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。     

EMX2、HOXA10与子宫内膜容受性

胚胎与子宫内膜的同步发育是着床的必要条件,而在着床窗期间,胚泡滋养外胚层侵入和妊娠建立的关键是子宫内膜具有容受性。近年从分子机制研究子宫内膜容受性取得很大进展,学者们发现了与子宫内膜容受性有关的一些基因。本文主要从同源结构域基因EMX2和HOXA10在子宫内膜的表达和对子宫内膜容受性的影响进行综述。     

上皮-间质转化和蜕膜化与子宫内膜容受性

人类生殖过程涉及各种复杂因素,其中胚胎能否着床是受孕过程中的关键步骤。顺利的着床需要有正常生命力的胚胎在适当的时间植入具有接受性的子宫内膜,且胚胎与内膜达到同步交流状态。着床过程中任何一个环节出现差错都会影响妊娠率,而子宫内膜容受性下降可能是导致不孕的主要原因之一。着床初期子宫内膜上皮细胞发生上皮-间质转化（epithelial-mesenchymal transition,EMT）为后续胚胎的植入做准备,随后子宫内膜间质细胞发生蜕膜化为胚胎提供良好的发育环境。这提示EMT与蜕膜化是维持子宫内膜容受状态的基本条件,而良好的子宫内膜容受性是胚胎植入并充分发育的生物学基础。     

人子宫内膜细胞共培养体系对早期鼠胚体外发育的影响

目的:研究人子宫内膜共培养体系对早期鼠胚体外发育的影响及移植后的妊娠情况。方法:将2-细胞小鼠胚胎与人子宫内膜细胞进行体外共培养,对照组为无营养细胞的单纯培养液,每日在显微镜下观察胚胎的发育情况。将培养到囊胚期的胚胎移植回小鼠的子宫腔,观察着床情况。结果:共培养体系中68.3％的2-细胞胚胎发育至桑椹胚期,50.8％发育至囊胚期,囊胚的孵化率为36.7％,胚胎的着床率为25.0％。而对照组只有24.8％的2-细胞胚胎发育至桑椹胚期,11.4％到达囊胚期,且其中大部分为早期囊胚即停止发育。另外对照组细胞碎片出现早且多,卵裂球不均匀,胚形态差,移植后胚胎的着床率仅为3.1％。结论:人子宫内膜细胞共培养体系可以促进小鼠胚胎的体外发育,改善胚胎的质量,提高着床率。     

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.     

sLe~x在小鼠胚泡植入早期子宫内膜中的表达及作用

目的:探讨唾液酸化的路易斯寡糖(sLex)在胚泡植入早期小鼠子宫内膜的表达及作用。方法:①应用免疫组化方法对sLex在小鼠妊娠早期子宫内膜的表达进行定位,免疫印迹法以另一侧为自身对照,检测妊娠d1-5小鼠子宫内膜sLeX的表达情况。②向妊娠小鼠一侧子宫角注射sLeX单克隆抗体,观察其对胚胎着床的影响。结果:妊娠d1-5小鼠子宫内膜组织中,sLeX表达量逐渐增加,在植入窗口期(d4)达高峰;子宫角注射抗体后对胚泡植入有明显的抑制作用。结论:在小鼠胚泡植入早期,sLex参与了胚泡的植入过程,在胚泡和子宫内膜的识别过程中发挥重要的作用。     

着床研究模型:离体胚泡与子宫内膜“共培养”

本工作用小鼠和豚鼠的离体胚泡与子宫内膜共培养方法,为研究着床与抗着床机理提供了一个体外研究模型。子宫内膜取自妊娠小鼠第5天上午8时前和妊娠豚鼠第4天下午,分别与小鼠或豚鼠胚泡共培养。经培养2～3天后可见胚泡从透明带逸出并附着于子宫内膜,部分植入内膜。     

子宫内膜容受性相关调控因子的时序表达及功能

胚胎着床是复杂的程序化的生理过程.子宫内膜仅在极短时期内允许胚泡植入,此时子宫内膜达到最大胚泡种植容受性.子宫内膜容受性形成过程中受多种细胞因子、蛋白分子调控,这些特定调控因子在子宫内膜发育分化过程中呈现较为显著的时空表达特征,对内膜容受性形成有重要作用.相关细胞因子(白血病抑制因子、白细胞介素)、蛋白分子(胎盘蛋白、半乳糖凝集素-1)及特定转录因子(同源框基因)在子宫内膜容受性形成过程中伴随短暂、瞬间性高表达,其表达量为增生期的4～5倍,该时期与着床窗期时间一致,呈现出时空特异性.表明这几类生物活性分子可能与子宫内膜容受性形成密切相关.     

