Flavivirus replication kinetics in early-term placental cell lines with different differentiation pathways

Background The uncontrollable spread of Zika virus (ZIKV) in the Americas during 2015–2017, and its causal link to microcephaly in newborns and Guillain-Barré syndrome in adults, led the World Health Organisation to declare it a global public health emergency. One of the most notable features of ZIKV pathogenesis was the ability of the virus to pass the placental barrier to infect the growing foetus. This pathogenic trait had not been observed previously for medically important flaviviruses, including dengue and yellow fever viruses. Methods In this study we evaluated the replication kinetics of ZIKV and the related encephalitic flavivirus West Nile strain Kunjin virus (WNVKUN) in early-term placental cell lines. Results We have observed that WNVKUN in fact replicates with a greater rate and to higher titres that ZIKV in these cell lines. Conclusions These results would indicate the potential for all flaviviruses to replicate in placental tissue but it is the ability to cross the placenta itself that is the restrictive factor in the clinical progression and presentation of congenital Zika syndrome.

An iTSC-derived placental model of SARS-CoV-2 infection unveils ACE2-dependent susceptibility in syncytiotrophoblasts

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing coronavirus disease 2019 (COVID-19) has caused a global health crisis. The primary site of infection is in the respiratory tract but the virus has been associated with a variety of complications involving the gastrointestinal and cardiovascular systems. Since the virus affects a variety of tissue types, there has been interest in understanding SARS-CoV-2 infection in early development and the placenta. The expression of ACE2 or TMPRSS2, both genes critical for viral entry, is present in placental-specific cell types such as extravillous trophoblasts (EVTs) and, especially, syncytiotrophoblasts (STs). The potential of SARS-CoV-2 to infect these placental cells and its effect on placental development and function is still unclear. Furthermore, it is crucial to understand the possible mechanism of vertical transmission of SARS-CoV-2 through the placenta. Here, we developed an in vitro model of SARS-CoV-2 infection of placental cell types using induced trophoblast stem cells (iTSCs). This model allowed us to show that STs but not EVTs are infected. Importantly, infected STs lack the expression of key differentiation genes, lack typically observed differentiated morphology and produce significantly lower human chorionic gonadotropin (HCG) compared to non-infected controls. We also show that an anti-ACE2 antibody prevents SARS-CoV-2 infection and restores normal ST differentiation and function. We highlight the establishment of a platform to study SARS-CoV-2 infection in early placental cell types, which will facilitate investigation of antiviral therapy to protect the placenta during early pregnancy and development.

Medicinal Plants for the Treatment of Mental Diseases in Pregnancy: An In Vitro Safety Assessment

Pregnancy is a critical period for medical care, during which the well-being of woman and fetus must be considered. This is particularly relevant in managing non-psychotic mental disorders since treatment with central nervous system-active drugs and untreated NMDs may have negative effects. Some well-known herbal preparations (phytopharmaceuticals), including St. Johnʼs wort, California poppy, valerian, lavender, and hops, possess antidepressant, sedative, anxiolytic, or antidepressant properties and could be used to treat mental diseases such as depression, restlessness, and anxiety in pregnancy. Our goal was to assess their safety in vitro, focusing on cytotoxicity, induction of apoptosis, genotoxicity, and effects on metabolic properties and differentiation in cells widely used as a placental cell model (BeWo b30 placenta choriocarcinoma cells). The lavender essential oil was inconspicuous in all experiments and showed no detrimental effects. At low-to-high concentrations, no extract markedly affected the chosen safety parameters. At an artificially high concentration of 100 µg/mL, extracts from St. Johnʼs wort, California poppy, valerian, and hops had minimal cytotoxic effects. None of the extracts resulted in genotoxic effects or altered glucose consumption or lactate production, nor did they induce or inhibit BeWo b30 cell differentiation. This study suggests that all tested preparations from St. Johnʼs wort, California poppy, valerian, lavender, and hops, in concentrations up to 30 µg/mL, do not possess any cytotoxic or genotoxic potential and do not compromise placental cell viability, metabolic activity, and differentiation. Empirical and clinical studies during pregnancy are needed to support these in vitro data.

Placental mammals acquired the functional region and domain in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologues across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: (1) mouse NRK is localised at the plasma membrane via the CNH domain, where the CIR inhibits CK2. (2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN, and (3) leads to the inhibition of AKT signalling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation.

Increased Placental Cell Senescence and Oxidative Stress in Women with Pre-Eclampsia and Normotensive Post-Term Pregnancies

Up to 11% of pregnancies extend to post-term with adverse obstetric events linked to pregnancies over 42 weeks. Oxidative stress and senescence (cells stop growing and dividing by irreversibly arresting their cell cycle and gradually ageing) can result in diminished cell function. There are no detailed studies of placental cell senescence markers across a range of gestational ages, although increased levels have been linked to pre-eclampsia before full term. This study aimed to determine placental senescence and oxidative markers across a range of gestational ages in women with uncomplicated pregnancies and those with a diagnosis of pre-eclampsia. Placentae were obtained from 37 women with uncomplicated pregnancies of 37–42 weeks and from 13 cases of pre-eclampsia of 31+2–41+2 weeks. The expression of markers of senescence, oxidative stress, and antioxidant defence (tumour suppressor protein p16INK4a, kinase inhibitor p21, interleukin-6 (IL-6), NADPH oxidase 4 (NOX4), glutathione peroxidases 1, 3, and 4 (GPx1, GPx3, and GPx4), placental growth factor (PlGF), and soluble fms-like tyrosine kinase-1 (sFlt-1)) genes was measured (quantitative real-time PCR). Protein abundance of p16INK4a, IL-6, NOX4, 8-hydroxy-2′-deoxy-guanosine (8-OHdG), and PlGF was assessed by immunocytochemistry. Placental NOX4 protein was higher in post-term than term deliveries and further increased by pre-eclampsia (p < 0.05 for all). P21 expression was higher in post-term placentae (p = 0.012) and in pre-eclampsia (p = 0.04), compared to term. Placental P16INK4a protein expression was increased post-term, compared to term (p = 0.01). In normotensive women, gestational age at delivery was negatively associated with GPx4 and PlGF (mRNA and protein) (p < 0.05 for all), whereas a positive correlation was seen with placental P21, NOX4, and P16INK4a (p < 0.05 for all) expression. Markers of placental oxidative stress and senescence appear to increase as gestational age increases, with antioxidant defences diminishing concomitantly. These observations increase our understanding of placental health and may contribute to assessment of the optimal gestational age for delivery.

