Placental SARS-CoV-2 distribution correlates with level of tissue oxygenation in COVID-19-associated necrotizing histiocytic intervillositis/perivillous fibrin deposition.

INTRODUCTION: Recent evidence supports the - rare - occurrence of vertical transplacental SARS-CoV-2 transmission. We previously determined that placental expression of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor, and associated viral cell entry regulators is upregulated by hypoxia. In the present study, we utilized a clinically relevant model of SARS-CoV-2-associated chronic histiocytic intervillositis/massive perivillous fibrin deposition (CHIV/MPFVD) to test the hypothesis that placental hypoxia may facilitate placental SARS-CoV-2 infection. METHODS: We performed a comparative immunohistochemical and/or RNAscope in-situ hybridization analysis of carbonic anhydrase IX (CAIX, hypoxia marker), ACE2 and SARS-CoV-2 expression in free-floating versus fibrin-encased chorionic villi in a 20-weeks' gestation placenta with SARS-CoV-2-associated CHIV/MPVFD. RESULTS: The levels of CAIX and ACE2 immunoreactivity were significantly higher in trophoblastic cells of fibrin-encased villi than in those of free-floating villi, consistent with hypoxia-induced ACE2 upregulation. SARS-CoV-2 showed a similar preferential localization to trophoblastic cells of fibrin-encased villi. DISCUSSION: The localization of SARS-CoV-2 to hypoxic, fibrin-encased villi in this placenta with CHIV/MPVFD suggests placental infection and, therefore, transplacental SARS-CoV-2 transmission may be promoted by hypoxic conditions, mediated by ACE2 and similar hypoxia-sensitive viral cell entry mechanisms. Understanding of a causative link between placental hypoxia and SARS-CoV-2 transmittability may potentially lead to the development of alternative strategies for prevention of intrauterine COVID-19 transmission.

Characterization of the temporal, cell-specific and interferon-inducible patterns of indoleamine 2,3 dioxygenase 1 (IDO1) expression in the human placenta across gestation.

INTRODUCTION: The human placenta performs multiple functions necessary for successful pregnancy, but the metabolic pathways and molecular mechanisms responsible for regulating placental development and functions remain incompletely understood. Catabolism of the essential amino acid tryptophan has numerous critical roles in normal physiology, including inflammation. The kynurenine pathway, which accounts for ∼90% of tryptophan breakdown, is mediated by indoleamine 2,3 dioxygenase 1 (IDO1) in the placenta. In pregnant mice, alterations of IDO1 activity or expression result in fetal resorption and a preeclampsia-like phenotype. Decreased IDO1 expression at the maternal-fetal interface has also been linked to preeclampsia, in utero growth restriction and recurrent miscarriage in humans. These collective observations suggest essential role(s) for IDO1 in maintaining healthy pregnancy. Despite these important roles, the precise temporal, cell-specific and inflammatory cytokine-mediated patterns of IDO1 expression in the human placenta have not been thoroughly characterized across gestation. METHODS: Western blot and whole mount immunofluorescence (WMIF) were utilized to characterize and quantify basal and interferon (IFN)-inducible IDO1 expression in 1st trimester (7-13 weeks), 2nd trimester (14-22 weeks) and term (39-41 weeks) placental villi. RESULTS: IDO1 expression is activated in the human placenta between the 13th and 14th weeks of pregnancy, increases through the 2nd trimester and remains elevated at term. Constitutive IDO1 expression is restricted to placental endothelial cells. Interestingly, different types of IFNs have distinct effects on IDO1 expression in the human placenta. DISCUSSION: Our collective results are consistent with potential role(s) for IDO1 in the regulation of vascular functions in placental villi.

Placental endothelin-converting enzyme-1 is decreased in preeclampsia.

Endothelin-converting enzyme-1(ECE-1) is a key regulatory enzyme in the processing of endothelin-1 (ET-1). We quantified and localized ECE-1 in normal and preeclamptic placentas. Normal (n=6) and preeclamptic (n=6) placentas were serially sectioned for immunofluorescence (IF). Cell type specific markers identified endothelial, trophoblast, macrophage, smooth muscle, and fibroblast cells. Quantitative analyses were performed by western blot and ELISA. IF identified ECE-1 expression within the stroma and villous space. Cellular localization of ECE-1 was limited to endothelial membranes. There was significantly less ECE-1 in preeclamptic placentas, suggesting ECE-1 is important for proper regulation of ET-1 within the placenta.

