Evidence of Placental Villous Inflammation and Apoptosis in Third-Trimester Symptomatic SARS-CoV-2 Maternal Infection.

PURPOSE: In view of conflicting reports on the ability of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to infect placental tissue, this study aimed to further evaluate the impact of inflammation and placental damage from symptomatic third-trimester maternal COVID-19 infection. MATERIALS AND METHODS: This case-control study included 32 placenta samples each from symptomatic COVID-19 pregnancy and normal non-COVID-19 pregnancy. The villous placental area's inflammatory expression [angiotensin converting enzyme-2 (ACE-2), transmembrane protease serine-2 (TMPRSS2), interferon-γ (IFN-γ), interleukin-6 (IL-6), and SARS-CoV-2 spike protein] and apoptotic rate were examined using immunohistochemistry and Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay. Comparison and correlation analysis were used based on COVID-19 infection, placental SARS-CoV-2 spike protein evidence, and maternal severity status. RESULTS: Higher expressions of TMPRSS2, IFN-γ, and trophoblast apoptotic rate were observed in the COVID-19 group (p<0.001), whereas ACE-2 and IL-6 expressions were not significantly different from the control group (p>0.05). Additionally, SARS-CoV-2 spike protein was detected in 8 (25%) placental samples of COVID-19 pregnancy. COVID-19 subgroup analysis revealed increased IFN-γ, trophoblast, and stromal apoptosis (p<0.01). Moreover, the results of the current study revealed no correlation between maternal COVID-19 severity and placental inflammation as well as the apoptotic process. CONCLUSION: The presence of SARS-CoV-2 spike protein as well as altered inflammatory and apoptotic processes may indicate the presence of placental disturbance in third-trimester maternal COVID-19 infection. The lack of correlation between placental disruption and maternal severity status suggests the need for more research to understand the infection process and any potential long-term impacts on all offsprings born to COVID-19-infected pregnant women.

Vertical transmission of African-lineage Zika virus through the fetal membranes in a rhesus macaque (Macaca mulatta) model.

Zika virus (ZIKV) can be transmitted vertically from mother to fetus during pregnancy, resulting in a range of outcomes including severe birth defects and fetal/infant death. Potential pathways of vertical transmission in utero have been proposed but remain undefined. Identifying the timing and routes of vertical transmission of ZIKV may help us identify when interventions would be most effective. Furthermore, understanding what barriers ZIKV overcomes to effect vertical transmission may help improve models for evaluating infection by other pathogens during pregnancy. To determine the pathways of vertical transmission, we inoculated 12 pregnant rhesus macaques with an African-lineage ZIKV at gestational day 30 (term is 165 days). Eight pregnancies were surgically terminated at either seven or 14 days post-maternal infection. Maternal-fetal interface and fetal tissues and fluids were collected and evaluated for ZIKV using RT-qPCR, in situ hybridization, immunohistochemistry, and plaque assays. Four additional pregnant macaques were inoculated and terminally perfused with 4% paraformaldehyde at three, six, nine, or ten days post-maternal inoculation. For these four cases, the entire fixed pregnant uterus was evaluated with in situ hybridization for ZIKV RNA. We determined that ZIKV can reach the MFI by six days after infection and infect the fetus by ten days. Infection of the chorionic membrane and the extraembryonic coelomic fluid preceded infection of the fetus and the mesenchymal tissue of the placental villi. We did not find evidence to support a transplacental route of ZIKV vertical transmission via infection of syncytiotrophoblasts or villous cytotrophoblasts. The pattern of infection observed in the maternal-fetal interface provides evidence of paraplacental vertical ZIKV transmission through the chorionic membrane, the outer layer of the fetal membranes.

Effect of SARS-CoV-2 infection in pregnancy on CD147, ACE2 and HLA-G expression.

