Twin pregnancies, crown-rump length and birthweight discordancy: the influence of chorionicity / Gestações gemelares, comprimento craniocaudal e discordância de peso ao nascimento: a influência da corionicidade

Abstract Objective The purpose of the present study was to analyze the influence of chorionicity in the biometric parameters crown-rump length (CRL), birthweight (BW), crown-rump length discordancy (CRLD) and birthweight discordancy (BWD), determine the correlation between these latter two in cases of intertwin discordancy, and to analyze the influence of chronicity in the presence of these discordancies with clinical relevance (> 10% and > 15%, respectively). Methods The present study was a retrospective study based on the twin pregnancy database of the Centro Hospitalar S. João (2010-2015), including 486 fetuses among 66 monochorionic (MC) and 177 dichorionic gestations (DC). The inclusion criteria were multiple pregnancies with 2 fetuses and healthy twin gestations. The exclusion criteria were trichorionic gestations and pregnancies with inconclusive chorionicity, multiple pregnancy with ≥ 3 fetuses and pathological twin gestations. Results No statistically significant difference was found in BW (p = 0.09) and in its discordancy (p = 0.06) nor in CRL (p = 0.48) and its discordancy (p = 0.74) between MCs and DCs. Crown-rump length discordancy and birthweight discordancy were correlated by the regression line "BWD = 0.8864 x CRLD + 0.0743," with r2 = 0.1599. Crown-rump length discordancy > 10% was found in 7.58% of monochorionic and in 13.56% of dichorionic twins. Birthweight discordancy > 15% was detected in 16.67% of monochorionic and in 31.64% of dichorionic twins. Conclusion No statistically significant influence of chorionicity was identified in both birthweight and birthweight discordancy, as in crown-rump length and crown-rump length discordancy. Birthweight discordancy was correlated to crown-rump length discordancy in 20% of cases.

Resumo Objetivo O objetivo do presente estudo foi analisar a influência da corionicidade nos parâmetros biométricos comprimento craniocaudal, peso ao nascimento, discordância de comprimento craniocaudal e discordância de peso ao nascimento, determinar a correlação entre estes dois últimos caso haja discordância intergemelar e analisar a influência da corionicidade na presença destas discordâncias com relevância clínica (> 10% e > 15%, respectivamente). Métodos O presente estudo foi um estudo retrospectivo baseado na base de dados de gestações gemelares do Centro Hospitalar S. João (2010-2015), incluindo 486 fetos de 66 gestações monocoriônicas e 177 dicoriônicas. Os critérios de inclusão foram gestações múltiplas de 2 fetos e gestações gemelares saudáveis. Os critérios de exclusão foram gestações tricoriônicas ou de corionicidade inconclusiva, gestações múltiplas com ≥ 3 fetos e gestações gemelares patológicas. Resultados Não se encontrou diferença estatisticamente significativa no peso ao nascimento (p =0,09) e sua discordância (p = 0,06) nem no comprimento craniocaudal (p = 0,48) e sua discordância (p = 0,74) entre gestações monocoriônicas e dicoriônicas. Considerando todas as gestações, as discordâncias de comprimento craniocaudal e peso ao nascimento foram correlacionadas pela reta de regressão "discordância de peso ao nascimento = 0.8864 x discordância de comprimento craniocaudal + 0.0743," com r2 = 0,1599. A discordância de comprimento craniocaudal > 10% descobriu-se em 7.58% das gestações monocoriônicas e em 13.56% das dicoriônicas. A discordância de peso ao nascimento > 15% detectou-se em 16.67% das gestações monocoriônicas e em 31.64% das dicoriônicas. Conclusão Não se identificou influência estatisticamente significativa no peso ao nascimento e sua discordância, bem como no comprimento craniocaudal e sua discordância. A discordância de peso ao nascimento correlacionou-se com a discordância de comprimento craniocaudal em 20% dos casos.

