。其抗着床ED_(50)大鼠和小鼠分别为1.03(0.66—1.62)和0.54(0.14—2.12)mg/kg,仓鼠为10.0(7.3—13.8)mg/kg。AF—113使幼年大鼠子宫增重的效价为EE 的1.9%;能抑制大鼠子宫内膜蜕膜瘤生长,抑制幼兔子宫内膜增生,但本身无孕激素活性;体外能与大鼠、小鼠和仓鼠子宫胞浆雌激素受体结合,其抑制~3[H]-雌二醇与雌激素受体相结合的IC_(50)分别为2.5±1.1×10~(-5),4.3±2.6×10~(-6)和5.0±0.3×10~(-5)M     

白血病抑制因子在小鼠早孕期子宫内膜表达规律及调节的研究

目的 :研究在小鼠胚泡着床中白血病抑制因子 (LIF)的生物学作用以及卵巢激素对LIF表达的调节。方法 :收集孕 1～ 8d小鼠子宫内膜 ,并以动情间期小鼠子宫内膜作为对照 ;将孕 2～ 4d小鼠随机分为 4组 ,分别灌服戊酸雌二醇 (10 0 0 μg/kg)、安宫黄体酮(10 0 0 μg/kg)、戊酸雌二醇 (10 0 0 μg/kg) +安宫黄体酮 (5 0 0 μg/kg)或植物油 ,孕 4d收集子宫内膜。采用流式细胞仪检测LIF在小鼠早期妊娠子宫内膜中的表达规律以及卵巢激素对LIF的调节作用。结果 :LIF呈时序性表达 ,孕第 1、2天表达量与未孕期相似 ,第 3天开始升高 ,第 4天达高峰 ,此后缓慢下降 ,第 7天降到未孕期水平 ;戊酸雌二醇对小鼠孕早期子宫内膜的LIF表达有上调作用。结论 :LIF可能是小鼠胚泡着床过程中的一个重要细胞因子 ,它在孕早期子宫内膜的表达受卵巢激素的调控     

LIF及其抗体对小鼠胚胎着床影响的体外研究

目的 :研究外源性白血病抑制因子 (LIF)及其抗体对小鼠胚胎着床的影响。方法 :将妊娠 4d的小鼠胚胎种植于已建立的子宫内膜体外模型上 ,观察不同浓度的LIF及其抗体对小鼠胚胎的粘附、植入及扩展情况。并用RT PCR法测定子宫内膜上皮细胞整合素β3的变化。结果 :不同浓度的LIF促进了胚胎的粘附 (P <0 .0 5 ) ,胚胎的扩展率降低。加入LIF抗体 (5 ,1 0 μg/ml)降低了胚胎的粘附率 (P <0 .0 5 ) ,同时LIF对整合素β3的表达有明显的促进作用 (P <0 .0 1 ) ,加入抗体后整合素β3的表达降低。结论 :LIF可能通过上调整合素β3的表达促进胚胎的粘附和植入     

Cycle and age-related changes in corticotropin-releasing hormone levels in human endometrium and ovaries

Corticotropin-releasing hormone (CRH) is synthesized in most female reproductive tissues such as the ovaries and the uterus. In the non-pregnant uterus ,it is mainly produced by epithelial cells of the endometrium. Recent in vitro experimental findings show that endometrial CRH is under the positive control of progesterone ,participating in the decidualization process of endometrial stroma and the progression of blastocyst implantation. CRH is also produced in the thecal compartment of the human ovary ,controlling ovarian steroid hormone biosynthesis. In the present study we compared the concentration of immunoreactive CRH (ir-CRH) in biopsies from proliferative and secretory human endometria ,and from pre- and postmenopausal human ovaries. We found that the concentration of ir-CRH was significantly higher in the secretory (92 ± 8 pg/mg protein; n = 10) than the proliferative (75 ± 9 pg/mg protein; n = 12; p < 0.05) endometria. This observation supports the experimental in vitro findings associating endometrial CRH in intrauterine phenomena of the secretory phase of the menstrual cycle (decidualization and implantation). Additionally ,we have shown that the concentration of ir-CRH was significantly higher in the premenopausal (125 ± 12 pg/mg protein; n = 14) than the postmenopausal (100 ± 12 pg/mg protein; n = 12; p < 0.05) ovaries, suggesting that ovarian CRH is related to normal ovarian function during the reproductive lifespan.     