INTRODUCTION: Recent studies reported a differential expression of both ACE2 and CD147 in pregnant women associated to SARS-CoV-2 placental infection. The aim of this study is to further investigate the placental SARS-CoV-2 infection and the potential effect on protein expression (ACE2, CD147, HLA-G and CD56). METHODS: The study was on three subgroups: i) 18 subjects positive for SARS-CoV-2 swab at delivery; ii) 9 subjects that had a positive SARS-CoV-2 swab during pregnancy but resulted negative at delivery; iii) 11 control subjects with physiological pregnancy and with no previous or concomitant SARS-CoV-2 swab positivity. None of the subjects were vaccinated for SARS-CoV-2 infection. The placenta samples were analyzed for SARS-CoV-2 NP (Nucleocapsid protein) positivity and the expression of ACE2, CD147, HLA-G and CD56. RESULTS: We observed a higher percentage of SARS-CoV-2 NP positive placenta samples in the group of SARS-CoV-2 PCR positive at delivery in comparison with SARS-CoV-2 PCR negative at delivery. The localization of SARS-CoV-2 NP positivity in placenta samples was mainly in syncytiotrophoblast (ST) of SARS-CoV-2 PCR positive at delivery group and in extra-villous trophoblast (EVT) of SARS-CoV-2 PCR negative at delivery group. CD147, HLA-G positivity was higher in ST of SARS-CoV-2 PCR positive at delivery group, while CD56-expressing immune cells were decreased in comparison with control subjects. DISCUSSION: We confirmed the ability of SARS-CoV-2 to infect placenta tissues. The simultaneous SARS-CoV-2 swab positivity at delivery and the positivity of the placenta tissue for SARS-CoV-2 NP seems to create an environment that modifies the expression of specific molecules, as CD147 and HLA-G. These data suggest a possible impact of SARS-CoV-2 infection during pregnancy, that might be worthy to be monitored also in vaccinated subjects.

The differential expression of toll like receptors and RIG-1 in the placenta of neonates with in utero infections.

Novel biomarkers of in utero infections are needed to help guide early therapy. The toll like receptors (TLRs) and retinoic acid-inducible gene 1 (RIG-1) are proteins involved in the initial reaction of the innate immune system to infectious diseases. This study tested the hypothesis that a panel of TLRs and RIG-1 in the placenta could serve as an early biomarker of in utero infections. The TLRs and RIG-1 expression as determined by immunohistochemistry was scored in 10 control placentas (normal delivery or neonatal damage from known non-infectious cause), 8 placentas from documented in utero bacterial infection, and 7 placentas from documented in utero viral infections blinded to the clinical information. The non-infected placentas showed the following profile: no expression (TLR1, TLR3, TLR4, TLR7, TLR8), moderate expression (TLR2), and strong expression (RIG-1). The bacterial and viral infection cases shared the following profile: no to mild expression (TLR 2, TLR7, and RIG1), moderate expression (TLR4), and strong expression (TLR1, TLR3, and TLR8). The histologic findings in the chorionic villi were equivalent in the infected cases and controls, underscoring the need for molecular testing by the surgical pathologist when in utero infection is suspected. The results suggest that a panel of TLRs/RIG-1 analyses can allow the pathologist and/or clinician to diagnose in utero infections soon after birth. Also, treatments to antagonize the effects of TLR1, 3, and 8 may help abrogate in utero neonatal damage.

Gardnerella vaginalis induces matrix metalloproteinases in the cervicovaginal epithelium through TLR-2 activation.

Lactobacillus-deficient cervicovaginal microbiota, including Gardnerella vaginalis, are implicated in cervical remodeling and preterm birth. Mechanisms by which microbes drives outcomes are not fully elucidated. We hypothesize that Gardnerella vaginalis induces matrix metalloproteinases through TLR-2, leading to epithelial barrier dysfunction and premature cervical remodeling. Cervicovaginal cells were treated with live Gardnerella vaginalis or Lactobacillus crispatus or their bacteria-free supernatants for 24 h. For TLR-2 experiments, cells were pretreated with TLR-2 blocking antibody. A Luminex panel was run on cell media. For human data, we conducted a case-control study from a prospective pregnancy cohort of Black individuals with spontaneous preterm (sPTB) (n = 40) or term (n = 40) births whose vaginal microbiota had already been characterized. Cervicovaginal fluid was obtained between 20 and 24 weeks' gestation. Short cervix was defined as < 25 mm by second trimester transvaginal ultrasound. MMP-9 was quantified by ELISA. Standard analytical approaches were used to determine differences across in vitro conditions, as well as MMP-9 and associations with clinical outcomes. Gardnerella vaginalis induced MMP-1 in cervical cells (p = 0.01) and MMP-9 in cervical and vaginal (VK2) cells (p ≤ 0.001 for all). TLR-2 blockade mitigated MMP-9 induction by Gardnerella vaginalis. MMP-9 in cervicovaginal fluid is higher among pregnant individuals with preterm birth, short cervix, and Lactobacillus-deficient microbiota (p < 0.05 for all). MMP-9 is increased in the cervicovaginal fluid of pregnant individuals with subsequent sPTB. Our in vitro work ascribes a potential mechanism by which a cervicovaginal microbe, commonly associated with adverse pregnancy outcomes, may disrupt the cervicovaginal epithelial barrier and promote premature cervical remodeling in spontaneous preterm birth.

