体外着床模型中人孵化后早胚细胞角蛋白和肌动蛋白的表达

目的:建立理想的体外胚胎着床模型,并检测模型中人孵化后早胚细胞角蛋白、肌动蛋白和hCG。方法:孵化后早胚与人子宫内膜蜕膜化的基质细胞共培养,观察胚泡在基质细胞层上的定位、黏附、铺展和侵入过程;用免疫荧光染色技术,测定共培养系统中的细胞角蛋白和肌动蛋白;用免疫荧光分析技术,测定培养液中的hCG水平。结果:胚泡和基质细胞共培养5h起,胚泡开黏附在基质细胞层上,最终侵入蜕膜化的基质细胞间。共培养48h后,细胞角蛋白仅仅在滋养层细胞中表达;肌动蛋白在人蜕膜化的基质细胞和滋养层细胞中均有表达。囊胚与子宫内膜基质细胞共培养的培养液中的hCG水平明显高于囊胚单独培养的(P<0.01)。结论:成功建立了一个能反映人胚泡黏附、铺展及侵入到人子宫基质细胞的体外着床模型,细胞角蛋白、肌动蛋白和hCG在着床早胚细胞中起相应变化。     

Evidence of embryo- and trophoblast-toxic cellular immune response(s) in women with recurrent spontaneous abortion.

OBJECTIVE: The purpose of this study was to determine whether reproductive antigens stimulate lymphocytes and macrophages from women with recurrent abortion to secrete factors that are toxic to preimplantation embryos or trophoblast cells in vitro. STUDY DESIGN: Mononuclear cells were isolated from 30 fertile controls and 300 nonpregnant women being evaluated for recurrent abortion. Supernatants generated from these cells after separate culture with sperm and trophoblast antigen extracts were added to two-cell mouse embryo cultures and trophoblast proliferation assays. Toxicity was assumed when the median percentage of embryos developing to blastocysts or trophoblast proliferation was less than or equal to 50% of control values. Both parametric and nonparametric statistical methods were used where appropriate. RESULTS: Mouse embryo development and/or trophoblast proliferation were significantly inhibited by supernatants from trophoblast and/or sperm antigen-activated peripheral blood leukocyte cultures from a majority of 300 women with recurrent abortion but not from 30 women with normal reproductive histories. The mouse blastocyst development assay was more sensitive than the trophoblast proliferation assay in determining toxic factor production. Embryo-toxic factors were produced by activated leukocyte cultures from 90% of 180 women with a history of recurrent abortion of unexplained etiology, whereas trophoblast-inhibitory factors were detected in 50% of women from the same group. The embryo-toxic factor(s) was heat labile, had a molecular weight(s) between 10 and 30 kd, and was absorbed out by passage through affinity columns containing anti-interferon gamma beads. CONCLUSION: We conclude that recurrent abortion in some women is associated with embryo- and/or trophoblast-toxic factor production in response to stimulation by sperm or trophoblast antigens and that the principal factor may involve the 18 kd, heat-labile, T-lymphocyte cytokine interferon gamma. This study suggests a new cause of recurrent abortion.     

Regulation of peri-attachment embryo development in the golden hamster: role of growth factors.

The molecular regulation of mammalian peri-implantation development is complex and difficult to study in vivo. We successfully cultured hamster blastocysts through hatching and peri-attachment stages, using a chemically defined medium, HECM-2h. Using this system, we showed that a species-specific, embryonic cysteine-like protease is involved in blastocyst hatching and that the process is modulated by growth factors. In particular, heparin binding-epidermal growth factor (HB-EGF) or leukemia inhibitory factor (LIF) enhance blastocyst hatching, and the former also improves attachment and trophoblast outgrowth. We observed interesting changing patterns of expression of mRNA and/or immunoreactive protein for EGF, HB-EGF, LIF and transforming growth factor-beta (TGF-beta) in the embryo and/or endometrial tissue, during peri-implantation development. Together, it appears that hamster blastocyst hatching, attachment and trophoblast outgrowth are regulated by autocrine and/or paracrine growth factors, produced by the embryo-endometrial tissues.     