Proteomics and bioinformatics analysis of altered protein expression in the placental villous tissue from early recurrent miscarriage patients.

INTRODUCTION: Recurrent miscarriage (RM) affects 5% of women, it has an adverse emotional impact on women. Because of the complexities of early development, the mechanism of recurrent miscarriage is still unclear. We hypothesized that abnormal placenta leads to early recurrent miscarriage (ERM). The aim of this study was to identify ERM associated factors in human placenta villous tissue using proteomics. Investigation of these differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying recurrent miscarriage (RM). METHODS: To gain more insight into mechanisms of recurrent miscarriage (RM), a comparative proteome profile of the human placenta villous tissue in normal and RM pregnancies was analyzed using iTRAQ technology and bioinformatics analysis used by Ingenuity Pathway Analysis (IPA) software. RESULTS: In this study, we employed an iTRAQ based proteomics analysis of four placental villous tissues from patients with early recurrent miscarriage (ERM) and four from normal pregnant women. Finally, we identified 2805 proteins and 79,998 peptides between patients with RM and normal matched group. Further analysis identified 314 differentially expressed proteins in placental villous tissue (≥1.3-fold, Student's t-test, p < 0.05); 209 proteins showed the increased expression while 105 proteins showed decreased expression. These 314 proteins were analyzed by Ingenuity Pathway Analysis (IPA) and were found to play important roles in the growth of embryo. Furthermore, network analysis show that Angiotensinogen (AGT), MAPK14 and Prothrombin (F2) are core factors in early embryonic development. We used another 8 independent samples (4 cases and 4 controls) to cross validation of the proteomic data. DISCUSSION: This study has identified several proteins that are associated with early development, these results may supply new insight into mechanisms behind recurrent miscarriage.

Increased placental phospholipase A2 gene expression and free F2-isoprostane levels in response to oxidative stress in preeclampsia.

Vasoactive eicosanoids such as thromboxane (TX) A2 and F2-isoprostanes (F2-isoPs) were shown to be increased in the preeclamptic placenta. Only one of the 64 possible isomers of F2-isoPs derived from the oxidation of arachidonic acid was investigated in the placenta so far. F2-isoPs are released from membrane phospholipids by phospholipases A2 (PLA2) and were shown to act on the TXA2 receptor (TBXA2R). However, the PLA2 deregulated in preeclampsia (PE) remains to be determined. In this study, we analyzed the concentrations of six isomers of F2-isoPs; 8-iso-PGF2α, 8-iso-15(R)-PGF2α, 15(R)-PGF2α, iPF2α-IV, iPF2α-VI, 5-iPF2α-VI and the concentrations of the stable metabolites of TXA2, TXB2, by high performance liquid chromatography coupled with tandem mass spectrometry in placentas of PE (n = 17) and normotensive (n = 15) pregnancies according to the biopsy site: peri-insertion or periphery. In the same biopsies, relative mRNA expression of PLA2G2A, PLA2G4A, PLA2G5, PLA2G7, the PLA2 receptor (PLA2R1), the TXA2 synthase and TBXAR2 were measured by quantitative RT-PCR. We observed similar concentrations of total F2-isoP isomers between groups whereas higher concentrations (>40%) of free F2-isoP were observed for all isomers (p ≤ 0.033) in PE than normotensive controls. As expected, we also observed higher placental concentrations of TXB2 in PE (p = 0.005). Interestingly, we concomitantly found higher mRNA expression of secretory PLA2G2A (p = 0.010), PLA2G5 (p = 0.038) and TBXA2R (p = 0.023) in PE than normotensive placentas. In sum, deregulated PLA2 could potentially be implicated in freeing F2-isoP which could participate in local hypertension observed in the PE placenta through the TX pathway.

Prenatal Diagnosis of Lysosomal Storage Disorders Using Chorionic Villi.

