miR-146a-5p-mediated suppression on trophoblast cell progression and epithelial-mesenchymal transition in preeclampsia

OBJECTIVE: This study aims to identify the effect of miR-146a-5p on trophoblast cell invasion as well as the mechanism in preeclampsia (PE). METHODS: Expression levels of miR-146a-5p and Wnt2 in preeclamptic and normal placentae were quantified. Trophoblast cells (HTR-8) were separately transfected with miR-146a-5p mimic, miR-146a-5p inhibitor, pcDNA3.1-Wnt2 or sh-Wnt2, and then the expression levels of miR-146a-5p, Wnt2, and epithelial-mesenchymal transition (EMT)-related proteins (Vimentin, N-cadherin and E-cadherin) were measured. Moreover, the proliferative, migratory and invasive capacities of trophoblast cells were detected, respectively. Dual luciferase reporter assay determined the binding of miR-146a-5p and Wnt2. RESULTS: Compared with normal placental tissues, the placentae from PE patients showed higher miR-146a-5p expression and lower Wnt2 expression. Transfection of miR-146a-5p inhibitor or pcDNA3.1-Wnt2 exerted pro-migratory and pro-invasive effects on HTR-8 cells and encouraged EMT in HTR-8 cells; transfection with miR-146a-5p mimic or sh-Wnt2 weakened the proliferative, migratory and invasive capacities as well as reduced EMT process of HTR-8 cells. Moreover, Wnt2 overexpression could partially counteract the suppressive effects of miR-146a-5p overexpression on the progression and EMT of HTR-8 cells. CONCLUSION: miR-146a-5p mediates trophoblast cell proliferation and invasion through regulating Wnt2 expression.

Pré-eclâmpsia: o que há de anômalo na placentação? / Preeclampsia: what is wrong with placentation

A pré-eclâmpsia (PE) constitui a principal causa de morte materna em diversos países do mundo e contribui significativamente para a prematuridade, o baixo peso fetal e o aumento da mortalidade neonatal. A placenta constitui o substrato anatômico etiopatogênico principal para a doença, inclusive em ambiente extra-uterino ou na ausência de embrião. O único tratamento efetivo para a PE consiste na interrupção da gravidez e remoção completa da placenta. Em muitos casos, esta medida precisa ser tomada prematuramente, visando garantir a vida da mãe, do bebê ou de ambos. Estudos clínicos, histológicos e laboratoriais demonstram alterações hemodinâmicas, histológicas, imunológicas e bioquímicas na placentação de mulheres portadoras de PE. Entender como essas alterações assumem proporções sistêmicas no organismo materno pode ser a chave para impedir a progressão abrupta e violenta da doença. Certamente, o entendimento de todo o processo fisiopatológico é necessário para qualquer proposta de predição, prevenção ou terapia que possa diminuir as altíssimas taxas de mortalidade atribuídas à PE.

Preeclampsia (PE) is the leading cause of maternal death in many countries worldwide and contributes significantly to prematurity, low fetal weight and increased neonatal mortality. The placenta seems to be the main etiopathogenic anatomical substrate for the disease even in extra-uterine environment or in the absence of the embryo. The only effective treatment for PE is the pregnancy interruption and complete placenta removal. In many cases, this action needs to be taken prematurely in order to ensure the life of the mother, baby or both. Clinical, histological and laboratory have shown hemodynamic, histological, immunological and biochemical abnormalities in placentation in women with PE. Understanding how these changes take on systemic proportions in the mother may be the key to prevent the abrupt progression of the disease. Indeed, an understanding of all physiological and the alterations in PE process is required for any action of prediction, prevention or therapy that can reduce the extremely high rates of mortality associated to PE.

Genotyping analysis of circulating fetal cells reveals high frequency of vanishing twin following transfer of multiple embryos

Detection of Circulating Fetal Trophoblastic Cells [CFTC] by single cell genotyping not only allows to identify fetal cells from maternal blood, but also to characterize their bi-parental genome. We have tested intact fetal trophoblastes recovered at 4th to 10th weeks of gestation [WG] from blood [10 ml per mother] of 13 women after In Vitro Fertilization [IVF] and transfer of one or several embryos. Large cells isolated from blood were individually microdissected and studied by genetic fingerprinting with a mean number of 3 Short Tandem Repeats [STR] markers, known to be informative by testing paternal and maternal blood DNA. CFTC were found in all mothers starting from the 5th WG. A mean number of 2.5 CFTC per ml of blood was found in all the analyzed samples collected at the different terms of pregnancy. All mothers who received the transfer of two or three embryos, including one who delivered twins and one with vanishing twin [identified by ultrasounds], were found to have CFTC with two or three different bi-parental genotypes, belonging to different embryos derived from the same parents. CFTC circulation is detectable starting from the 5th WG. A "vanishing twin" phenomenon frequently develops after IVF and transfer of multiple embryos, being undetectable by ultrasounds and revealed by genetic CFTC fingerprinting