Immunopotentiation reverses the embryotoxic effect of serum from women with pregnancy loss.

OBJECTIVE: To assess the effect of sera of women with habitual abortions (AB) on attachment and spreading of mouse blastocysts in vitro. DESIGN: Expansion, attachment, and spreading were the mouse blastocyst parameters utilized. Deoxyribonucleic acid (DNA) synthesis and cell markers expression were also assayed by autoradiography analysis and the indirect immunofluorescent technique. SETTING: Sera were drawn from patients attending a habitual AB clinic in a tertiary care university hospital. PARTICIPANTS: Thirty-nine serum samples were drawn from habitually aborting women and the effect compared with 17 control AB sera. INTERVENTION: Habitually aborting women were immunized with paternal leucocytes; 18 post-immunization sera were also assessed. OUTCOME AND RESULTS: After 48 hours, there was delayed attachment and spreading (4% of test blastocysts spread as compared with 50.5% of controls). This was more profound after 72 hours culture (7.5% spread as compared with 72.8% of controls). Experimental sera were capable of reducing DNA synthesis, cytokeratin, fibronectin, or placental alkaline phosphatase expression by blastocyst cells. Leucocyte immunization of women with habitual ABs, clearly reversed the embryotoxic effect of the sera and enhanced cell markers expression. CONCLUSIONS: These data suggest that immunopotentiation may improve blastocyst survival in utero.     

The effects of serum from women with miscarriages on the in vitro development of mouse preimplantation embryos

It is generally believed that some human miscarriages result from embryotoxic factors existing in the sera. To study the embryotoxicity of such sera, 3.5-day-old mouse blastocysts were cultured for 72 h on 80% sera from different groups of women. After 72 h there was no blastocystic development in 53.2% of the cases grown on sera from women after two or more miscarriages, and none in 33.6% of the blastocysts grown on sera from women after one miscarriage, as compared with 8.2% and 12% respectively on control sera. Sera from women with miscarriages were divided into 'high risk' (50% or more embryotoxicity) and 'low risk' (less than 50% embryotoxicity) sera. The 'high risk' sera from two or more miscarriages caused an average of 72.1% undevelopment, while the 'low risk' sera (less than 50% embryotoxicity) from the same group caused 33.6% undevelopment. The 'high risk' sera from one miscarriage were embryotoxic to 55.8% of the blastocysts and the 'low risk' sera from the same group caused only 8.7% undevelopment. No significant differences were found in the mean serum concentrations of folic acid, zinc and copper of many of the experimental groups, in comparison with controls. The embryotoxic factor/s which exist in the 'high risk' sera from women with miscarriages are still not known.     

Leptin Expression in Primary Trophoblast Cells in Response to Incubation with the Serum of Preeclamptic Women

Objective. We investigated whether the increase of leptin expression in preeclamptic placentas is additionally influenced by soluble maternal factors under hypoxic and nonhypoxic conditions. Methods. Term trophoblast cells were isolated and stimulated with sera from preeclamptic women under normoxic or hypoxic conditions. Levels of leptin mRNA and protein were evaluated by real-time RT-PCR or ELISA and Western blot analysis. Results. Leptin concentrations were increased in the serum of patients with preeclampsia and gestational diabetes. Hypoxia, insulin, and dexamethasone induced leptin expression in trophoblast cells. The incubation with sera from preeclamptic women led to a small, though, significant, increase of leptin gene expression. The effect of preeclamptic serum on leptin gene expression in trophoblast cells was lost under hypoxia. The serum of women with gestational diabetes did not increase leptin expression neither in normoxic nor hypoxic primary trophoblast cells. Conclusion. Our results can not exclude a soluble maternal factor in the serum of women with preeclampsia accounting for increased leptin expression in placental tissue in addition to hypoxia. However, an important biological role of this small increase in nonhypoxic conditions does not seem very likely.     