Prenatal enzymatic diagnosis for an array of lysosomal storage disorders (LSDs) can be performed accurately, provided that a confirmed diagnosis by biochemical/molecular study in the index case is available and a strict defined protocol, specific to each individual disorder is followed. The present chapter describes the protocols for reliable and accurate prenatal enzymatic diagnoses by fluorometric and spectrophotometric methods of lysosomal storage disorders: Gaucher, Fabry, Pompe, Niemann Pick A/B, Tay Sach, Sandhoff, GM1, Mucoplysaccharidoses, Wolman, Krabbe, Metachromatic leukodystrophy, and Batten diseases using uncultured chorionic villi samples. The biological reference intervals for enzyme levels in normal and affected fetuses are given for interpretation of prenatal results. It is imperative to establish normal reference interval in each laboratory to take into account the local environment, technical variations, and different ethnicities. Besides, enzyme activity in the fetus should be represented as percentage of the mean activity of enzyme of normal fetuses. The pitfalls and challenges in prenatal diagnosis as well as technical problems in performing enzyme assays are also discussed to help the reader in standardization and performing the assays for correct diagnosis.

Expression of PAPPA2 in human fetomaternal interface and involvement in trophoblast invasion and migration.

Pregnancy-associated plasma protein-A 2 (PAPPA2) is a placental-enriched gene that is important for normal human placentation and defects in the gene can cause complications in pregnancy. Yet the exact expression pattern and role of PAPPA2 in the human fetomaternal interface are not clear. In this study, in situ hybridization (ISH) and immunohistochemistry (IHC) were employed to examine the spatial and temporal expression of PAPPA2 in the human fetomaternal interface. IHC results exhibited wide expression of PAPPA2 in the fetomaternal interface, with placental syncytiatrophoblast (STB) and extravillous trophoblast (EVT) showing strong expression and the cytotrophoblast (CTB) showing weak expression of PAPPA2. These results were confirmed by ISH. Quantitative reverse transcription-polymerase chain reaction and western blot showed the elevation of PAPPA2 in first trimester EVT differentiation and term CTB spontaneous syncytialization. PAPPA2-siRNA transfection significantly depressed the invasion and migration ability of a trophoblast cell line (HTR8/SVneo) in a transwell migration and Matrigel invasion model compared to a negative control siRNA (P < 0.05), also revealing that matrix metalloproteinase 9 (MMP9) secretion is downregulated. This was confirmed using a human first trimester placental villi explant culture model. Our results reveal the spatial and temporal expression of PAPPA2 in the human fetomaternal interface and show the positive regulatory role of PAPPA2 in human trophoblast invasion and migration through the secretion of MMP9.

Inherited metabolic disorders: prenatal diagnosis of lysosomal storage disorders.

OBJECTIVE: To offer accurate prenatal diagnosis of lysosomal storage disorders in early pregnancy. METHOD: Prenatal enzymatic diagnoses of Gaucher, Fabry, Pompe, Niemann Pick A/B, Tay Sach, Sandoff, GM1, mucoplysaccharidoses, Wolman, Krabbe, Metachromatic leukodystrophy and Batten diseases were made in uncultured chorionic villi samples by fluorometric/spectrophotometric methods. RESULTS: Of 331 prenatal enzymatic diagnosis, 207 fetuses (67%) were normal and 124 (37%) were affected. The interpretation of affected, normal and carrier fetuses was done using their respective reference ranges as well as % enzyme activity of normal mean. The prenatal molecular confirmation was feasible in 43 biochemically diagnosed fetuses. Of the 207 normal reported fetuses, post natal enzymatic confirmation was done in 23 babies, clinical status of another 165 babies was assessed as unaffected via questionnaire on telephone and 19 were lost to follow-up. In affected pregnancies, 123 opted for termination of which 44 were confirmed enzymatically after abortion. A single false positive was determined to be a carrier by prenatal mutation analysis and carried to term. CONCLUSION: We recommend uncultured chorionic villi for reliable prenatal enzymatic diagnosis of various lysosomal storage disorders on account of the low rate of false positive (0.5%) and false negative (2.2%) results.

Altered ß1,6-GlcNAc and bisecting GlcNAc-branched N-glycan on integrin ß1 are associated with early spontaneous miscarriage in humans.