Histochemical study of early embryo implantation in rats / Estudio histoquímico de la implantación temprana en ratas

Embryo implantation is the process that results in attachment of the conceptus to the uterine wall. In this histochemical study, we investigated the early stage of embryo implantation in rats by morphological analysis and by the detection of total proteins and glycosaminoglycans using hematoxylin-eosin, toluidine blue at pH 4.0 (TB), and Xylidine Ponceau at pH 2.5 (XP). In non-pregnant females, the uterine layers could be clearly distinguished and showed the normal histology of the organ. In pregnant females, an increase in the number of cells and a reduction in the interstitial space were observed in the endometrium close to the implantation sites. The blastocyst was partially inserted in the endometrium, with the observation of the inner cells mass around the blastocyst cavity surrounded by trophoblastic cells. TB staining revealed mild metachromatic basophilia, which was more evident in the endometrial stroma around the implantation site. Histochemical staining with XP was also more intense in the stroma close to the site of implantation. On the other hand, histochemical staining with either TB or XP was more discrete at sites distant from the conceptus. This study demonstrated changes in the endometrial stroma in areas adjacent to the site of embryo implantation, with variations in glycosaminoglycans and proteins as demonstrated by the detection of anionic and cationic radicals, respectively.

La implantación embrionaria es el proceso que resulta en la unión del embrión a la pared uterina. En este estudio histoquímico, se determinó la fase inicial de implantación del embrión en ratas mediante el análisis morfológico y por la detección de proteínas totales y glicosaminoglicanos con hematoxilina-eosina, azul de toluidina a pH 4,0 (TB) y xilidina Ponceau a un pH de 2,5 (XP). En las hembras no preñadadas, las capas del útero pueden ser claramente distinguidas y mostraron la histología normal del órgano. En las hembras preñadas, se observa un incremento en el número de células y una reducción en el espacio intersticial del endometrio para cerrar los sitios de implantación. El blastocisto se implanta parcialmente en el endometrio, con presencia de masa de células internas en torno a la cavidad del blastocisto, rodeado por las células trofoblásticas. La tinción TB reveló leve basofilia metacromática, lo cual fue más evidente en el estroma endometrial alrededor del sitio de implantación. Tinción histoquímica con XP también fue más intensa próximo del estroma en el sitio de implantación. Por otro lado, la tinción histoquímica, ya sea con la tuberculosis o XP fue más leve en los lugares distantes del embrión. Este estudio demostró cambios en el estroma del endometrio en las zonas adyacentes al sitio de la implantación del embrión, con variaciones en glucosaminoglicanos y proteínas, como lo demuestra la detección de radicales aniónicos y catiónicos, respectivamente.

Co-localization and interaction of human organic anion transporter 4 with caveolin-1 in primary cultured human placental trophoblasts

The human organic anion transporter 4 (hOAT4) has been identified as the fourth isoform of OAT family. hOAT4 contributes to move several negatively charged organic compounds between cells and their extracellular milieu. The functional characteristics and regulatory mechanisms of hOAT4 remain to be elucidated. It is well known that caveolin plays a role in modulating proteins having some biological functions. To address this issue, we investigated the co-localization and interaction between hOAT4 and caveolin-1. hOAT4 and caveolin-1 (mRNA and protein expression) were observed in cultured human placental trophoblasts isolated from placenta. The confocal microscopy of immuno-cytochemistry using primary cultured human trophoblasts showed hOAT4 and caveolin-1 were co-localized at the plasma membrane of the cell. This finding was confirmed by Western blot analysis using isolated caveolae-enriched membrane fractions and immune-precipitates from the trophoblasts. When synthesized cRNA of hOAT4 along with scrambled- or antisense-oligodeoxynucleotide (ODN) of Xenopus caveolin-1 were co-injected to Xenopus oocytes, the [3H]estrone sulfate uptake was significantly decreased by the co-injection of antisense ODN but not by scrambled ODN. These findings suggest that hOAT4 and caveolin-1 share a cellular expression in the plasma membrane and caveolin-1 up-regulates the organic anionic compound uptake by hOAT4 under the normal physiological condition.