Screening test for mouse blastocysts as an index of the vitality of embryos

Summary We attempted to investigate the viability of mouse blastocysts in vitro, comparing with embryo transfer to recipient mice. Despite normal appearances of the blastocysts, the rate of trophoblastic outgrowth of blastocysts fertilized in vitro was lower than that of blastocysts fertilized in vivo. However, the rate of transferred blastocysts developing into fetuses did not differ significantly between in vivo and in vitro fertilized blastocysts. It is considered that blastocyst hatching, attachment, and trophoblastic outgrowth in vitro can serve as indices for the vitality of individual embryos.     

A search for antibodies in term maternal sera to solubilized syncytiotrophoblast surface components. A passive haemagglutination assay yields negative evidence

Using a passive haemagglutination assay, agglutinins against trophoblast components were detected in term maternal sera at titres above those of non-pregnant control sera. These agglutinin titres were independent of the family relationship between individual sera and trophoblast samples. They were unaffected by adsorption with excess packed fetal or paternal red blood cells or paternal lymphocytes. The agglutinins of both normal control and term maternal sera were found to elute in the macroglobulin fraction of serum having a molecular weight of greater than 350 kDa and were shown to be a component of euglobulin precipitates. The titres of the agglutinins were unaffected by adsorption using solid phase antibodies against human IgM and IgG from the sera. It was concluded that these agglutinins were not a maternal antibody response against trophoblast surface components.     

不育、习惯性流产妇女血清对2－细胞鼠胚体外生长的影响

本文报道２０例原发不育妇女、２５例习惯性流产妇女以及１１例正常生育妇女血清作ＢＷＷ培养基添加剂分别对昆明小白鼠的８１３个、１０８５个和３８１个２—细胞鼠胚进行体外培养的比较研究。结果显示：各组７２ｈ囊胚形成百分率分别为４８．７０±１３．５０％，３７．３２±２０．３５％和５９．０５±７．３３％。不育组、习惯性流产组囊胚形成率与正常生育组囊胚形成百分率经统计学处理分别为Ｐ＜０．０５，Ｐ＜０．０２，均有显著性差异。这些结果提示：部分不育与习惯性流产妇女血清中可能存在有抑制早期胚胎发育的因子，因此检测血清中鼠胚生长抑制因子，有助于分析免疫性妊娠失败的病因。     

sLe~X抗体对小鼠胚泡在E-选择素上黏附和扩展的影响

目的:探讨E-选择素及其配体在着床中的作用。方法:建立小鼠胚泡的体外培养模型,在倒置相差显微镜下观察不同条件下小鼠胚泡在细胞外基质E-选择素上黏附、扩展的改变。结果:小鼠胚泡能在一定浓度E-选择素上黏附、扩展,E-选择素对胚泡的脱透明带有促进作用。结论:确定了E-选择素体外实验的最佳作用浓度。小鼠胚泡在特定浓度的E-选择素上能黏附、扩展,但这种作用能被sLe~X 抗体所阻断,并呈浓度依赖性。     

Quantitative analysis of the spreading of the mouse trophoblastin vitro: a model for early invasion

The outgrowth of the mouse blastocyst in culture represents an in vitro model of trophoblastic invasion. In the present study weanalysed trophoblast spreading by time lapse video microscopy. Trophoblast spreading consists of (1) the migration and (2) the giant cell transformation of trophoblast cells, (3) the proliferation of ectoplacental cone (EPC) cells and (4) the subsequent transformation of EPC cells into the secondary giant cells. During migration, ruffling of the trophoblast cell membrane is followed by the formation of lamellipodia. The mean surface areas of the spreading trophoblast, measured in more than 100 cultured blastocysts, increased linearly from 48 to 96 h of culture, while the linear migratory speed at the periphery of the outgrowth declined as the time of culture advanced. The EPC cells increased in size approximately eightfold during the giant cell transformation. The apparent nuclear: cytoplasmic ratios, i.e., ratios between the size of nucleus and that of the cytoplasm, measured as the surface areas on the photomicrographs, of EPC cells increased between 40–46 h of culture, but a sharp decline in the ratio occurred between 50 and 51 h of culture, reflecting either the sudden and tremendous increase in the cellular volume and/or spreading of the cytoplasm. The rates of trophoblast spreading varied considerably among the blastocysts of different genetic constitution examined (ICR, C57BL/6, C3H/He and (B6 × C3)F1. It was fastest in blastocysts obtained from matings of males and females of (B6 × C3)F1, and slowest in the C57BL/6 embryos. The differences in the rate of outgrowth observed may not simply be ascribed to difference in the developmental speed of the early embryos, because the rate of outgrowth reached a plateau at about 96–120 h and no ‘catch-up’ was observed by leaving the blastocysts in culture longer. Our results strongly suggest the possible presence of genetic regulatory mechanisms underlying trophoblast outgrowth; further analysis of the phenomenon may provide clues to understand the molecular mechanisms of trophoblastic invasion during the early phase of implantation, hopefully leading to improved success rates of in vitro fertilization-embryo transfer.     