An investigational study of a dual-layer, chorion-free amnion patch as a protective barrier following lumbar laminectomy in a sheep model.

The inherent properties of the human amniotic membrane (HAM) suggest its potential for use as a physical barrier during surgery to protect neural elements and vessels from the surrounding environment. The objective of this study was to evaluate the effect of a dual-layer, chorion-free amnion patch (DLAM; ViaShield®, Globus Medical Inc., Audubon, PA, USA) processed from HAM as a protective barrier following lumbar laminectomy in a sheep model. A multiplex immunoassay was performed to quantify the inherent cytokines present in the amnion after processing. Twelve skeletally mature female crossbred Suffolk sheep were randomly divided into two equal post-operative periods (4 and 10 weeks). Each sheep underwent a laminectomy at L3 and L5, and one of the surgical sites randomly received the DLAM treatment. At each postsurgical time point, the extent of epidural fibrosis and neurohistopathological responses at the laminectomy sites was assessed based on epidural fibrosis-dura tenacity scores and decalcified histology, respectively. Immunoassay results showed that inflammatory mediators and immunomodulatory cytokines were present in the amnion after processing, but no proangiogenic cytokines were detected. At 10 weeks, tissue tenacity was significantly less in the DLAM treatment group when compared with the operative control (1.2 ± 0.4 vs. 2.8 ± 0.4, p < 0.05), demonstrating the ability of DLAM to act as a barrier and cover the dura. Gross observations showed fewer fibroblasts in the DLAM group in comparison with the control at both post-operative time points. Fibroblast infiltration analysis indicated that at both 4 and 10 weeks, there were significantly more infiltrated fibroblasts in the operative control sites than in the DLAM-treated sites, expressed as a percentage of the total number of fibroblasts present (4 weeks: 72.3 ± 10.2% vs. 10.8 ± 10.1%, p < .05; 10 weeks: 84.9 ± 15.8% vs. 43.1 ± 11.6%, p < .05). Additionally, fibroblasts travelled further into the dura in the operative control group compared with the DLAM-treated group at both time points. In conclusion, this study found that DLAM reduced fibroblast infiltration and tissue tenacity following lumbar laminectomy in a sheep animal model. These findings support the potential use of DLAM in clinical practice as a protective barrier for neural elements and anterior vessels.

Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro.

The endometrium extracellular matrix (ECM) is essential for embryo implantation. Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and forms large ECM aggregates, can influence fundamental physiological phenomena, such as cell proliferation, adhesion and migration. The present study investigated the possible role of versican in human embryo implantation. Versican V1 expression and secretion in human endometrial epithelial cells (EECs) was most prominent in the mid-secretory phase. Versican expression in EECs significantly increased after treatment with estrogen and progesterone, but not by estrogen alone. We also established versican V1-overexpressing Ishikawa (endometrial cancer cell line) cells (ISKW-V1), versican V3-overexpressing (ISKW-V3) and control GFP-overexpressing (ISKW-GFP) Ishikawa cells. By the in vitro implantation model, the attachment ratio of BeWo (choriocarcinoma cell line) spheroids to the monolayer of ISKW-V1, but not of ISKW-V3, was found significantly enhanced compared with attachment to the ISKW-GFP monolayer. The conditioned medium derived from ISKW-V1 (V1-CM) also promoted the attachment of BeWo spheroids to the ISKW monolayer. However, this attachment-promoting effect was abolished when V1-CM was pretreated with chondroitinase ABC, which degrades chondroitin sulfate. Therefore, out of the ECM components, versican V1 may facilitate human embryo implantation.

Differences in the Expression of TLR-2, NOD2, and NF-κB in Placenta Between Twins.