抗孕激素RU486和ZK98734对双池培养的颗粒细胞和内泡膜细胞分泌功能的影响

本文观察了RU486和ZK98734对培养在羊膜双池培养系统中的猪卵巢颗粒细胞和内泡膜细胞在甾体激素生成过程中的影响。生长在羊膜两书侧的颗粒细胞和内泡膜细胞在加入或不加FSH、LH及不同浓度RU486或ZK98734的条件下培养48h。用RIA测定内、外池培养液中的孕酮（P）和雌二醇（E2）的浓度并与单独培养的结果相比较，同时亦与大鼠颗粒细胞的结果作比较。结果表明：1．有FSH刺激时，两种抗孕激素均明显抑制双池培养中颗粒细胞的P和E2产量；2．不论有或无LH刺激，两种抗孕激素均显著抑制双池培养中内泡膜细胞的P产量；3．双池培养系统模拟了两种卵巢细胞在体内的旁分泌调节关系，比单独培养更加合理；4与大鼠相比，猪卵巢细胞体外培养是研究避孕药对卵巢功能影响的适合模型。     

黄体酮阴道环在家兔的药代动力学研究

目的:提供研制可供哺乳期妇女避孕的黄体酮阴道环的药理学依据。方法:将18只去卵巢后2周的新西兰雌兔随机分为3组:阴道环低剂量组(175mg,A组)、高剂量组(350mg,B组)及肌注组(C组),分别在放置阴道环及肌注黄体酮前后不同时点取静脉血,用磁性分离酶联免疫法测定兔血清中黄体酮的浓度,用PK-Graph程序计算药代动力学参数。结果:黄体酮阴道环和黄体酮注射剂在兔体内的药代动力学符合二室开放模型。主要药代动力学参数分别是A组:Tmax:2.23±1.3h,Cmax:47.64±23.58ng/ml,T1/2:818.08±511.77h,AUC:16115.11±8398.88ng·ml·h-1;B组:Tmax:2.03±1.33h,Cmax:74.04±24.57ng/ml,T1/2:730.31±306.49h,AUC:28751.95±7151.95ml·h-1;C组:Tmax:1.54±0.77h,Cmax:138.88±60.96ng/ml,T1/2:2.55±0.89h。其中A组的AUC(56d)明显低于B组,二者有显著性差异(P<0.05);A组和B组的Cmax均低于C组,差异显著(P<0.05);A组和B组的T1/2均明显高于C组(P<0.01),但A、B组间无差异。结论:与传统的黄体酮注射剂相比,黄体酮阴道环的药代动力学参数呈现明显的长效缓释特征。应可成为安全长效且使用方便的哺乳期避孕药具。     

No advantage of fresh blastocyst versus cleavage stage embryo transfer in women under the age of 39: a randomized controlled study

Purpose

Is there a difference in implantation and pregnancy rates between embryos transferred electively at cleavage or blastocyst stage in infertile women ≤?38 years with at least four zygotes on day 1 post retrieval?

Methods

A randomized clinical trial was conducted in a single tertiary care hospital with a sample size of 194 patients in each arm for a total population of 388 women. Patients less than 39 years of age with more than three fertilized oocytes and less than four previous assisted reproductive technology (ART) attempts were inclusion criteria.

Results

The two groups were similar for age, years of infertility, indication to treatment, basal antimüllerian hormone and FSH, number of previous ART cycles, primary or secondary infertility, type of induction protocol, days of stimulation, total gonadotrophin dose, and estradiol (E2) and progesterone (P) levels at trigger. No statistically significant differences were found in terms of number of retrieved oocytes, inseminated oocytes, fertilization rate, canceled transfers (7.73% in blastocyst and 3.61% in cleavage stage group), and cycles with frozen embryos and/or oocytes. Although a higher number of fertilized oocytes were in the blastocyst stage group (6.18?±?1.46 vs 5.89?±?1.54, p?=?0.052), a statistically greater number of embryos/randomized cycle were transferred at cleavage stage (1.93?±?0.371) compared with the number of transferred blastocysts (1.80?±?0.56), probably due to the number of embryos not reaching blastocyst stage (3.09%). The implantation rate (28.37 vs 25.67%), pregnancy rate per cycle (36.06 vs38.66%), transfer (39.66 vs 40.11%), spontaneous abortions (19.72% vs 12.00%), delivery rate per cycle (27.84 vs 32.99%), and transfer (30.17 vs 34.22%) were not significantly different between the blastocyst and cleavage stage groups. The twin delivery rate was higher in the blastocyst stage group, although not significant (42.59 vs 28.12%). The mean numbers of frozen blastocyst (2.30?±?1.40 vs 2.02?±?1.00) and frozen oocytes (7.09?±?3.55vs 6.79?±?3.26) were not significantly different between the two groups.