Antibody reactivity against trophoblast and trophoblast products

Sensitive immunoassays have been applied to WHO reference bank sera from fertile and infertile women in order to assess any naturally occurring antibody reactive with isolated human placental trophoblast membranes or two separate trophoblast protein products (hCG and SP1). A very low incidence of antibody reactive with solubilised trophoblast membrane was detected, and no significant antibody to either hCG or SP1 could be detected. Infertile states represented within this serum bank appear unlikely to involve adverse immune reactions to trophoblast.     

Human NK cell activity is not inhibited by pregnancy and cord serum factors and female steroid hormones in vitro

The effects of female sex steroid hormones and of pregnancy and cord sera on human NK cell activity against the NK sensitive K562 cells were studied. The cytotoxicity of adult peripheral blood lymphocytes was measured after incubation of peripheral blood effector cells in vitro with estrone, 17 beta-estradiol, estriol, or progesterone at concentrations ranging from 1 to 1000 nM. The cytotoxic activity of effector cells was not altered by pre-treatment of cells with steroid hormones, and the presence of the steroids in the NK assays caused minor changes only at the highest concentrations. Similarly, the effects of sera obtained from pregnant women during the third trimester and from cord blood were studied. Compared to normal pooled AB serum, pregnancy and cord sera had no effect on NK cell activity. On the contrary, the presence of fetal calf serum in the culture medium consistently resulted in a higher NK activity than that obtained with AB serum, cord serum, pregnancy serum, or the medium alone. The augmenting effect of human leukocyte interferon on NK cell activity was not inhibited by these sera. It is concluded that female sex steroid hormones and pregnancy and cord sera are not inhibitory in vitro to normal human NK cells.     

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.     

Effects of human sera and human serum albumin on mouse embryo culture

Human proteins normally used to supplement human in vitro fertilization—embryo transfer (IVF-ET) culture media were tested for their effects on mouse embryo development from the zygote stage. These proteins included follicular and luteal-phase maternal sera, fetal cord sera, and both human and bovine serum albumin. Our results revealed that both maternal and fetal cord sera did not permit mouse blastocyst formation. Furthermore, predialysis of the human maternal sera and removal of IgG by protein A column chromatography did not improve their support of mouse embryonic development to the blastocyst stage. Similar detrimental effects were observed with maternal sera from term-pregnant IVF-ET patients. Interestingly, these serum samples had supported the in vitro growth of the human zygotes which resulted in these patients' pregnancies. Only some batches of human serum albumin supported mouse blastocyst formation, whereas all sources of bovine serum albumin were effective in this regard. These results raise the question of the suitability of the mouse embryo culture system as a quality control for the testing of protein supplements for human IVF-ET.     

小鼠囊胚在子宫内膜体外共培养模型上着床的超微结构观察

目的:进一步证明已建立的体外着床模型的可行性。方法:将妊娠d4的小鼠囊胚培养在已建立的体外子宫内膜共培养模型上,电镜观察胚胎植入时囊胚与子宫内膜上皮细胞相互关系的超微结构。结果:小鼠囊胚能正常脱透明带、黏附和扩展。扫描电镜见黏附在子宫内膜上的囊胚呈椭圆形或扁平形;胚胎表面具微绒毛;在胚胎与子宫内膜细胞接触点可见胚胎滋养细胞黏附于胞饮突上。透射电镜观察显示,胚胎滋养细胞通过微绒毛黏附于上皮细胞上,有些滋养细胞通过微绒毛开始植入,局部滋养细胞与上皮细胞形成紧密连接。胚胎滋养细胞与上皮细胞间形成许多镶嵌连接。超微结构显示粗面内质网、线粒体、溶酶体和核糖体非常丰富。结论:小鼠囊胚在子宫内膜体外模型上生长发育良好,该胚胎与子宫内膜共培养系统是研究胚胎着床机理的理想体外模型。     