Dizygotic twins share the same type of genetic relationship as non-twin siblings. Whereas monozygotic (MZ) twins are considered to have identical genetic material, they still differ. There is a number of reasons for early MZ twin discordance, including differences in the in utero environment, stochasticity, genetic mosaicism, and epigenetic factors. During gestation, the efficient innate immune system is of utmost importance. Our study was based on immunohistochemical evaluation of the differences in innate immune protein expression (TLR-2, NOD2, and NF-κB) in the 95 placentas between twins. Our study revealed statistical significant differences between diamniotic-dichorionic and monoamniotic-dichorionic twins. Monoamniotic-monochorionic twins exhibited no significant differences in protein expressions. To identify epigenetic factors causing the differences between twins, we made a series of comparisons with clinical data. The study revealed more cases with infections, miscarriages, in vitro fertilization, and premature rupture of membranes within the group with higher differences level of NF-κB, NOD2 and TLR-2 between twins. In case of twin-to-twin transfusion syndrome, there were no significant differences in innate immune protein expressions between twins. These results show that dissimilar genetic material and separate in utero environment promote discordance in innate immune protein expressions between twins. Moreover, additional blood flow between twins may be favorable in life-threatening conditions ensuring similar microenvironment.

Placental weight and birth weight to placental weight ratio in monochorionic and dichorionic growth-restricted and non-growth-restricted twins

OBJECTIVE: The aim of the present study was to compare the placental weight and birth weight/placental weight ratio for intrauterine growth-restricted and non-intrauterine growth-restricted monochorionic and dichorionic twins. METHODS: This was a retrospective analysis of placentas from twin pregnancies. Placental weight and the birth weight/placental weight ratio were compared in intrauterine growth-restricted and non-intrauterine growth-restricted monochorionic and dichorionic twins. The association between cord insertion type and placental lesions in intrauterine growth-restricted and non-intrauterine growth-restricted monochorionic and dichorionic twins was also investigated. RESULTS: A total of 105 monochorionic (intrauterine growth restriction=40; non-intrauterine growth restriction=65) and 219 dichorionic (intrauterine growth restriction=57; non-intrauterine growth restriction=162) placentas were analyzed. A significantly lower placental weight was observed in intrauterine growth-restricted monochorionic (p=0.022) and dichorionic (p<0.001) twins compared to non-intrauterine growth-restricted twins. There was no difference in the birth weight/placental weight ratio between the intrauterine growth restriction and non-intrauterine growth restriction groups for either monochorionic (p=0.36) or dichorionic (p=0.68) twins. Placental weight and the birth weight/placental weight ratio were not associated with cord insertion type or with placental lesions. CONCLUSION: Low placental weight, and consequently reduced functional mass, appears to be involved in fetal growth restriction in monochorionic and dichorionic twins. The mechanism by which low placental weight influences the birth weight/placental weight ratio in intrauterine growth-restricted monochorionic and dichorionic twins needs to be determined in larger prospective studies.

Understanding the Impact of Preservation Methods on the Integrity and Functionality of Placental Allografts.