STUDY QUESTION: Do N-acetylglucosaminyltransferase (GnT-V) and N-acetylglucosaminyltransferase III (GnT-III) play an important role in early spontaneous miscarriage (ESM) in humans. SUMMARY ANSWER: The dynamic balance between GnT-V and GnT-III expression in chorionic villi differed between early normal pregnancy and ESM and was associated with altered ß1,6-N-acetylglucosamine (ß1,6-GlcNAc) and bisecting N-acetylglucosamine (bis-GlcNAc) branched N-glycans on integrin ß1. WHAT IS KNOWN ALREADY: GnT-V contributes to metastasis, while GnT-III is recognized as a metastasis suppressor. It has been reported that GnT-V contributes to placentation in the early phase of pregnancy, possibly regulating trophoblast invasion. However, the expressions of GnT-V and GnT-III in ESM have not been reported. STUDY DESIGN, SIZE, DURATION: Villous samples from 6 to 9 weeks of gestation were collected in the First Affiliated Hospital of Chongqing Medical University from May 2013 to September 2014 from 60 normal pregnant women undergoing elective termination of pregnancy and from 40 patients with a clinical diagnosis of ESM. PARTICIPANTS, MATERIALS, SETTING, METHODS: Quantitative PCR and western blots were used to examine the GnT-V and GnT-III mRNA (Mgat5 and Mgat3) and protein expression, respectively, of chorionic villi in both the ESM group and the normal group from week 6 to week 9. We used immunofluorescence and immunohistochemistry to detect the location of GnT-V and GnT-III. Lectin fluorescence and histochemistry were used to test the location of ß1,6-GlcNAc and bis-GlcNAc branching in the normal and ESM groups. To assess the functional capacity of GnT-V and GnT-III in the chorionic villi between the two groups, we used an enzyme-linked immunosorbent assay kit to measure the activity of these enzymes. Using co-precipitated integrin α5ß1 followed by phytohaemagglutinin (PHA)-L and PHA-E blotting, we investigated whether GnT-V and GnT-III could modify the N-glycosylation profile in terms of the ß1,6-GlcNAc and bis-GlcNAc structures in integrin α5ß1 during the first trimester in both groups. MAIN RESULTS AND THE ROLE OF CHANCE: In the normal group expression and activity of GnT-V and the concentration of its product, ß1,6-GlcNAc were higher at week 9 than at weeks 6, 7 and 8 (P < 0.05). In contrast, the expression and activity of GnT-III and the concentration of its product, bis-GlcNAc were higher at week 6 than at weeks 7, 8 and 9 (P < 0.05). Compared with the normal group, the ESM group exhibited a lower expression of GnT-V and ß1,6-GlcNAc (P < 0.05) and a higher expression of GnT-III and bis-GlcNAc (P < 0.05) with consistent changes in enzymatic activity. Immunofluorescence showed that GnT-V was located mainly in the cytoplasm of syncytiotrophoblasts (STBs) and chorionic villous cytotrophoblasts (CTBs), in both the ESM group and the normal group. ß1,6-GlcNAc N-glycan was mainly located outside of the STB and CTB layer in normal villi and was expressed only rarely in the ESM villi. GnT-III was expressed primarily in the cytoplasm of STBs and expressed only very weakly in the CTBs of normal villi, whereas it was highly expressed in both the STBs and CTBs in the ESM group. bis-GlcNAc was primarily located outside of the STBs in the normal villi, whereas it was expressed much more abundantly outside of both the STBs and CTBs in the ESM group at each week of gestation. Moreover, decreased ß1,6-GlcNAc-branched N-glycans and increased bis-GlcNAc-branched N-glycans on integrin ß1 (P < 0.05) were observed in the ESM group. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide a new insight for studying the mechanism of clinical ESM in humans and it might be valuable for the clinical diagnosis and treatment of ESM. LIMITATIONS, REASONS FOR CAUTION: The study lacks experiments in vitro to disclose the precise mechanism by which GnT-V and GnT-III regulate ESM. In some cases, degradation of the tissues after the miscarriage event cannot be ruled out. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the National Natural Science Foundation of China (31271546). The authors have no competing interests.

Caspase expression in placental terminal villi in spontaneous and induced pregnancy.

Placental levels of caspase 1, 8, and 10 were studied by the immunohistochemical method in full-term spontaneous and induced pregnancies. The levels of these caspases in the placental terminal villi were significantly higher in induced pregnancy. The changes were more marked after intracytoplasmatic injection of spermatozoa.

Elevated expression levels of matrix metalloproteinase-9 in placental villi and tissue inhibitor of metalloproteinase-2 in decidua are associated with prolonged bleeding after mifepristone-misoprostol medical abortion.