P–421 Down-expression of glycolytic pathway-related protein A1 is associated with the pathogenesis of recurrent pregnancy loss

Study question How does glycolytic pathway-related protein A1 (GPRPA1) relate to the pathogenesis of recurrent pregnancy loss (RPL)? Summary answer GPRPA1 was found as a substrate of ITI-H4 to modulate the inflammatory response and was down-expressed in the sera of RPL patients. What is known already Thus far, the pathogenesis of RPL was not fully understood. In a previous study, the short isoform ITI-H4 cleaved by kallikrein B1 was detected in the sera of RPL patients and would be an important inflammatory factor for RPL by increasing pro-inflammatory cytokines. GPRPA1, a new binding partner of ITI-H4, was known to relate with pre-eclampsia and human decidualization by regulating angiogenesis and glycolysis. Also, GPRPA1 affects placental cell motility and cancer cell proliferation. Study design, size, duration Through immunoprecipitation (IP) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analyses, we found new binding partners of ITI-H4. Of these, GPRPA1 was selected, and direct binding between GPRPA1 and ITI-H4 was confirmed by IP and GST pull-down assay. Differential expression of GPRPA1 in sera and cellular functions of GPRPA1 in the placental cell line were investigated by molecular and cellular analyses. Participants/materials, setting, methods The Flag-tagged full-length ITI-H4 and the short isoform ITI-H4 were transfected into HEK293T cells and IP has proceeded with the Flag antibody. Spots showing differential expression were analyzed by MALDI-TOF/MS analysis and peptide sequence alignment was performed. The binding between GPRPA1 and ITI-H4 was confirmed using IP and GST pull-down assay. The effects of GPRPA1 on cellular functions in the placental cell were investigated by CCK–8 assay, invasion assay, and colony-forming assay. Main results and the role of chance Through IP, MALDI-TOF/MS analysis, and peptide sequence alignment, we found new substrates of ITI-H4 related to glycolysis, T cell activation, and production of thyroid hormones. Of these, we selected GPRPA1 which is secreted in the serum to utilize a serum biomarker of RPL. GPRPA1 directly binds to the full-length ITI-H4 and also binds to the short isoform ITI-H4 shown by IP and GST pull-down assay. Besides, GPRPA1 as a protein kinase increases serine phosphorylation of ITI-H4 and inhibits the cleavage by KLKB1. GPRPA1 is expressed significantly lower in the sera of PRL patients than the control group and knockdown of GPRPA1 negatively regulates cell motility in the placental cell. Therefore, down-expressed GPRPA1 would be one of the causes of RPL and can be utilized as a serum biomarker of RPL. Limitations, reasons for caution Additional in vivo study is needed to specifically investigate the effect of GPRPA1 on the pathogenesis of RPL. Wider implications of the findings: By investigating the cellular functions of GPRPA1 in the placental cell, we found that it is an important key factor for the pathogenesis of RPL, and down-regulation of GPRPA1 can be utilized as a biomarker of RPL. Trial registration number Not applicable

Cell wall polymers in the Phaeoceros placenta reflect developmental and functional differences across generations

The placenta of hornworts is unique among bryophytes in the restriction of transfer cells that are characterized by elaborate wall labyrinths to the gametophyte generation. During development, cells around the periphery of the sporophyte foot elongate, forming smooth-walled haustorial cells that interdigitate with gametophyte cells. Using immunogold labeling with 22 antibodies to diverse cell wall polymers, we examined compositional differences in the developmentally and morphologically distinct cell walls of gametophyte transfer cells and sporophyte haustorial cells in the placenta of Phaeoceros. As detected by Calcofluor White fluorescence, cellulose forms the cell wall scaffolding in cells on both sides of the placenta. Homogalacturonan (HG) and rhamnogalacturonan I (RG-I) pectins are abundant in both cell types, and haustorial cells are further enriched in methyl-esterified HGs. The abundance of pectins in placental cell walls is consistent with the postulated roles of these polymers in cell wall porosity and in maintaining an acidic apoplastic pH favorable to solute transport. Xyloglucan hemicellulose, but not mannans or glucuronoxylans, are present in cell walls at the interface between the two generations with a lower density in gametophytic wall ingrowths. Arabinogalactan proteins (AGPs) are diverse along the plasmalemma of placental cells and are absent in surrounding cells in both generations. AGPs in placental cell walls may play a role in calcium binding and release associated with signal transduction as has been speculated for these glycoproteins in other plants. Callose is restricted to thin areas in cell walls of gametophyte transfer cells. In contrast to studies of transfer cells in other systems, no reaction to the JIM12 antibody against extensin was observed in Phaeoceros.