INTRODUCTION: Human placental membranes (hPMs) have a long history in treating burns and wounds. The composition of hPMs includes structural matrix, growth factors, and neonatal cells, all of which contribute to their regenerative potential. However, most hPM products are devitalized after dehydration and irradiation. We compared the functionality of single-layer viable cryopreserved human amniotic membrane (vCHAM) with multilayer devitalized dehydrated human amnion/chorion membrane (dHACM) in wound-relevant models to determine the effect of different processing methods on hPMs. METHODS: Viable cryopreserved human amniotic membrane and dHACM were compared with fresh hPM for structural integrity and viability. Viable cell persistence in vCHAM over time was evaluated in vitro and in vivo in a diabetic chronic wound mouse model. Proliferation of cells within fresh hPM and vCHAM was evaluated with bromodeoxyuridine and Ki-67 staining, and proliferation of isolated cells in culture was evaluated. Growth factor release over time and in vitro response to chronic wound stimuli (tumor necrosis factor α, lipopolysaccharide, and hypoxia) were used to compare the functionality of vCHAM and dHACM. RESULTS: The structure and thickness of fresh hPM were retained in vCHAM but were compromised in dHACM. Similar to fresh hPM, vCHAM contained viable cells, whereas dHACM did not. Cells in vCHAM remained viable after 4 and 7 days in culture and in an in vitro chronic wound environment and after 4 and 8 days in vivo after application to a mouse chronic wound. Staining for bromodeoxyuridine and Ki-67 did not reveal proliferative cells within fresh hPM and vCHAM. However, isolated cells proliferated in culture. Viable cryopreserved human amniotic membrane increased platelet-derived growth factor BB, hepatocyte growth factor, and epidermal growth factor levels over time and responded to chronic wound stimuli in vitro by significantly increasing levels of vascular endothelial growth factor and prostaglandin E2. Dehydrated human amnion/chorion membrane showed no significant accumulation of growth factors and did not respond to chronic wound stimuli. CONCLUSIONS: These results indicate that vCHAM retains intact, native matrix, and viable, active cells and responds to chronic wound stimuli in vitro. The inclusion of multiple layers of hPM does not compensate for structural degradation and loss of viability caused by dehydration as evidenced by a lack of functional response by dHACM. The clinical significance of these results remains to be answered.

The expression of neurogenic markers after neuronal induction of chorion-derived mesenchymal stromal cells.

OBJECTIVES: Chorion is a tissue of early embryologic period that is discarded after delivery. It might be the potential source of mesenchymal stromal cells (MSCs) that can be used for research and eventually for therapeutic studies. At present, the biological properties and the differentiation capacity of chorion-derived MSCs are still poorly characterised. The objective of this study is to characterise and explore the differentiating potential of chorion-derived MSCs towards the neuronal lineages. METHODS: Chorionic membrane was digested with enzyme and cultured in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum. The expression of MSC markers was examined using flow cytometry. The adipogenic, osteogenic and neurogenic differentiation were examined by culturing in appropriate induction media. The expression of neuronal markers was determined by immunofluorescence and quantitative real time-PCR. RESULTS: Chorion-derived MSCs were easily expanded up to 20 passages. They were positive for MSC markers (CD73, CD90 and CD105), and negative for haematopoietic markers (CD34 and CD45). Chorion-derived MSCs could differentiate into several mesodermal-lineages including adipocytes and osteoblasts. Moreover, chorion-derived MSCs could differentiate into neuronal-like cells as characterised by cell morphology and the presence of neural markers including MAP-2, glial fibrillary acidic protein (GFAP) and beta-tubulin III. DISCUSSION: Chorion-derived MSCs can be readily obtained and expanded in culture. These cells also have transdifferentiation capacity as evidenced by their neuronal differentiation potential. Therefore, chorion can be used as an alternative source of MSCs for stem cell therapy in nervous system disorders.

Side population cells in the human decidua of early pregnancy exhibit stem/progenitor cell-like characteristics.

It is proposed that human decidua contains a population of stem cells that are responsible for the proliferation ability during the process of embryo implantation and placenta formation and that factors in the crosstalk between the decidua and chorion may mediate decidual stem cell differentiation. This study analysed the phenotype of side population (SP) cells and investigated the clonogenicity and differentiation ability of SP cells in human decidua of early pregnancy. Serum-free culture-conditioned media of human decidua and chorion were obtained from decidua and chorion explant culture. Decidual SP cells were isolated by fluorescence-activated cell sorting. Different inducing media were added and the functional differentiation of decidual SP cells was examined. Decidual SP cells were negative for the mature decidual cell marker CD13 and prolactin and negative for CD34 and CD45 expression. Decidual SP cells formed clones after culture in colony-forming medium and they could form clones again. Differentiated cells expressing CD13 and prolactin were observed and stroma-like structures expressing CD13 were obtained. These results indicate that decidual SP cells are enriched for stem cell activity. Oestradiol, progesterone and factors in culture-conditioned media of human decidua and chorion induced their proliferation and differentiation.