OBJECTIVE: To determine whether the expression levels of matrix metalloproteinases 2 and 9 (MMP-2 and -9) and tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and -2) in the villi and the decidua are associated with prolonged bleeding after medical abortion. DESIGN: Case-controlled study. SETTING: University hospital. PATIENT(S): Mifepristone-misoprostol medical abortion patients were divided into two groups (20 women each) based on the length of time (>14 or ≤14 days) of bleeding after the abortion. INTERVENTION(S): Discharged villi and deciduas were collected. MAIN OUTCOME MEASURE(S): The expression levels of MMP-2 and -9 and TIMP-1 and -2 in the villi and deciduas were assessed with semiquantitative immunohistochemistry. RESULT(S): The median semiquantitative immunohistochemistry staining index (SI) scores for MMP-9 expression in the villi were elevated in the bleeding group compared with the control group (median SI scores 0.31 and 0.03, respectively). TIMP-2 expression was elevated in the decidua in the bleeding group compared with the control group (median SI scores 1.00 and 0.20, respectively). No significant differences were observed between the two groups in the expression levels of MMP-2 in the villi or of MMP-2, MMP-9, or TIMP-1 or of the ratios of MMP-9/TIMP-1 or MMP-2/TIMP-2 in the decidua. CONCLUSION(S): Elevated expression levels of MMP-9 in the villi and of TIMP-2 in the decidua were associated with prolonged bleeding after medical abortion.

Phospholipase C gamma and ERK1/2 mitogen activated kinase pathways are differentially modulated by Trypanosoma cruzi during tissue invasion in human placenta.

Chagas' disease is caused by the haemophlagelated protozoan Trypanosoma cruzi (T. cruzi). During congenital transmission the parasite breaks down the placental barrier, however studies about the physiopathology of this process are scarce. Different signal transduction pathways are involved during cell invasion of the parasite. However, the possible role of those processes during tissue infection has not been studied. In the present study we analyzed the modulation of two signal transduction pathways, PLC-γ and ERK1/2 MAPK, during ex vivo infection of human placental chorionic villi explants. Chorionic villi from healthy woman placentas were incubated in the presence or absence of 10(5) or 10(6)T. cruzi trypomastigotes (DM28c strain) with or without specific inhibitors for each pathway. Effective infection was tested determining parasite DNA by PCR. The activation of PLC-γ and ERK1/2 MAPK signaling pathways was determined by western blotting and immunofluorescence. The low concentration of T. cruzi trypomastigotes activates both signaling pathways; however, the high concentration of parasite induces a modest activation of the PLC-γ pathway and impairs the ERK1/2 MAPK pathway activation. Interestingly, inhibition of any of those signaling pathways did not prevent parasite infection, as it was previously shown in cell cultures. We conclude that both signal transduction pathways are modulated during ex vivo T. cruzi infection of human placental chorionic villi explants.

Role of matrix metalloproteinases 2 and 9 in ex vivo Trypanosoma cruzi infection of human placental chorionic villi.

BACKGROUND: Chagas' disease is caused by the haemophlagelated protozoan Trypanosoma cruzi (T. cruzi). During congenital transmission the parasite breaks down the placental barrier. In the present study we analyzed the participation of matrix metalloproteases (MMPs) in the extracellular matrix (ECM) remodeling during T. cruzi ex vivo infection of human placental chorionic villi explants. METHODS: Chorionic villi from healthy woman placentas were incubated in the presence or absence of 105 or 106 T. cruzi trypomastigotes (Y strain) with or without the MMPs inhibitor doxycycline. Effective infection was tested measuring parasite DNA by real time PCR (qPCR). MMP-2 and MMP-9 expression were determined by western blotting and immunohistochemistry and their activities were measured by zymography. The effect of MMPs on ECM structure was analyzed histochemically. RESULTS: T. cruzi induces the expression and activity of MMP-2 and MMP-9 in chorionic villi. Inhibition of the MMPs prevents the tissue damage induced by T. cruzi and partially decreases the ex vivo infection of the chorionic villi. CONCLUSION: MMPs are partially responsible for the ECM changes observed in human chorionic villi during T. cruzi infection and participate in tissue invasion. On the other hand, MMPs may be part of a local placental antiparasitic mechanism.

Upregulation of HtrA4 in the placentas of patients with severe pre-eclampsia.