Conclusions

Fresh blastocyst-stage transfer versus cleavage-stage transfer did not show any significant difference in terms of implantation and pregnancy rate in this selected group of patients. A high twin delivery rate in both groups (35.59%) was registered, and although not significant, they were higher in the blastocyst transfer group (42.59 vs 28.12%). Our conclusion supports considering single embryo transfer (SET) policy, even in cleavage stage in patients younger than 39 years with at least four zygotes.

Trial registration

ClinicalTrials.gov registration number NCT02639000

     

环氧司坦对大鼠止孕作用的药理研究

本文研究了环氧司坦(epostane)对妊娠大鼠的止孕作用。环氧司坦48mg/kg灌胃给药,最佳给药时间为妊娠第10～11天。大鼠妊娠第10天,环氧司坦以48mg/kg灌胃给药或96mg/kg肌肉注射给药,均可使大鼠完全止孕。灌胃给药的ED_(50)为22.3±0.05mg/kg,95%可信限为17.3～28.8mg/kg;肌肉注射给药的ED_(50)为29.6±0.06mg/kg,95%可信限为22.0～39.5mg/kg。二者之间无显著差异(P>0.05)。环氧司坦对3β-羟甾脱氢酶具有抑制作用,黄体酮或消炎痛均可拮抗环氧司坦对大鼠的止孕作用,绒毛膜促性腺激素(hCG)不能拮抗环氧司坦对大鼠止孕作用。环氧司坦48mg/kg灌胃给药可使血浆孕酮水平下降,但无雌激素活性。     

Progesterone antagonist lilopristone: a potent abortifacient in the common marmoset

The effects of a progesterone antagonist ZK 98.734 (lilopristone) on implantation, early pregnancy, and midpregnancy were studied in the common marmoset, Callithrix jacchus jacchus. Treatment (5 mg/da intramuscularly for 3 consecutive days) on day (n = 8) after the midcycle peak in estradiol levels in mated animals induced a premature drop in plasma progesterone levels and shortened the ovarian cycle length. Treatment on day 20 (n = 5) or day 40 (n = 5) induced a drop in progesterone levels and decidual collapse. In three animals treated on day 40, vaginal bleeding was observed within 46 hours of the initiation of treatment. Treatment on day 80 resulted in expulsion of the fetuses with a mean induction abortion interval of 39 hours (range, 20 to 48 hours). The progesterone antagonistic effects of ZK 98.734 could be a result of the decrease in progesterone synthesis by the corpus luteum and/or placenta in addition to the interference with the progesterone binding to its cellular receptors in the target organ. Our study suggests that ZK 98.734 has potential for fertility regulation. Clinical trials for postcoital contraception, induction of menstruation, and early abortifacient effects are warranted.     

Chorionic gonadotropin receptors and immunoreactive chorionic gonadotropin in implantation of the rabbit blastocyst

To determine the temporal relationship between immunoreactive chorionic gonadotropin, chorionic gonadotropin receptors, and implantation of the rabbit blastocyst, (1) immunoreactive chorionic gonadotropin in plasma, uterine flushings and blastocysts; (2) chorionic gonadotropin receptors on blastocysts (day 5 and 6) and embryonic and interembryonic segments of the uterus (day 7); and (3) chorionic gonadotropin receptors on the endometrium and myometrium (day 1 through 6) were measured. Binding of I125-labeled beta-subunit of human chorionic gonadotropin (hCG) by cell membranes from blastocysts increased significantly from 6.0 +/- 1.1 fmol/mg of protein (mean +/- SE) on day 5 (N = 6) to 16.1 +/- 1.3 fmol/mg of protein on day 6 (n = 6) (P less than 0.001). Immunoreactive chorionic gonadotropin levels in blastocyst fluid increased from 0.3 ng/ml of day 5 to 0.65 ng/ml on day 6. Specific binding of I125-labeled hCG was absent in endometrial and myometrial cell membranes before implantation (days 1 to 6) but was found in decidual cells from embryonic segments on day 7. Plasma immunoreactive chorionic gonadotropin or chorionic gonadotropin--like material increased from 6 ng/ml of day 1 to 52 ng/ml on day 7. Uterine flushings had chorionic gonadotropin levels of 0.4 ng/ml of day 2, which increased slowly to 1.0 ng/ml on day 7. Intrauterine instillation of hCG into nonpregnant uterine horns demonstrated transuterine absorption of hCG with plasma hCG levels showing a dose-related response. Our findings demonstrate that (1) immunoreactive chorionic gonadotropin or chorionic gonadotropin--like material is detectable in plasma, uterine flushings, and blastocyst fluid before implantation, (2) chorionic gonadotropin receptors are present on the blastocyst cells before implantation, and (3) the uterus can absorb chorionic gonadotropin from its lumen.