Chorionic enhancer is dispensable for regulated expression of the human renin gene.

We tested the hypothesis that a transcriptional chorionic enhancer (CE), previously identified to increase human renin expression in choriodecidual cells is required to mediate tissue-specific, cell-specific, and regulated expression of human renin in transgenic mice. Recombineering was used to delete the CE upstream of the renin gene alone or in combination with the kidney enhancer (KE) in a large artificial chromosome construct containing the entire human renin gene and extensive flanking sequences. Deletion of the CE had no qualitative or quantitative effect on the tissue-specific expression of human renin, nor on the cellular localization of human renin in the kidney or placenta. Combined deletion of both the CE and KE caused a decrease in the level of renal renin expression consistent with the established role of the KE. We also considered the possibility that the CE is a downstream enhancer of the KiSS1 gene, which lies directly upstream of renin and is also expressed in the placenta. Deletion of the CE alone, or the CE and KE together, had no effect on the level of KiSS1 expression in the placenta. These data provide convincing evidence that the CE is silent in vivo, at least in the mouse. The absence of a phenotype caused by deletion of the CE is consistent with the observation that the sequence is not evolutionarily conserved.

Modeling trophoblast differentiation using equine chorionic girdle vesicles.

The chorionic girdle of the equine conceptus is comprised of specialized trophoblast cells which, at day 36-38 of equine pregnancy, gain an invasive phenotype and invade the endometrium to form endometrial cups. Studies of equine endometrial cups remain difficult to perform because of the invasive techniques required to obtain cup tissue and because sampling requires termination of the pregnancy. In this study we developed a system to model trophoblast differentiation and trophoblast-immune interactions in vitro and in vivo. We utilized a method of culturing chorionic girdle pieces in serum-free medium to promote spontaneous formation of vesicle structures enriched for terminally differentiated binucleate cells that secreted equine chorionic gonadotrophin (eCG). Immunohistochemical staining and scanning electron microscopy showed that the cells of the vesicles closely resembled the outer layers of chorionic girdle immediately prior to invasion. Chorionic girdle vesicles were harvested after 72h in culture and ectopically transplanted via injection into the vulvar mucosa of recipient mares. At 7, 14, 21 and 28days after transplantation, biopsies of the injection sites were obtained. Immunohistochemical labeling of cryostat sections of the biopsies with a panel of monoclonal antibodies to horse trophoblast molecules demonstrated survival, differentiation, and presence of trophoblast cells for at least 21days. Serial sections of the biopsies labeled with antibodies to the equine lymphocyte surface markers CD4 and CD8, together with lymphocyte microcytotoxicity assays, revealed that the recipients mounted both cellular and humoral antibody immune responses to the transplanted trophoblast cells. This new method for culturing equine chorionic girdle trophoblast cells, and for transplanting trophoblast vesicles to ectopic sites, should allow identification of key aspects of trophoblast differentiation and the interactions that occur between invasive trophoblast and the maternal immune system.

Hematopoietic cells.

Murine embryonic stem cells (mESC) readily form embryoid bodies (EBs) that exhibit hematopoietic differentiation. Methods based on EB formation or ESC coculture with murine bone marrow stromal cell lines have revealed pathways of both primitive and definitive hematopoietic differentiation progressing from primitive mesoderm via hemangioblasts to endothelium and hematopoietic stem and progenitor cells. The addition of specific hematopoietic growth factors and morphogens to these cultures enhances the generation of neutrophils, macrophages, megakaryocyte/platelets, and hemoglobinized mature red cells. In addition, selective culture systems have been developed to support differentiation into mature T lymphocytes, natural killer cells, B cells, and dendritic cells. In most cases, culture systems have been developed that support equivalent differentiation of various human ESC (hESC). The major obstacle to translation of ESC hematopoietic cultures to clinical relevance has been the general inability to produce hematopoietic stem cells (HSC) that can engraft adult, irradiated recipients. In this context, the pattern of ES hematopoietic development mirrors the yolk sac phase of hematopoiesis that precedes the appearance of engraftable HSC in the aorta-gonad-mesonephros region. Genetic manipulation of mESC hematopoietic progeny by upregulation of HOXB4 or STAT5 has led to greatly enhanced long- or short-term multilineage hematopoietic engraftment, suggesting that genetic or epigenetic manipulation of these pathways may lead to functional HSC generation from hESC.