胞外囊泡对改善子宫内膜容受性的作用与研究现状

子宫内膜容受性是影响胚胎顺利植入的关键因素,子宫内膜容受性异常将导致胚胎着床失败和胎盘发育异常。胚胎植入过程中,母体和胚胎的胞外囊泡的有序产生和精确协调确保了胚胎的顺利植入。间充质干细胞来源的胞外囊泡可改善病理状态下的子宫内膜容受性。     

子宫内膜容受性的调控及其特征标记的研究进展

胚泡着床是一复杂的生理过程.着床只发生在具有接受性的子宫内膜,子宫对胚泡的接受性叫容受性.子宫内膜容受性的调控机制尚不清楚,涉及卵巢甾体激素,细胞因子和黏附分子等的自分泌及旁分泌机制.综述子宫内膜容受性的调节机制及特征性标记.     

In vivo hatching phenomenon of mouse blastocysts during implantation

Purpose: To determine whether the blastocyst zona shedding process within the murine uterine cornus in vivo is due to a global lytic process caused by uterine proteolytic enzyme, or is triggered by the blastocyst hatching process as observed in vitro.Methods: Fifty-one female ICR mice aged 5–8 weeks were used for this study. From 8:00 p.m. of the 4th day postcoitus to 7:00 p.m. of the 5th day postcoitus, the uterine cornua of 51 mice were isolated at 30-min intervals. Blastocysts within the uterine cornua were flushed out with a balanced solution under the dissecting microscope. The stages of blastocyst development and the proportion of hatching or hatched blastocysts and the discarded zona pellucida (ZP) were inspected and counted.Results: A total of 672 blastocysts were recovered from the uterine horns of the 51 mice. They were divided into six groups according to the blastocyst developmental stages (before or after ZP escape; before or after the initiation of implantation). Group I represents the earliest embryonic stage and Group VI represents the most advanced blastocyst developmental stage during the peri-implantation period. The empty ZP recovery rates (number of discarded ZP/all hatched blastocysts) were 52.3%, 21.5%, 17.2%, 6.6%, 1.6%, and 0% in Groups I–VI, respectively. Five hatching blastocysts out of 199 embryos in Group I were found and a 100% of ZP recovery rate was obtained from 6 of 19 mice in Groups I and II.Conclusions: The present study shows that active blastocyst hatching occurs in vivo because both hatching blastocysts and empty ZP can be found within the uterine cornua of ICR mice before implantation. The empty ZP recovery rate declined significantly, along with a progression of embryo development and implantation, implying that intrauterine zona lytic activity occurs during the peri-implantation stage.     

A sequence of events in the uterus prior to implantation in the mouse

I reviewed a series of events in the mouse uterus before implantation on Day 4 of pregnancy (the sperm positive day is counted as Day 1). Major events are spacing of embryos along the uterine horns, shedding of the zona pellucida, and closure of the uterine lumen. How subtle they may be, there appear to exist interactions between intrauterine blastocysts and the uterus which is regulated by ovarian steroids. Spacing of embryos along the uterine horn is not random, but they are rather evenly distributed along the entire horn. The mechanism of even distribution of embryos needs clarification, although studies indicate that adrenergic nerve activity, prostaglandins, and other molecules appear to be involved. Shedding of the zona pellucida involves trypsin-like proteinase lysis of the zona. Through the opening created by zona lysis, blastocyst gets out of the zona by repeating expansion-collapse movements. Closure of rat uterine lumen is reported to be the result of absorption of uterine fluid through uterine glands. This needs to be confirmed in other species of rodents. Since these events influence blastocyst implantation, we need more detailed information on their regulatory mechanisms in order to improve the rate of healthy implantation of transferred embryo.     

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.     