RNA-Seq of amniotic fluid cell-free RNA: a discovery phase study of the pathophysiology of congenital cytomegalovirus infection.

BACKGROUND: Congenital cytomegalovirus infection is the most common perinatal infection and a significant cause of sensorineural hearing loss, cerebral palsy, and neurodevelopmental disability. There is a paucity of human gene expression studies examining the pathophysiology of cytomegalovirus infection. OBJECTIVE: This study aimed to perform a whole transcriptomic assessment of amniotic fluid from pregnancies with live fetuses to identify differentially expressed genes and enriched Gene Ontology categories associated with congenital cytomegalovirus infection. STUDY DESIGN: Amniotic fluid supernatant was prospectively collected from pregnant women undergoing amniocentesis for suspected congenital cytomegalovirus infection because of first-trimester maternal primary infection or ultrasound features suggestive of fetal infection. Women who had received therapy to prevent fetal infection were excluded. Congenital cytomegalovirus infection was diagnosed via viral polymerase chain reaction of amniotic fluid; cytomegalovirus-infected fetuses were paired with noninfected controls, matched for gestational age and fetal sex. Paired-end RNA sequencing was performed on amniotic fluid cell-free RNA with the Novaseq 6000 at a depth of 30 million reads per sample. Following quality control and filtering, reads were mapped to the human genome and counts summarized across genes. Differentially expressed genes were identified using 2 approaches: voomWithQualityWeights in conjunction with limma and RUVSeq with edgeR. Genes with a false discovery rate <0.05 were considered statistically significant. Differential exon use was analyzed using DEXSeq. Functional analysis was performed using gene set enrichment analysis and Ingenuity Pathway Analysis. Manual curation of differentially regulated genes was also performed. RESULTS: Amniotic fluid samples were collected from 50 women; 16 (32%) had congenital cytomegalovirus infection confirmed by polymerase chain reaction. After excluding 3 samples without matched controls, 13 cytomegalovirus-infected samples collected at 18 to 23 weeks and 13 cytomegalovirus-negative gestation-matched controls were submitted for RNA sequencing and analysis (N=26). Ten of the 13 pregnancies with cytomegalovirus-infected fetuses had amniocentesis because of serologic evidence of maternal primary infection with normal fetal ultrasound, and 3 had amniocentesis because of ultrasound abnormality suggestive of cytomegalovirus infection. Four cytomegalovirus-infected pregnancies ended in termination (n=3) or fetal death (n=1), and 9 resulted in live births. Pregnancy outcomes were available for 11 of the 13 cytomegalovirus-negative controls; all resulted in live births of clinically-well infants. Differential gene expression analysis revealed 309 up-regulated and 32 down-regulated genes in the cytomegalovirus-infected group compared with the cytomegalovirus-negative group. Gene set enrichment analysis showed significant enrichment of multiple Gene Ontology categories involving the innate immune response to viral infection and interferon signaling. Of the 32 significantly down-regulated genes, 8 were known to be involved in neurodevelopment and preferentially expressed by the brain. Six specific cellular restriction factors involved in host defense to cytomegalovirus infection were up-regulated in the cytomegalovirus-infected group. Ingenuity Pathway Analysis predicted the activation of pathways involved in progressive neurologic disease and inflammatory neurologic disease. CONCLUSION: In this next-generation sequencing study, we revealed new insights into the pathophysiology of congenital cytomegalovirus infection. These data on the up-regulation of the intraamniotic innate immune response to cytomegalovirus infection and the dysregulation of neurodevelopmental genes may inform future approaches to developing prognostic markers and assessing fetal responses to in utero therapy.

Remdesivir impairs mouse preimplantation embryo development at therapeutic concentrations.