The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential.

The chorio-allantoic placenta forms through the fusion of the allantois (progenitor tissue of the umbilical cord), with the chorionic plate. The murine placenta contains high levels of hematopoietic stem cells, and is therefore a stem cell niche. However, it is not known whether the placenta is a site of hematopoietic cell emergence, or whether hematopoietic cells originate from other sites in the conceptus and then colonize the placenta. Here, we show that the allantois and chorion, isolated prior to the establishment of circulation, have the potential to give rise to myeloid and definitive erythroid cells following explant culture. We further show that the hematopoietic potential of the allantois and chorion does not require their union, indicating that it is an intrinsic property of these tissues. These results suggest that the placenta is not only a niche for, but also a source of, hematopoietic cells.

Oligomannurarate sulfate, a novel heparanase inhibitor simultaneously targeting basic fibroblast growth factor, combats tumor angiogenesis and metastasis.

Inhibitors of tumor angiogenesis and metastasis are increasingly emerging as promising agents for cancer therapy. Recently, heparanase inhibitors have offered a new avenue for such work because heparanase is thought to be critically involved in the metastatic and angiogenic potentials of tumor cells. Here, we report that oligomannurarate sulfate (JG3), a novel marine-derived oligosaccharide, acts as a heparanase inhibitor. Our results revealed that JG3 significantly inhibited tumor angiogenesis and metastasis, both in vitro and in vivo, by combating heparanase activity via binding to the KKDC and QPLK domains of the heparanase molecule. The JG3-heparanase interaction was competitively inhibited by low molecular weight heparin (4,000 Da) but not by other glycosaminoglycans. In addition, JG3 abolished heparanase-driven invasion, inhibited the release of heparan sulfate-sequestered basic fibroblast growth factor (bFGF) from the extracellular matrix, and repressed subsequent angiogenesis. Moreover, JG3 inactivated bFGF-induced bFGF receptor and extracellular signal-regulated kinase 1/2 phosphorylation and blocked bFGF-triggered angiogenic events by directly binding to bFGF. Thus, JG3 seems to inhibit both major heparanase activities by simultaneously acting as a substrate mimetic and as a competitive inhibitor of heparan sulfate. These findings suggest that JG3 should be considered as a promising candidate agent for cancer therapy.

Cadmium accumulation in zebrafish (Danio rerio) eggs is modulated by dissolved organic matter (DOM).

Experiments were conducted to investigate factors influencing the accumulation of cadmium (Cd(2+)) into zebrafish (Danio rerio) eggs. The accumulation of (109)Cd was affected by: (1) concentration, (2) time, (3) presence of dissolved organic material (DOM), (4) different origin of DOM and (5) different parts of fish eggs. Over a 5-h exposure, zebrafish eggs showed a steady increase in Cd-accumulation. DOM-concentrations over 15ppm carbon (C) decreased Cd-uptake significantly. Both samples of DOM, brown water marsh (LM) and a eutrophic pond (SP), at 16.9ppmC, reduced the Cd-accumulation in the chorion, perivitelline liquid and the embryo. Cd was mainly accumulated in the egg's outer shell chorion (61%) and only small amounts passed through the chorion into the perivitelline liquid (38%) and embryo (1%). In the presence of LM-DOM, the accumulation of Cd into the egg components was decreased by 43% (chorion), 52% (perivitelline liquid) and 52% (embryo), respectively, compared with the control group. Similarly, the presence of SP-DOM reduced the Cd-accumulation by 29% (chorion), 61% (perivitelline liquid) and 60% (embryo), respectively, compared with the controls. DOM-concentration should be taken into consideration when determining ecotoxicological effects of Cd on fish populations.

