Subplacental development in Galea spixii / Desenvolvimento subplacentário do preá (Galea spixii)

Animal models are essential to understand healthy human placentation. Guinea pig related rodents became on focus for such purposes. In particular, processes of trophoblast invasion are similar. The latter is associated with a specialized area, the subplacenta. Since previous results showed differences between the guinea pig and its close relative Galea spixii, we aimed to study subplacental development with more detail. We investigated 16 pregnant females of 14 to 55 days of gestation by means of histology, morphometrics, immunohistochemistry and electron microscopy. The overlap between the fetomaternal blood systems resulted as intimate, suggesting some exchange processes. Proliferation was revealed by three independent methods, being most active in early and mid-gestation, which was in accordance to former results. Though degeneration of tissues took place, the subplacenta was maintained towards term with access to the fetal vascularization, supporting a hypothesis about the release of substances to the fetal unit in advanced gestation. In contrast to other species, the extraplacental trophoblast showed a shift from syncytial streamers to giant cells during mid-gestation. Views on placentation in caviomorphs were influenced by the guinea pig, but our data supported recent studies that the subplacenta had a much greater placidity. In regard to subplacental grow, degeneration and likely also exchange processes, Galea and other species showed a more basal pattern of caviomorphs than the guinea pig. Such differences should be considered, when choosing most adequate animal models for special purposes in comparison to human placentation.(AU)

Modelos animais são essenciais para entender a placenta humana sadia. Neste sentido os roedores relacionados ao porquinho da índia tornaram-se foco para tal entendimento. Em particular, os processos de invasão trofoblástica são semelhantes. O último está associado a uma área especializada, a subplacenta. Uma vez que os resultados anteriores mostraram diferenças entre o porquinho da índia e seu relativo o preá, buscamos estudar o desenvolvimento subplacentário com mais detalhes. Pesquisamos 16 fêmeas gestantes de 14 a 55 dias de gestação por meio de histologia, morfometria, imuno-histoquímica e microscopia eletrônica. A sobreposição entre os sistemas sanguíneos materno e fetal apresentou-se com íntima relação, sugerindo alguns processos de troca. A proliferação foi revelada por três métodos independentes, sendo mais ativos no início e metade da gestação, o que corroborou com os resultados anteriores. Embora a degeneração dos tecidos tenha ocorrido, a subplacenta foi mantida até o termo gestacional com acesso à vascularização fetal, apoiando uma hipótese sobre a liberação de substâncias para a unidade fetal em gestação avançada. Em contraste com outras espécies, o trofoblasto extraplacentário mostrou uma mudança de flâmulas sinciciais para células gigantes durante a metade da gestação. As visualizações sobre a placentação em caviomorfos foram influenciadas pelo porquinho da índia, mas nossos dados apoiaram estudos recentes de que a subplacenta apresentava uma plasticidade muito maior. Em relação ao crescimento subplacentário, a degeneração e provavelmente também os processos de troca, o preá e outras espécies apresentaram um padrão mais basal de caviomorfos do que o porquinho da índia. Tais diferenças devem ser consideradas, ao escolher os modelos animais mais adequados para fins especiais em comparação com a placentação humana.(AU)

Effect of fetal-maternal immune tolerance on pregnancy-induced hypertension / 中国免疫学杂志