Blastocyst-endometrium interaction: intertwining a cytokine network

The successful implantation of the blastocyst depends on adequate interactions between the embryo and the uterus. The development of the embryo begins with the fertilized ovum, a single totipotent cell which undergoes mitosis and gives rise to a multicellular structure named blastocyst. At the same time, increasing concentrations of ovarian steroid hormones initiate a complex signaling cascade that stimulates the differentiation of endometrial stromal cells to decidual cells, preparing the uterus to lodge the embryo. Studies in humans and in other mammals have shown that cytokines and growth factors are produced by the pre-implantation embryo and cells of the reproductive tract; however, the interactions between these factors that converge for successful implantation are not well understood. This review focuses on the actions of interleukin-1, leukemia inhibitory factor, epidermal growth factor, heparin-binding epidermal growth factor, and vascular endothelial growth factor, and on the network of their interactions leading to early embryo development, peri-implantatory endometrial changes, embryo implantation and trophoblast differentiation. We also propose therapeutical approaches based on current knowledge on cytokine interactions.

Effect of (Ala8,13,18)-magainin II amide on human trophoblast cells in vitro.

Magainins are cationic peptides with anti-bacterial and anti-tumor properties. The anti-nidatory function of a synthetic analogue of magainin, (Ala8,13,18)-magainin II amide, has earlier been reported, and it has been indicated that placental trophoblast cells could be a target of magainin resulting in its contragestational action. The aim of the present study was to examine the effect of (Ala8,13,18)-magainin II amide (100 ng/ml and 1000 ng/ml) on attachment efficiency, viability, differentiation in terms of hCG secretion and invasive function of isolated first trimester, human placental trophoblast cells grown on rat-tail collagen type-I matrix in primary cell culture. In the present experimental model, magainin was not found to affect human trophoblast cell functions in vitro.

Differential Expressions of Fas and Fas Ligand in Human Placenta

To investigate the expressions of Fas and Fas ligand (FasL) in human placenta, we studied the expressions of Fas and FasL in placenta with RT-PCR, immunoblotting and immunostaining. We observed amplified products of Fas and FasL transcripts, the band of Fas (52 kDa) and multiple bands of FasL (42-52 kDa) in pla-centa. Fas and FasL localized mainly on fetal vessels and on syncytiotrophoblasts respectively. The differential distribution of Fas and FasL in human placenta may reflect intrinsic expressions of them by trophoblasts during differentiation. The increased expression of Fas in trophoblasts may promote apoptosis of placenta in pathologic condition such as preeclampsia.

Comportamiento clínico y seguimiento de la enfermedad del trofoblasto gestacional abril-1996-dicembre-1999 en el Hospital Bertha Calderón

El presente estudio es de tipo descriptivo decorte longitudinal realizado en el período de abril de 1996 a diciembre de 1999. Se estudiaron 118 pacientes que se ingresaron al servicio de complicaciones I y Oncología: fueron ingresadas por presentar cualquier entidad clínica de la Enfermedad Trofoblastica gestacional; a estas pacientes se les dió seguimiento durante el tiempo que duró el estudio en el área de consulta externa del Hospital Berta Calderón. Se analizó cada uno de los casos tomando como parámetros, el abordaje clínico y de laboratorio. La enfermedad de Trofoblasto afecta especialmente a las mujeres jóvenes en edad reproductiva, menor de 25 años no existiendo diferencia respecto a la aparición de la enfermedad con la gestación. En su mayoría consultaron por sangrado transvaginal asociado con dolor bajo vientre o hiperemesis gravídica. Acudieron con el útero de mayor tamaño de acuerdo a su edad gestacional. El 77 porciento de las pacientes fueron diagnósticadas por ultrasonido pélvico. La cuantificación de la Hormona Gonodotropina Coriónica Humana en el 64 porciento de los casos no fue realizada; siendo la causa principal el factor económico. El 97 porciento de los casos se evacuó por legrado por aspiración eléctrico, confirmándose histopatológicamente como Mola Hidatidiforme completa en el 86 porciento de los casos 2.5 como coriocarcino, 1.6 porciento como Mola Parcial y Mola Invasiva respectivamente. En cuanto a la estadificación de riesgo se encontró 7 casos de bajo riesgo, y 3 de alto riesgo. Se trato a las pacientes de bajo riesgo con mono quimioterapia (Methodrexate o Actinomicina D). Los altos riesgos 3 caso; 2 de ellos se trataron con poli quimoterapia (MAC o EMACO), que además se asoció con histerectomía abdominal, una por sangrado profuso y otra por que el foco tumoral In útero. El tercero caso se le indico la poli quimoterapia, pero no se le aplicó por malas condiciones de la pacientes y falleció antes de aplicarse el tratamiento. Se considera que el tratamiento indicado fue satisfactorio en las pacientes que acudieron a su consulta y que los niveles de Hormona Gondotropina Coriónica Humana llegaron a niveles normales. El 54 porciento de las pacientes estudiadas acudieron por lo menos a un control de seguimiento en la consulta externa; el 6 porciento de éstos acudieron a sus 8 controles...