Remdesivir (RDV) is the first antiviral drug to be approved by the US Food and Drug Administration for the treatment of COVID-19. While the general safety of RDV has been studied, its reproductive risk, including embryotoxicity, is largely unknown. Here, to gain insights into its embryotoxic potential, we investigated the effects of RDV on mouse preimplantation embryos cultured in vitro at the concentrations comparable to the therapeutic plasma levels. Exposure to RDV (2-8 µM) did not affect the initiation of blastocyst formation, although the maintenance of the cavity failed at 8 µM due to increased cell death. While exposure to 2-4 µM permitted the cavity maintenance, expressions of developmental regulator genes associated with the inner cell mass (ICM) lineage were significantly diminished. Adverse effects of RDV depended on the duration and timing of exposure, as treatment between the 8-cell to early blastocyst stage most sensitively affected cavity expansion, gene expressions, and cell proliferation, particularly of the ICM than the trophectoderm lineage. GS-441524, a major metabolite of RDV, did not impair blastocyst formation or cavity expansion, although it altered gene expressions in a manner differently from RDV. Additionally, RDV reduced the viability of human embryonic stem cells, which were used as a model for the human ICM lineage, more potently than GS-441524. These findings suggest that RDV is potentially embryotoxic to impair the pluripotent lineage, and will be useful for designing and interpreting further in vitro and in vivo studies on the reproductive toxicity of RDV.

Decidual immune response following COVID-19 during pregnancy varies by timing of maternal SARS-CoV-2 infection.

While COVID-19 infection during pregnancy is common, fetal transmission is rare, suggesting that intrauterine mechanisms form an effective blockade against SARS-CoV-2. Key among these is the decidual immune environment of the placenta. We hypothesize that decidual leukocytes are altered by maternal SARS-CoV-2 infection in pregnancy and that this decidual immune response is shaped by the timing of infection during gestation. To address this hypothesis, we collected decidua basalis tissues at delivery from women with symptomatic COVID-19 during second (2nd Tri COVID, n = 8) or third trimester (3rd Tri COVID, n = 8) and SARS-CoV-2-negative controls (Control, n = 8). Decidual natural killer (NK) cells, macrophages and T cells were evaluated using quantitative microscopy, and pro- and anti-inflammatory cytokine mRNA expression was evaluated using quantitative reverse transcriptase PCR (qRT-PCR). When compared with the Control group, decidual tissues from 3rd Tri COVID exhibited significantly increased macrophages, NK cells and T cells, whereas 2nd Tri COVID only had significantly increased T cells. In evaluating decidual cytokine expression, we noted that IL-6, IL-8, IL-10 and TNF-α were significantly correlated with macrophage cell abundance. However, in 2nd Tri COVID tissues, there was significant downregulation of IL-6, IL-8, IL-10, and TNF-α. Taken together, these results suggest innate and adaptive immune responses are present at the maternal-fetal interface in maternal SARS-CoV-2 infections late in pregnancy, and that infections earlier in pregnancy show evidence of a resolving immune response. Further studies are warranted to characterize the full scope of intrauterine immune responses in pregnancies affected by maternal COVID-19.

The impact on colostrum oxidative stress, cytokines, and immune cells composition after SARS-CoV-2 infection during pregnancy.

Background: Limited data are available regarding the differences between immunological, biochemical, and cellular contents of human colostrum following maternal infection during pregnancy with coronavirus 2 disease (COVID-19). Objective: To investigate whether maternal COVID-19 infection may affect immunological, biochemical, and cellular contents of human colostrum. Methods: Using a case-control study design, we collected colostrum from 14 lactating women with a previous diagnosis of COVID-19 during pregnancy and 12 without a clear diagnosis during September 2020 to May 2021. Colostrum samples were analysed for some enzymes and non-enzymatic oxidative stress markers (SOD, CAT, GPx, MDA, GSH, GSSG, H2O2, MPO) and for IL-1ß, IL-6, tumour necrosis factor (TNF)-α, protein induced by interferon gamma (IP)-10, IL-8, IFN-λ1, IL12p70, IFN-α2, IFN-λ2/3, granulocyte macrophage colony stimulating factor (GM-CSF), IFN-ß, IL-10 and IFN-γ, along with IgA and IgG for the SARS-CoV-2 S protein. We perform immunophenotyping to assess the frequency of different cell types in the colostrum. Results: Colostrum from the COVID-19 symptomatic group in pregnancy contained reduced levels of H2O2, IFN-α2, and GM-CSF. This group had higher levels of GSH, and both NK cell subtypes CD3-CD56brightCD16-CD27+IFN-γ+ and CD3-CD56dimCD16+CD27- were also increased. Conclusion: The present results reinforce the protective role of colostrum even in the case of mild SARS-Cov-2 infection, in addition to demonstrating how adaptive the composition of colostrum is after infections. It also supports the recommendation to encourage lactating women to continue breastfeeding after COVID-19 illness.