Objective:To analyze the levels of immune cytokine in peripheral blood(PB) and the expressions of immune related transcription factors in placenta cytotrophoblast,which in patients with pregnancy-induced hypertension(PIH).And to explore the effect of fetal-maternal immune tolerance on pregnancy-induced hypertension.Methods:50 cases patients with PIH were selected as the observation group,and 40 healthy people were chosen as the control group.Enzyme-linked immunoassay(ELISA) was performed to detect the levels of IFN-γ,IL-4,IL-17 and TGF-β in peripheral blood.Quantitative Real-time PCR(RT-qPCR) and Western blot were used to analyze the mRNA expressions and protein levels of T-bet,GATA-3,RORC and Foxp3 in placenta cytotrophoblast,respectively.Results:①Compared with the control group,the IFN-γ and IL-17 levels in peripheral blood in the observation group were obviously higher and the TGF-β level was greatly lower(P0.05),but the ratios of IFN-γ/ IL-4 and IL-17/TGF-β in the observation group were larger than that in the control group(P<0.05).②Compared with the control group,the mRNA expressios and protein levels of T-bet and RORC in the observation group were increased significantly(P<0.05),but that of GATA-3 and Foxp3 were decreased greatly(P<0.05).Thus,the ratios of T-bet/GATA-3 and RORC/Foxp3 in the observation group were significantly larger than that in the control group(P<0.05).Conclusion:PIH is occurred with the imbalance of Th1/Th2 and Th17/Treg,and it might be associated with the mechanism of fetal-maternal immune tolerance disorder that induced by Th cells.

Regulation of Growth and Function of Lifeline Placenta.

Placenta is an association of fetal and maternal tissues which develops during pregnancy. Placenta is often called lifeline, because it is the link between mother and growing fetus. It serves variety of functions, which include transport of nutrients to growing fetus, waste products from fetus, exchange of gases and also immunological protection to the fetus. It has a unique ability to function as a hypothalamo-pituitary-gonadal-axis as it can produce a variety of peptide, protein and steroid hormones. Thus, it is an autonomous unit capable of regulating its own growth and function.

The levels of NO decrease induced apoptosis in human placental trophoblast cells through oxidative stress / 中国药理学通报

Aim To investigate the possible mecha-nisms of the levels of NO decrease induced apoptosis in human placental trophoblast cells. Methods Human placental trophoblast cells ( HTR-8 ) were cultured in 5 ml DMEM-F12 culture medium with 37℃ 5% CO2 . Then, the old culture medium was discarded and re-placed with 10,100,500,1 000 μmol·L-1 L-NAME, and the group without L-NAME was set as the control group, cultured for 48h. The effects of L-NAME on the survival of cells were detected by methylthiazolyldiphe-nyl tetrazolium bromide ( MTT); the content of NO in cells was tested by nitrate reductive enzymatic;trans-mission electron microscopy, flow cytometry analysis and Annexin-V FITC dyeing were used to test the effects of L-NAME on apoptosis in HTR-8 cells;restore Fe3+ colorimetric assay was applied for detection of to-tal antioxidant capacity ( T-AOC ) , xanthine oxidase for detection of superoxide dismutase ( SOD) activity, and thiobarbituric acid colorimetry for determination of content of MDA. Results Compared with the control group, the survival rate of HTR-8 cells and the levels of NO in 100,500,1 000 μmol·L-1 L-NAME group were significantly reduced(P<0.05,P<0.01). Flow analysis and Annexin-V FITC staining showed that L-NAME could induce cell apoptosis in a dose-dependent manner. The number of cell apoptosis was negatively correlated with the content of NO ( r = -0.5210 ) in HTR-8 cells. Transmission electron microscopy results showed that compared with the control group, the ex-perimental group's cell nucleus shape was irregular, nuclear pyknosis in irregular shape, the chromatin ag-glutination or side the collection, mitochondrial swell-ing or enrichment, crest fracture or dissolved, even vanished, forming the vacuole, especially in 100 μmol ·L-1 L-NAME group, the apoptotic bodies obviously appeared. At the same time, T-AOC, SOD levels in HTR-8 cells decreased ( P <0.05 ) , and the MDA content increased ( P<0.05 ) . The number of cell ap-optosis was negatively correlated with the level of T-AOC ( r= -0.3212 ) , SOD ( r= -0.2779 ) in HTR-8 cells , while positively correlated with the content of MDA(r=0.2807). Conclusion Oxidative stress may play an important role in the levels of NO decrease in-duced apoptosis in human placental trophoblast cells.