Evaluation of serum-associated embryotoxicity in women with reproductive disorders

Purpose Our purpose was to determine whether some cases of infertility may be due to serological factors inhibiting development of the embryo.Method We examined the effect of infertile women's sera on the expansion, attachment, and spreading of mouse blastocysts in culture. Cell marker expression was also assayed by an indirect immunofluorescence technique. Serum samples from 75 infertile women were compared to the effect of 24 control AB sera.Results After 72 hr, blastocyst spreading was significantly different depending on whether cultured in sera from women with unexplained infertility, anovulatory infertility, diethylstilbesterol exposure or controls. Neither sera from women with mechanical infertility (14) nor sera from women with endometriosis (8) affected blastocyst growth in culture.Conclusions Inhibitory sera were capable of reducing cytokeratin expression but had no effect on placental alkaline phosphatase or concanavalin A expression by blastocyst cells. It can be inferred that the inhibitory effect of sera from women with certain types of infertility might be due to damage to the cytoskeleton. This in vitro assay may predict the success or failure of IVF.     

Efficient blastomere biopsy for mouse embryo splitting for future applications in human assisted reproduction

The objective of the current study was to establish a safe, efficient biopsy procedure for embryo splitting using the mouse model for future applications in human assisted reproduction. From mouse embryos at the 2-, 4-, 6- and 8-cell stage, half the number of blastomeres were microsurgically biopsied and transferred into empty mouse zonae pellucidae. Twin embryonic development was monitored during in-vitro culture. Blastocyst developmental rate using 2-, 4-, 6-, and 8-cell splitting was 74.4, 75.0, 66.7 and 38.4 respectively, with corresponding hatching rates of 94.9, 97.5, 92.7 and 83.8%. Blastocysts from 2-, 4-, and 6-cell splitting resulted in elevated hatching rates compared with non-operated blastocysts (87.5%), due to the Tyrode-assisted hatching effect. Blastocyst morphology was superior from 2- and 4-cell splitting when compared with 6- and 8-cell splitting. Furthermore, outgrowth of twin blastocysts from 2- and 4-cell splitting showed well-developed colonies with trophoblast cells and clusters of ICM cells, whereas those obtained from 6- and 8-cell splitting frequently formed small-sized colonies. Due to the high twinning success rate obtained under the experimental conditions employed in this study, it appears that with further modifications and proper safeguards, such embryo splitting efforts could have potential applications in humans.     

ELISA test for the detection of an immunological blocking factor in human pregnancy serum

We have previously shown that progesterone-treated lymphocytes of healthy pregnant women can produce a 34 kDa protein, progesterone-induced blocking factor (PIBF) capable of blocking lymphocyte function in vitro. Lymphocytes of women with idiopathic threatened pre-term delivery failed to produce this factor. On the assumption that the PIBF appears in the serum of healthy pregnant women but not in that of women at risk for immunologically based abortion of pre-term delivery, an ELISA has been designed for detecting the blocking factor in serum samples. Sera obtained at the time of delivery, as well as those from women with pre-term deliveries or miscarriages, contained significantly less PIBF than sera of 209 healthy pregnant women. Data obtained by testing sera of women at 16 weeks gestation whose pregnancies ended in spontaneous abortion showed that the predictive value of the test depended on the time interval between blood sampling and the onset of abortion. In 11 of 13 women with uterine contractions the outcome of pregnancy was predictable by normal or lower than normal levels of PIBF in the sera taken at the time the symptoms were presented. The data suggest that determination of PIBF in pregnancy serum might contribute to the diagnosis of immunologically bases pre-term disruption of pregnancy.