Identification of placenta growth factor determinants for binding and activation of Flt-1 receptor.

Placenta growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family and represents a key regulator of angiogenic events in pathological conditions. PlGF exerts its biological function through the binding and activation of the seven immunoglobulin-like domain receptor Flt-1, also known as VEGFR-1. Here, we report the first detailed mutagenesis studies that provide a basis for understanding molecular recognition between PlGF-1 and Flt-1, highlighting some of the residues that are critical for receptor recognition. Mutagenesis analysis, performed on the basis of a structural model of interaction between PlGF and the minimal binding domain of Flt-1, has led to the identification of several PlGF-1 residues involved in Flt-1 recognition. The two negatively charged residues, Asp-72 and Glu-73, located in the beta3-beta4 loop, are critical for Flt-1 binding. Other mutations, which bring about a significant decrease in PlGF binding activity, are Gln-27, located in the N-terminal alpha-helix, and Pro-98 and Tyr-100 on the beta6 strand. The mutation of one of the two glycosylated residues of PlGF, Asn-84, generates a PlGF variant with reduced binding activity. This indicates that, unlike in VEGF, glycosylation plays an important role in Flt-1 binding. The double mutation of residues Asp-72 and Glu-73 generates a PlGF variant unable to bind and activate the receptor molecules on the cell surface. This variant failed to induce in vitro capillary-like tube formation of primary endothelial cells or neo-angiogenesis in an in vivo chorioallantoic membrane assay.

Expression of extracellular matrix metalloproteinase inducer in human placenta and fetal membranes at term labor.

Matrix metalloproteinases (MMPs) are the main mediators of extracellular matrix (ECM) degradation during human parturition. However, the mechanisms involved in regulation of MMP production during parturition remain poorly understood. Recently, an extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to play a key role, as a local regulator, in stimulating MMP production in cancer systems. Whether EMMPRIN is expressed and stimulates MMP production in human placenta and fetal membranes is presently unknown. In this study, we investigated the expression of EMMPRIN at the levels of mRNA and protein in human term placenta and fetal membranes with or without labor. Western blot analysis showed that EMMPRIN protein was detected in term placenta and fetal membranes at two molecular masses of 40 and 65 kDa (glycosylated protein) and one of approximately 30 kDa (nonglycosylated protein). The ratio of 65 kDa EMMPRIN to total EMMPRIN significantly increased (P < 0.05) in term labor chorio-decidua and amnion compared with nonlabor chorio-decidua and amnion. Immunohistochemical analysis revealed that EMMPRIN was expressed in placental syncytiotrophoblast, amniotic epithelial cells, trophoblast cells of chorion laeve, and decidua parietalis. EMMPRIN was also detected at the mRNA level using RT-PCR in cultured placental syncytiotrophoblast, amniotic epithelial cells, and chorionic trophoblast cells. We conclude that human placenta and fetal membranes express EMMPRIN, with the potential to stimulate MMP production, thereby facilitating fetal membrane rupture and leading to detachment of the placenta and fetal membranes from the maternal uterus at the time of parturition.

[Characterization of choriodecidual space as an effector molecule-rich environment that induces rupture of fetal membranes during labor]. / Caracterización del espacio coriodecidual como un microambiente rico en moléculas efectoras que inducen la rotura de las membranas corioamnióticas durante el trabajo de parto.