Effect of Human Cytomegalovirus on Invasive Capability of Early Pregnant Extravillous Cytotrophoblasts / 华中科技大学学报（医学）（英德文版）

The effect of human cytomegalovirus (HCMV) on invasive capability of early pregnant extravillous cytotrophoblasts (EVTs) was investigated in vitro.Primary EVTs were obtained by complex phosphoesterasum digestion and gradient centrifugation from villous tissue aseptically taken from healthy pregnant women.Cytokeratin7 (CK7),vimentin (Vim) and c-erbB-2 were immunocytochemically detected to identify source of cells,and HCMVpp65 antigen was assayed to determine the infection state of primary EVTs by immunocytochemical staining.The EVTs were divided into two groups:control group and HCMV group,and the expression of c-erbB-2,matrix metalloproteinase-2 (MMP-2) and MMP-9 proteins was detected in two groups by immunocytochemistry and Western blotting.Enzymic activity changes of MMP-2 and MMP-9 were tested by gelatin zymography in primary EVTs infected with HCMV.The invasion of primary EVTs was detected by cell invasion assay in vitro after they were infected by HCMV.The cell source identification showed that the cells obtained were highly-pure primary EVTs,and primary EVTs could be infected by HCMV.Primary EVTs could express c-erbB-2,MMP-2 and MMP-9 proteins,and as compared with control group,the protein expression was decreased significantly in HCMV groups (P＜0.05).Primary EVTs could secrete active MMP-2 and MMP-9 in vitro,and the activity of two MMPs was decreased significantly in HCMV groups (P＜0.05).The in vitro cell invasion assay showed that the number of primary EVTs permeating Matrigel in HCMV group was decreased (P＜0.05).We are led to conclude that HCMV can infect primary EVTs and inhibit their invasion capability,suggesting that the impaired EVT's invasion capability might be related to the abnormal expression of c-erbB-2,MMP-2and MMP-9 proteins.

Effects of preeclampsia serum on activity and invasive ability of cultured cytotrophoblasts of first trimester of pregnancy / 上海交通大学学报（医学版）

Objective To investigate the effects of preeclampsia serum on activity and invasive ability of cultured cytotrophoblasts of first trimester of pregnancy. Methods Cytotrophoblasts of normal 6 to 8-week pregnancy were cultured by tissue explants adherent method,and were incubated with serum of women with normal pregnancy(normal group)and preeclampsia(preeclampsia group),respectively for 24 h.The activity of cytotrophoblasts was examined by CCK-8,invasive ability was determined by Transwell invasion assay,and expression of MMP-2,MMP-9 and PAI-1 mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR). Results The activity and invasive ability of cytotrophoblasts in preeclampsia group were lower than those in normal group(P<0.05,P<0.01).Compared with normal group,the expression of MMP-2 and MMP-9 mRNA of cytotrophoblasts was significantly lower(P<0.01),and the expression of PAI-1 mRNA was significantly higher(P<0.01).In both groups,the expression of MMP-2 mRNA was negatively related to that of PAI-1 mRNA(r=-0.985,P<0.01;r=-0.933,P<0.05),while there was no correlationship between the expression of MMP-9 mRNA and that of PAI-1 mRNA. Conclusion The preeclampsia serum may affect the invasive ability of cytotrophoblasts by regulating the expression of MMP-2,MMP-9 and PAI-1.

Effect of Leptin on Cytotrophoblast Proliferation and Invasion / 华中科技大学学报（医学）（英德文版）

The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated.Immunohistochemistry was used to determine the placental expression of leptin in first-trimester preg-nancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, inva-siveness and migration was assessed by MTT, Transwell invasion assay and migration assay respec-tively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that: (1) Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear mem-brane of cytotrophoblast; (2) Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly (P<0.01); (3) After culture with different concen-trations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls (P<0.05). It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotro-phoblast invasion and migration activity.