Extended uterine receptivity for blastocyst implantation and full-term fetal development in mice with vitrified–warmed ovarian tissue autotransplantation

Purpose

Our previous study demonstrated that vitrified?Cwarmed ovarian tissue autotransplantation (VOAT) into estrus cycle-ceased ovariectomized mice restored fertility to achieve full-term fetal development for transferred embryos, while less steroidogenesis in the corpus luteum was observed in VOAT mice. It has been reported that the window of uterine receptivity for blastocyst implantation is extended at lower estrogen levels. Therefore, we hypothesized that duration of the window in VOAT mice could be extended.

Methods

Blastocysts were transferred into VOAT mice on day 5 of pseudopregnancy. Immunohistochemical analysis was performed to examine the potential in VOAT ovarian tissues.

Results

The rate of live birth pups from embryos transferred on day 5 of pseudopregnant VOAT mice was not different from that of embryos transferred on day 4 of pseudopregnancy in VOAT mice, while embryo transfer on day 5 into intact mice showed no pregnancy. Immunohistochemical analysis of the corpus luteum of day 8 pseudopregnant VOAT mice with uteri having decidualization induced on day 5 showed less steroidogenesis and blood vessel formation as compared to intact mice.

Conclusions

Uterine receptivity was extended in VOAT mice. Less steroidogenesis and blood vessel formation in the transferred ovarian tissues may be associated with the extended uterine receptivity.     

Cyclic adenosine monophosphate-induced changes in the surface morphology of diapausing blastocysts and the effects on implantation.

Estrogen induces blastocyst implantation in diapausing female mice, increases uterine levels of adenyl cyclase and cyclic adenosine monphosphate (cylic AMP), and stimulates synthesis of ribonucleic acid and protein in both the uterus and blastocyst. Furthermore, the surface ultrastructure of trophoblast cells seen with scanning electron microscopy (SEM) changes with activation of the diapausing blastocyst by estrogen. Cyclic AMP-activated blastocysts were investigated with the use of SEM and compared to blastocysts activated by estrogen. Intraperitoneal administration of cyclic AMP was generally ineffective, whereas administration of cyclic AMP intraluminally in the uterus effectively mimicked the early estrogen activation. These findings are discussed in relation to other known activation changes in preimplantation blastocysts and with regard to similar findings after administration of various antiestrogens.     

Molecular and cellular events involved in the completion of blastocyst implantation

Blastocyst implantation is an interactive process between the embryo and the uterus. The synchronization of embryonic development with uterine differentiation to a receptive state is essential for a successful pregnancy. The period of uterine receptivity for implantation is limited. Although implantation involves the interaction of numerous signaling molecules, our understanding of the hierarchical mechanisms that coordinate with the embryo–uterine dialogue is not yet sufficient to prevent infertility caused by implantation failure. This review highlights our knowledge on uterine receptivity and hormonal regulation of blastocyst implantation in mice. We also discuss the adhesion molecules, cross-linker proteins, extracellular proteins, and matricellular proteins involved in blastocyst implantation. Furthermore, our recent study reveals that selective proteolysis in an activated blastocyst is associated with the completion of blastocyst implantation after embryo transfer. A better understanding of uterine and blastocyst biology during the peri-implantation period would facilitate further development of reproductive technology.     

Heparin binding-epidermal growth factor improves blastocyst hatching and trophoblast outgrowth in the golden hamster

The effect of heparin binding-epidermal growth factor (HB-EGF) on the in-vitro development of hamster 8-cell embryos was investigated. Supplementation of HB-EGF to culture medium accelerated zona escape of blastocysts (63 +/- 9% compared with 33 +/- 9% after 36 h; P < 0.05). Complete zona escape of blastocysts persisted even after 48 h (61 +/- 11% versus 30 +/- 4%) and 60 h (75 +/- 6% versus 42 +/- 8%). Addition of anti-HB-EGF antibody drastically reduced the percentage of zona escaped-blastocysts (30.0 +/- 5.0% versus 92.3 +/- 2.8%; P < 0.05). Interestingly, a significant increase in the area of trophoblast outgrowth occurred in the presence of HB-EGF (116 x 10(3) +/- 8 x 10(3) microm(2) versus 74 x 10(3) +/- 8 x 10(3) microm(2) at 48 h; P < 0.05). This, however, was not due to an increased number of trophectodermal cells in HB-EGF-treated blastocysts. Immunoreactive HB-EGF was localized in blastocysts and uterine sections, visible by intense immunostaining in the luminal epithelium, particularly on the apical surface. Moreover, the expression of HB-EGF in the uterus was maximum on day 4 of pregnancy, coinciding with the timing of zona escape and implantation. The receptor of HB-EGF, viz. EGF receptor was also detected in blastocysts and the luminal epithelium of day 4 pregnant uterus. These results show that HB-EGF improves blastocyst hatching and trophoblast outgrowth in hamsters.     