High temperature requirement A (HtrA) family proteins are serine proteases that may serve in the quality control of misfolded or mislocalized proteins. Recently, possible involvements of HtrA1 in the normal development of the placenta and in the pathogenesis of pre-eclampsia were reported. In this study, we characterized HtrA4, a previously uncharacterized HtrA protein family member, in pre-eclampsia. Elevated expression levels of placental HtrA4 in pre-eclampsia patients were observed by qRT-PCR. Western blotting also showed an increased production of HtrA4 at the protein level in pre-eclamptic placentas. In normal chorionic villi, HtrA4 protein was more abundant in the cytoplasm of cytotrophoblasts than in syncytiotrophoblasts. In contrast, the amount of HtrA4 protein in syncytiotrophoblasts was dramatically increased in pre-eclamptic placentas. Circulating HtrA4 was detected at higher levels in sera from women with pre-eclampsia than from those with normotensive pregnancies. Serum HtrA4 levels were higher in patients with early onset and inversely correlated with the weights of the newborn and placenta. Furthermore, serum levels correlated with serum PAPP-A and PAPP-A2 levels, indicating a functional role for HtrA4 in the common pathway. These data suggest that increased HtrA4 may be involved in the onset of pre-eclampsia, and elevated levels in sera imply a potential application as a biomarker for this disorder.

Caspase-mediated apoptosis of trophoblasts in term human placental villi is restricted to cytotrophoblasts and absent from the multinucleated syncytiotrophoblast.

Human placental villi are surfaced by a multinucleated and terminally differentiated epithelium, the syncytiotrophoblast, with a subjacent layer of mononucleated cytotrophoblasts that can divide and fuse to replenish the syncytiotrophoblast. The objectives of this study were i) to develop an approach to definitively identify and distinguish cytotrophoblasts from the syncytiotrophoblast, ii) to unambiguously determine the relative susceptibility of villous cytotrophoblasts and syncytiotrophoblast to constitutive and stress-induced apoptosis mediated by caspases, and iii) to understand the progression of apoptosis in villous trophoblasts. Confocal microscopy with co-staining for E-cadherin and DNA allowed us to clearly distinguish the syncytiotrophoblast from cytotrophoblasts and identified that many cytotrophoblasts are deeply interdigitated into the syncytiotrophoblast. Staining for specific markers of caspase-mediated apoptosis indicate that apoptosis occurs readily in cytotrophoblasts but is remarkably inhibited in the syncytiotrophoblast. To determine if an apoptotic cell or cell fragment was from a cytotrophoblast or syncytiotrophoblast, we found co-staining with E-cadherin along with a marker for apoptosis was essential: in the absence of E-cadherin staining, apoptotic cytotrophoblasts would easily be mistaken as representing localized regions of apoptosis in the syncytiotrophoblast. Regions with perivillous fibrin-containing fibrinoid contain the remnants of trophoblast apoptosis, and we propose this apoptosis occurs only after physical isolation of a region of the syncytium from the main body of the syncytium. We propose models for the progression of apoptosis in villous cytotrophoblasts and for why caspase-mediated apoptosis does not occur within the syncytium of placental villi.

In vitro study on human cytomegalovirus affecting early pregnancy villous EVT's invasion function.

BACKGROUND: Human cytomegalovirus (HCMV) is the most common pathogen in uterus during pregnancy, which may lead to some serious results such as miscarriage, stillbirth, cerebellar malformation, fetus developmental retardation, but its pathogenesis has not been fully explained. The hypofunction of extravillous cytotrophoblast (EVT) invasion is the essential pathologic base of some complications of pregnancy. c-erbB-2 is a kind of oncogene protein and closely linked with embryogenesis, tissue repair and regeneration. Matrix metalloproteinase (MMP) is one of the key enzymes which affect EVT migration and invasion function. The expression level changes of c-erbB-2, MMP-2 and MMP-9 can reflect the changes of EVT invasion function. RESULTS: To explore the influence of HCMV on the invasion function of EVT, we tested the protein expression level changes of c-erbB-2, MMP-2 and MMP-9 in villous explant cultured in vitro infected by HCMV, with the use of immunohistochemistry SP method and western blot. We confirmed that HCMV can reproduce and spread in early pregnancy villus; c-erbB-2 protein mainly expressed in normal early pregnancy villous syncytiotrophoblast (ST) remote plasma membrane and EVT, especially remote EVT cell membrane in villous stem cell column, little expressed in ST proximal end cell membrane and interstitial cells; MMP-2 protein primarily expressed in early pregnancy villous EVT endochylema and rarely in villous trophoblast (VT), ST and interstitial cells; MMP-9 protein largely expressed in early pregnancy villous mesenchyme, EVT and VT endochylema. Compared with control group, the three kinds of protein expression level in early pregnancy villus of virus group significantly decreased (P < 0.05). CONCLUSION: HCMV can infect villus in vitro and cause the decrease of early pregnancy villous EVT's invasion function.