Efecto de los retinoides en la diferenciación del trofoblasto humano en cultivo / Effect of retinoids on the differentiation of human trophoblast culture

Objetivo: El presente estudio analiza el efecto de diferentes retinoides sobre la función y diferenciación celular del trofoblasto humano en cultivo. Material y métodos: Se realizó un estudio experimental in vitro, en donde se utilizaron vellosidades coriónicas y células de citotrofoblasto obtenidas de placentas de término de embarazos normales, que fueron cultivadas y estimuladas con concentraciones fisiológicas diferenciales de retinol (ROL), ácido 9-cis retinoico (CRET) y ácido all-trans retinoico (TRET). Se analizaron los cambios en la actividad de la metaloproteinasa-9 (MMP-9) y la metaloproteinasa-2 (MMP-2), además de documentar las condiciones óptimas para el cultivo. En una segunda fase, se estimularon células de citotrofoblasto (CTB) aisladas y se documentaron los cambios morfológicos de formación de sincicio in vitro al ser estimulados con los retinoides mencionados. Resultados: Los explantes de placenta respondieron en forma dosis dependiente, en la actividad de MMP-2 y MMP-9 cuando fueron incubados en presencia de ROL y en menor proporción cuando se incubaron con CRET y TRET. El CTB aislado y estimulado con ROL y CRET formó sincicio en un lapso de 48 horas, en tanto que los estimulados con TRET lo hicieron hasta el 4o. día de incubación. Conclusiones: La placenta y el trofoblasto aislado responden a los diferentes retinoides. La respuesta del CTB al ROL indica que el CTB cuenta con la maquinaria metabólica para transformar el retinol en CRET y TRET que tienen actividad biológica. El efecto de estos compuestos se pudiera expresar en la función de implantación y en la placenta en desarrollo.

Objective: In the current study we investigate the effect of different retinoids on the function and cellular differentiation of human cytotrophoblast (CTB) cells in culture. Material and Methods: Experimental in vitro study that analyzed explants of placenta from term pregnancy were cultured and stimulated with different physiological concentrations of retinol (ROL), 9-cis retinoic acid (CRET) and all-trans retinoic acid (TRET). The optimal conditions of CTB culture and changes in metalloproteinase-9 (MMP9) and metalloproteinase-2 (MMP2) secretion stimulated by retinoids were analyzed by zymography. Isolated CTB cells were stimulated with retinoids and morphological changes of in vitro syncytio formation were observed by light microscopy. Results: The explants of placenta had a dose-dependent increment in the activity of MMP-2 and MMP-9 when they were incubated in presence of ROL and in less proportion when they were incubated with CRET and TRET. For isolated CTB cells, CRET stimulates the differentiation to syncytio within 48 h of incubation, whereas in the absence of TRET differentiation started after the fourth day incubation. Conclusions: The placenta and isolated CTB cells have the capacity to respond to ROL, this fact suggest the presence of a metabolic pathway necessary to transform retinol to the biological active retinoids like CRET and TRET. The effect of these retinoids might be expressed in the function of implantation and developing placenta.

Effects of Magnesium sulfate on the expression of caspase-3 and caspase-9 of cytotrophoblasts under hypoxic condition / 대한산부인과학회잡지

OBJECTIVE: To determine the effect of magnesium sulfate (MgSO4) on the expression of caspase-3 and caspase-9 of cytotrophoblasts in vitro under normal and hypoxic condition as assessed immunoblot analyses. METHODS: Normal cytotrophoblasts were isolated from second trimester placentas and cultured in several physiologically relevant concentrations of MgSO4 under standard tissue culture condition (20% O2) or hypoxic condition (1-2% O2). Cytotrophoblasts apoptosis was estimated by TUNEL staining and by immunoblotting for Caspase-9 and Caspase-3. RESULTS: The apoptotic index was highest in cytotrophoblasts cultured under hypoxic conditions for 36 hour in the absence of MgSO4 and was showed decreasing tendency by the addition of MgSO4 under the same condition. The expression of Caspase-9 did not change under both standard condition and hypoxic condition with increasing MgSO4 concentrations, but the expression of Caspase-3 decreased under hypoxic condition with increasing of MgSO4 concentrations. CONCLUSION: MgSO4 might protect cytotrophoblasts from apoptosis under hypoxic condition.