Fragmentos de vellosidades coriónicas en muestras cervicales / Villous chorionic fragments in cervix samples

Extendidos cervicales de tres casos de postparto y un caso de postaborto fueron procesados con la técnica de Papanicolaou. El método de Estrepto-Avidina-Biotina se aplicó para detectar Gonofotrofina coriónica. En los cuatro casos fueron observados estructuras tridimensionales, ramificadas, con una zona central amorfa cubierta por células aplanadas de núcleo delgado, interpretadas como fragmentos de vellosidades coriónicas. En uno de los casos estos fragmentos estaban acompañados por células deciduales, en otro por células multinucleadas interpretadas como sincicio.trofoblasto, mientras que otra paciente presentaba células con características de citotrofoblasto. En coincidencia con las referencias bibliográficas, no se detectó Gonadotrofina coriónica en ninguno de los casos estudiados. el reconocimiento de fragmentos de vellosidades coriónicas podría facilitar la identificación de células placentarias atípicas que pueden ser confundidas con células displásicas o malignas, un error reconocido en este tipo de materiales

Collagenase-IV in human trophoblast invasion and differentiation.

Trophoblast cells are unique with respect to their functions and responsibilities. These cells demonstrate three sequential phenotypes, proliferation and invasion into the endometrium, differentiation to form syncytia and endocrine secretions. Equipped with these properties placental trophoblasts are endowed with a variety of functions, like implantation of the blastocyst to the endometrium, providing nutrition to the developing embryo and also transmitting extraordinary array of signals for the embryonic development. Experimental evidences and logical extrapolation suggest that these functions are precisely controlled by growth factors, cytokines and hormones produced either by the trophoblast themselves or by the utero-placental unit. Any error in this control mechanism has extremely adverse consequences. The cells also synthesize a large number of enzymes, amongst which collagenase type IV secretion is involved in digestion of underlying basement membrane necessary for the process of invasion. Our results implicate the enzyme in the functional differentiation of the trophoblast as well. Inhibitors to this enzyme inhibit trophoblast differentiation as monitored by secretion of hCG and progesterone, the two markers of trophoblastic differentiation. In contrast, BeWo cells, a choriocarcinoma cell line which does not differentiate spontaneously, undergo increased proliferation when challenged with EGF. The results indicate the possibility of invasive and differentiative phenotypes to be coupled. Exact molecular involvements in this coupling process are looked into.

Effects of dipyrone on the placenta of albino rats: morphological and biochemical aspects

The effect of dipyrone (N-2,3-dimethyl-5-oxo-1-phenyl-3-pyrazolin-4-yl)-methyllamino methanesulfonate sodium monohydrate) on the placenta of 2 BAW albino rats was studied through Karyometry of trophoblastic giant cells and DNA, RNA and total protein determinations. The animals received a single daily dose of 50 mg/Kg body weight during different periods of pregnancy: from the 9th to the 12th, 11th to the 14th, 13th to the 16th, 15th to the 18th and 17th to the 20 thday. Control animals received a single daily dose of 0.5ml distilled water at the same time. Karyometric results showed a statistically significant increase in nuclear volumes of placental cells of rats receiving dipyrone during the first three periods, when compared to control groups. In the two groups that received the drug nearer to term there was no significant difference. Regarding DNA, RNA and total protein determinations, there was a statistically significant difference, for all of them, in the rats that received the drug from the 9th to the 12 th day of pregnancy when compared to the control group. There was no significant difference in the groups that received the drug after that period. The results show that dipyrone had a blocking effect on cell division and that this effect happens mainly in the initial period of placental development