Inhibition of choriodecidual cytokine production and inflammatory gene expression by selective I-kappaB kinase (IKK) inhibitors.

BACKGROUND AND PURPOSE: Inflammation of the extraplacental membranes plays a key role in the pathogenesis of preterm labour. The aim of this study was to screen a number of commercially available small molecule nuclear factor-kappa B inhibitors to identify candidates suitable for clinical evaluation as anti-inflammatory agents for the prevention of preterm birth. EXPERIMENTAL APPROACH: Nine inhibitors were evaluated across a range of concentrations for their ability to inhibit lipopolysaccharide (LPS)-stimulated cytokine production in primary term choriodecidual cells in culture without affecting cell viability. Expression of 112 inflammation- and apoptosis-related genes was evaluated using boutique oligonucleotide arrays. KEY RESULTS: Two IKKbeta inhibitors were found to be highly effective and non-toxic inhibitors of choriodecidual cytokine production: parthenolide and [5-(p-fluorophenyl)-2-ureido] thiophene-3-carboxamide (TPCA-1). Both compounds also inhibited LPS-stimulated nuclear translocation of p65/RelA. Expression of 38 genes on the arrays (34%) was significantly (P < 0.05) inhibited by TPCA-1 or parthenolide. Of the 14 genes significantly stimulated by LPS, all were inhibited by TPCA-1 and 12 were inhibited by parthenolide. Overall, gene expression was more robustly inhibited by TPCA-1 than parthenolide; however, expression of two genes was only inhibited by parthenolide. Neither compound significantly altered the expression profile of anti-apoptosis genes on the arrays. CONCLUSIONS AND IMPLICATIONS: These studies provide evidence that pharmacological inhibition of IKKbeta activity holds promise as a potential strategy for the prevention and/or treatment of inflammation-driven preterm birth. TPCA-1 appeared the most promising compound among those tested in this study. Different inhibitors may have subtly different effect profiles despite having similar modes of action.

Chorionic villi ultrastructure in the prenatal diagnosis of glycogenosis type II.

OBJECTIVE: To perform the ultrastructural examination of a chorionic villi biopsy as a predictor of foetal involvement in the infantile form of glycogenosis type II (Pompe disease). METHODS: Ultrastructural, biochemical and genetic analyses were performed on chorionic villi biopsies of three consecutive pregnancies in a woman with a previous child affected by Pompe disease. RESULTS: In the only affected foetus, glycogen storage was observed in fibrocytes and endothelial cells of a chorionic villi sample at 11 week's gestation. Severe multi-organ involvement was demonstrated in the tissues of the aborted foetus. No abnormal material was found in the chorionic samples of two subsequent pregnancies, and a healthy boy and girl were born at term and remain unaffected. Both exhibited a partial reduction in acid maltase and were carriers of the maternal mutation. CONCLUSIONS: Ultrastructural findings correlated with biochemical and genetic results, providing a clear and early indicator of the definite diagnosis for future pregnancy management or an early therapeutic approach.

Fusion of villous trophoblast can be visualized by localizing active caspase 8.

Villous cytotrophoblast differentiation and subsequent fusion with the overlying syncytiotrophoblast depend on multiple factors such as growth factors, cytokines, hormones, protein kinases, transcription factors, structural membrane proteins and proteases. Caspase 8, an aspartate-specific cysteine protease, is mainly known for its role in programmed cell death, but was also demonstrated to be crucial for villous trophoblast differentiation. This study aimed to localize active caspase 8 in the villous trophoblast layer of human first trimester placenta. To this end, immunofluorescence double staining was performed, using a monoclonal rabbit antibody against cleaved caspase 8 in combination with antibodies against cytokeratin 7, chorionic gonadotropin beta subunit (beta hCG), beta-actin, placental protein 13 (PP13), alpha-fodrin and Ki-67. Immunofluorescence revealed cleaved caspase 8 in one out of 422 villous cytotrophoblasts resting on the basement membrane, in one out of 759 perinuclear regions within the syncytiotrophoblast and in few trophoblasts located between these two layers. Double staining of cleaved caspase 8 and Ki-67 antigen revealed that caspase 8 is activated only in cytotrophoblasts which have left the cell cycle. The staining suggests that caspase 8 is activated in villous cytotrophoblasts just prior to fusion of these cells and escorts the nuclei from the mononucleated to the syncytial state.