Effects of Magnesium Sulfate on the Apoptosis and Invasion of Cytotrophoblasts under Hypoxic Condition / 대한산부인과학회잡지

OBJECTIVE: To determine the effect of magnesium sulfate (MgSO4) on the apoptosis and invasion of cytotrophoblasts in vitro under normal and hypoxic condition as assessed immunoblot analyses of Bcl-2/Bax, invasion assay and immunohistochemical staining of integrin alpha1. METHODS: Normal cytotrophoblasts were isolated from second trimester placentas and cultured in several physiologically relevant concentrations of MgSO4 under control tissue culture condition (20% O2) or hypoxic condition (1-2% O2). Apoptosis of cytotrophoblasts was estimated by immunoblotting for Bcl-2/Bax, invasiveness was estimated by invasion assay and immunohistochemical staining of Integrin alpha1. RESULTS: The expression of Bcl-2 did not change under standard condition, but it decreased under hypoxic condition with increasing of MgSO4 concentrations. The expression of Bax did not change under both standard condition and hypoxic condition with increasing MgSO4 concentrations. The invasiveness of cytotrophoblasts significantly decreased under both control and hypoxic conditions with increasing of MgSO4 concentrations. The expression of Integrin alpha1 immumohistochemical staining significantly decreased under control condition and showed decreasing tendency under hypoxic condition with increasing of MgSO4 concentrations. CONCLUSION: MgSO4 might induce cytotrophoblasts to the apoptosis and inhibit invasion of cytotrophoblasts under hypoxic condition.

Lipopolysaccharide regulates TNF-? expression in cytotrophoblasts / 中国免疫学杂志

Objective:To explore whether lipopolysaccharide regulate TNF-? expression in cytotrophoblasts.Methods:Cytotrophoblasts were obtained from the villous tissues of normal abortions and cultured in serum-free FD medium.Lipopolysaccharide was added into the culture at concentrations of 0,25,50,100 and 200 ng/ml respectively.Levels of TNF-? were detected with confocal immunofluorescence technique and enzyme-linked immunosorbent assay.Results:Both confocal immunofluorescence technique and enzyme-linked immunosorbent assay confirmed that lipopolysaccharide promoted TNF-? expression in cytotrophoblasts. TNF-? levels in the medium with LPS at the concentrations above were(179.95?35.58),(334.75?74.09),(390.36?79.14),(447.72?27.81) and(482.37?39.14) pg/ml,respectively.The differences of TNF-? levels in the medium had statistical meanings and the value of P was less than 0.01.Conclusion:Lipopolysaccharide can induce TNF-? expression in cytotrophoblasts.

In Vitro Culture of Human Endometrial and Cytotrophoblast Cells / 대한산부인과학회잡지

Implantation is a cascade of process which fertilized conceptal tissue move into maternalendometrium. Recently, with development of life science, the informations about humanimplantation process have been important topic with regard to the reproductive medicaltechnology. However, the precise mechanism and the factors involved in the process is notclearly understood, because the human experiments have been inhibited due to ethical andreligious views to the point. To study the human implantation process, we develop the modelto culture the human endometrium and trophoblast in vitro. The microscopic findings ofcultured monolayer of endometrial glandular epithelium and stroma in respond to co-culturedtrophoblast cells were observed. As results, trophoblasts didn't change the microscopicmorphology of endometrial glandular layer. However, trophoblast cells destructed the higherconcentration of epidermal growth factor in culture media. These results suggests thatepidermal growth factor may be a important factor for trophoblast invasion to stromal layerand trophoblast may invade the glandular layer by different mechanism to endometrial stroma.