The Role of Innate Immune System in the Human Amniotic Membrane and Human Amniotic Fluid in Protection Against Intra-Amniotic Infections and Inflammation.

Intra-amniotic infection and inflammation (IAI) affect fetal development and are highly associated with preterm labor and premature rupture of membranes, which often lead to adverse neonatal outcomes. Human amniotic membrane (hAM), the inner part of the amnio-chorionic membrane, protects the embryo/fetus from environmental dangers, including microbial infection. However, weakened amnio-chorionic membrane may be breached or pathogens may enter through a different route, leading to IAI. The hAM and human amniotic fluid (hAF) respond by activation of all components of the innate immune system. This includes changes in 1) hAM structure, 2) presence of immune cells, 3) pattern recognition receptors, 4) cytokines, 5) antimicrobial peptides, 6) lipid derivatives, and 7) complement system. Herein we provide a comprehensive and integrative review of the current understanding of the innate immune response in the hAM and hAF, which will aid in design of novel studies that may lead to breakthroughs in how we perceive the IAI.

The road (not) taken - Placental transfer and interspecies differences.

Species differences are among the main reasons for the high failure rate of preclinical studies. A better awareness and understanding of these differences might help to improve the outcome of preclinical research. In reproduction, the placenta is the central organ regulating fetal exposure to a substance circulating in the maternal organism. Exact information about placental transfer can help to better estimate the toxic potential of a substance. From an evolutionary point of view, the chorioallantoic placenta is the organ with the highest anatomical diversity among species. Moreover, frequently used animal models in reproduction belong to rodents and lagomorphs, two groups that are characterized by the generation of an additional type of placenta, which is crucial for fetal development, but absent from humans: the inverted yolk sac placenta. Taken together, the translatability of placental transfer studies from laboratory animals to humans is challenging, which is supported by the fact that numerous species-dependent toxic effects are described in literature. Thus, reliable human-relevant data are frequently lacking and the toxic potential of chemicals and pharmaceuticals for humans can hardly be estimated, often resulting in recommendations that medical treatments or exposure to chemicals should be avoided for safety reasons. Although species differences of placental anatomy have been described frequently and the need for human-relevant research models has been emphasized, analyses of substances with species-dependent placental transfer have been performed only sporadically. Here, we present examples for species-specific placental transfer, including that of nanoparticles and pharmaceuticals, and discuss potential underlying mechanisms. With respect to the COVID 19-pandemic it might be of interest that some antiviral drugs are reported to feature species-specific placental transfer. Further, differences in placental structure and antibody transfer may affect placental transfer of ZIKA virus.

Diagnostic predictability of miR-4535 and miR-1915-5p expression in amniotic fluid for foetal morbidity of infection.

INTRODUCTION: Clinical prediction of foetal inflammatory response syndrome (FIRS) is highly necessary. We have previously reported that miR-4535 and miR-1915-5p are potential biomarkers for severe chorioamnionitis based on the results of microRNA array analysis. Therefore, we evaluated the relationship between foetal morbidity of infection and miR-4535, miR-1915-5p, interleukin (IL)-6, or 16S rDNA copy number levels in amniotic fluid from pregnant women with chorioamnionitis. METHODS: Amniotic fluid from 57 pregnant women with preterm premature membrane rupture or threatened premature labour were collected. Infants with WBC counts <5000/µL or >20,000/µL, CRP >0.5 mg/mL, or IgM >20 mg/mL at birth received a diagnosis of suspicious foetal infection, and those requiring antibiotic administration for >5 days were considered infected newborns. miR-4535, miR-1915-5p, and IL-6 levels and 16S rDNA copy number were evaluated. Mann-Whitney U test and Dunn's test were used for comparison. The area under the curve (AUC) and Youden index were calculated to examine the diagnostic accuracy of foetal morbidity of infection. RESULTS: miR-4535, miR-1915-5p, 16S rDNA, and IL-6 were significantly higher in patients with severe chorioamnionitis than in patients with chorionitis or sub-chorionitis (P < 0.05). miR-4535 and miR-1915-5p levels were significantly associated with WBC counts <5000/µL or >20,000/µL, CRP >0.5 mg/mL, or IgM >20 mg/mL (P < 0.05). AUC values of miR-4535 and miR-1915-5p indicated moderate or low accuracy for foetal morbidity of infection, while those of IL-6 and 16S rDNA seemed unreliable. DISCUSSION: MiR-4535 and miR-1915-5p levels in amniotic fluid may be considered clinically predictive for foetal morbidity of infection.