OBJECTIVE: To identify a specific microenvironment in direct contact with fetal membranes where effector molecules acumulate, aiming to degrade the components of its extracellular matrix during labor. TYPE OF STUDY: Experimental, analytic, longitudinal and prospective. MATERIALS AND METHODS: Blood samples were collected from maternal, fetal and choriodecidual compartments, and mononuclear cells were isolated. Part of these cells was stained with antibodies to determine leukocyte subpopulations by flow cytometry. The other part was cocultured for 12 h with amniochorion explants. After coculture, MMP-9 was identified on the mononuclear cells by immunofluorescence. IL-1beta and TNF-alpha were determined in the supernatants by ELISA. Three independent experiments were carried out with duplicates and analyzed with Mann-Whitney's U test. RESULTS: Although there were no significant differences in the mononuclear cell subpopulations from the three compartments, MMP-9 production was higher in choriodecidual cells than in those of the maternal and fetal compartments. Furthermore, IL-1beta and TNF-alpha were significantly more abundant in cocultures with choriodecidual cells compared with the other two compartments. CONCLUSIONS: During labor, choriodecidual cell subpopulations are not phenotypically different from those of the maternal or fetal compartments, but they are regarding MMP-9 production, which suggests that the environment surrounding chorioamniotic membranes enhances the synthesis of this enzyme, thus promoting degradation of connective tissue.

Developmental changes at the materno-embryonic interface in early pregnancy of the alpaca, Lamos pacos.

This study analyses the manner in which trophoblast cells adhere to uterine epithelium and the subsequent interactions that contribute to the establishment of epitheliochorial placentation in the alpaca Lama pacos. Specimens at the luteal and follicular phases and at 22, 26, 30 and 45 days of pregnancy (op) were processed for morphological studies. On day 15 op, the blastocysts are completely free within the uterine lumen, with implantation starting around day 20. On days 22 and 26 of gestation, the trophoblast is apposed to the epithelial surface of the uterus, with areas of contact and adhesion by means of complex interdigitation. Implantation sites occur prevalently in the left uterine horn, but an expanded trophoblast also occupies large extensions of the right horn, where the maternofetal interaction shows peculiar areas of apposition. As development continues, attachment areas become more extensive. On days 30 and 45, many secretory granules can be seen in the uterine epithelium, while giant multinucleate cells appear interposed between the remaining trophoblast cells, showing intense alkaline phosphatase activity, deposits containing iron and PAS-positive granules. Placental lactogen hormone is not present within the cytoplasm of the binucleate or multinucleate trophoblast cells. By day 30 of gestation, the trophoblast layer is lined by an extraembryonic connective tissue that by day 45 is well vascularized, thus indicating the starting point of placental formation. Fetal and maternal capillaries indent the epithelium and the trophoblast, narrowing the specialized areas of exchange, which occur along the entire maternofetal interface, characterizing the diffuse nature of this placenta.

Baicalein and baicalin are potent inhibitors of angiogenesis: Inhibition of endothelial cell proliferation, migration and differentiation.

In recent studies, we have shown that baicalein and baicalin, 2 major flavonoids of Scutellaria baicalensis Georgi, exhibit anticancer activity against several cancers in vitro. In our present study, we assessed their potential as anti-angiogenic agents in vivo employing chicken chorioallantoic membrane (CAM) assay and in vitro human umbilical vein endothelial cells (HUVECs) culture. When CAMs were treated with either baicalein or baicalin for 48 hr, the angiogenic response induced by basic fibroblast growth factor (bFGF) was markedly reduced in a dose-dependent manner. Further characterization showed that both flavonoids exhibited dual antiproliferative (at low dose) and apoptogenic (at high dose) effects on HUVECs. In biochemical analysis, treatment of HUVECs with baicalein and baicalin for 24 hr resulted in a dose-dependent decrease in the matrix metalloproteinase (MMP)-2 activity. Moreover, the migration of endothelial cells and the differentiation of endothelial cells into branching networks of tubular structures in vitro were also inhibited by these 2 flavonoids in a dose-dependent manner. Baicalein is more potent than baicalin in anti-angiogenesis in vivo as well as in vitro. Taken together, the results of our study provide evidence that baicalein and baicalin possess an anti-angiogenesis potential that is a previously unrecognized biologic activity.