The effect of indomethacin on the implantation and metabolism of blastocysts

The experiments were performed in order to reveal the role of prostaglandins in the implantation process, using indomethacin, a inhibitor of prostaglandin synthesis. Indomethacin was administered to mice on various days of pregnancy, and then the number of implantation sites and the uterine weight per the number of implantation sites were determined on day 10 of pregnancy. Furthermore [3H]uridine incorporation of blastocysts incubated in the medium containing indomethacin with/without prostaglandin F2 alpha was evaluated in vitro. The results were as follows. Indomethacin reduced the number of implantation sites in the uterus and the uterine weight per the number of implantation sites (embryonal growth). Indomethacin inhibited the [3H]uridine incorporation of blastocysts, but prostaglandin F2 alpha counteracted the effect of indomethacin. These results suggested that prostaglandins play a role in inducing implantation and embryonal growth, and that blastocysts might synthesize prostaglandins.     

Progesterone receptor requires a co-chaperone for signalling in uterine biology and implantation

Embryo implantation is absolutely dependent on the preparation of the uterus to the receptive stage and attainment by the blastocyst of implantation competency. Co-ordinated effects of progesterone and oestrogen are essential for these processes and determine the window of implantation. In rodents, a generalized stromal edema occurs before blastocyst attachment followed by uterine luminal closure. This leads to apposition of the blastocyst trophectoderm against the luminal epithelium and ultimately attachment. Progesterone is essential for luminal closure, which must occur for successful implantation. More importantly, progesterone is critical for almost every stage of pregnancy, including ovulation, fertilization, implantation, decidualization and pregnancy maintenance. Progesterone exerts its effects on target tissues primarily via nuclear progesterone receptor (PR) whose optimal activity is potentiated by an immunophilin co-chaperone, FK-506 binding protein 4 (FKBP52). While mice lacking PR are infertile due to complete failure of ovulation, fertilization, and implantation, female mice with targeted deletion of the Fkbp52 gene are infertile specifically because of implantation failure resulting from compromised uterine receptivity. This review highlights the evolution of knowledge about progesterone signalling during early pregnancy. Future studies are likely to provide a better understanding of FKBP52-PR signalling in promoting uterine receptivity for implantation and may reveal new targets for improving infertility.     

Aneuploidy rates and blastocyst formation after biopsy of morulae and early blastocysts on day 5

Purpose

Studies have demonstrated high implantation rates after trophectoderm biopsy of day 5 expanded blastocysts. However, biopsy of cleavage stage embryos may adversely affect embryo development and implantation. No studies have assessed the utility of day 5 morulae and early blastocyst biopsy. This study sought to better understand these slower embryos’ aneuploidy rates and implantation potential.

Methods

This was a retrospective review of all autologous IVF cycles utilizing PGS at a single academic infertility center.

Results

The biopsy of day 5 morulae and early blastocysts provided 22 % additional euploid blastocysts available for fresh day 6 transfer compared to day 5 biopsy of only expanded blastocysts. Aneuploidy did correlate with embryo stage on day 5, even after controlling for maternal age, with 16 % of morulae and 35 % of blastocysts being euploid. The majority (83 %) of euploid morulae progressed to the blastocyst stage by day 6. Experience transferring slower developing embryos is limited, but preliminary pregnancy and implantation rates appear similar to euploid embryos biopsied as expanded blastocysts.

Conclusions

The biopsy of all non-arrested embryos on day 5 provides genetic information for all blastocysts on day 6, increasing the pool of euploid blastocysts available for fresh transfer and avoiding the need to cryopreserve developmentally competent embryos without genetic information.