Expression of Immune Checkpoint Receptors in Placentae With Infectious and Non-Infectious Chronic Villitis.

Pregnancy is an immunological paradox whereby maternal immunity accepts a genetically unique fetus (or fetuses), while maintaining protective innate and adaptive responses to infectious pathogens. This close contact between the genetically diverse mother and fetus requires numerous mechanisms of immune tolerance initiated by trophoblast cell signals. However, in a placental condition known as villitis of unknown etiology (VUE), there appears to be a breakdown in this tolerance allowing maternal cytotoxic T-cells to traffic into the placenta to destroy fetal villi. VUE is associated with several gestational complications and an increased risk of recurrence in a subsequent pregnancy, making it a significant obstetrical diagnosis. The cause of VUE remains unclear, but dysfunctional signaling through immune checkpoint pathways, which have a critical role in blunting immune responses, may play an important role. Therefore, using placental tissue from normal pregnancy (n=8), VUE (n=8) and cytomegalovirus (CMV) infected placentae (n=4), we aimed to identify differences in programmed cell death 1 (PD-1), programmed death ligand-1 (PD-L1), LAG3 and CTLA4 expression between these etiologies by immunohistochemistry (IHC). Results demonstrated significantly lower expression of PD-L1 on trophoblast cells from VUE placentae compared to control and CMV infection. Additionally, we observed significantly higher counts of PD-1+ (>100 cells/image) and LAG3+ (0-120 cells/image) cells infiltrating into the villi during VUE compared to infection and control. Minimal CTLA4 staining was observed in all placentae, with only a few Hofbauer cells staining positive. Together, this suggests that a loss of tolerance through immune checkpoint signaling may be an important mechanism leading to the activation and trafficking of maternal cells into fetal villi during VUE. Further mechanistic studies are warranted to understand possible allograft rejection more clearly and in developing effective strategies to prevent this condition from occurring in utero.

Maternal Neutrophil Depletion Fails to Avert Systemic Lipopolysaccharide-Induced Early Pregnancy Defects in Mice.

Maternal infection-induced early pregnancy complications arise from perturbation of the immune environment at the uterine early blastocyst implantation site (EBIS), yet the underlying mechanisms remain unclear. Here, we demonstrated in a mouse model that the progression of normal pregnancy from days 4 to 6 induced steady migration of leukocytes away from the uterine decidual stromal zone (DSZ) that surrounds the implanted blastocyst. Uterine macrophages were found to be CD206+ M2-polarized. While monocytes were nearly absent in the DSZ, DSZ cells were found to express monocyte marker protein Ly6C. Systemic endotoxic lipopolysaccharide (LPS) exposure on day 5 of pregnancy led to: (1) rapid (at 2 h) induction of neutrophil chemoattractants that promoted huge neutrophil infiltrations at the EBISs by 24 h; (2) rapid (at 2 h) elevation of mRNA levels of MyD88, but not Trif, modulated cytokines at the EBISs; and (3) dose-dependent EBIS defects by day 7 of pregnancy. Yet, elimination of maternal neutrophils using anti-Ly6G antibody prior to LPS exposure failed to avert LPS-induced EBIS defects allowing us to suggest that activation of Tlr4-MyD88 dependent inflammatory pathway is involved in LPS-induced defects at EBISs. Thus, blocking the activation of the Tlr4-MyD88 signaling pathway may be an interesting approach to prevent infection-induced pathology at EBISs.

Role of ACE2 in pregnancy and potential implications for COVID-19 susceptibility.

In times of coronavirus disease 2019 (COVID-19), the impact of severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 infection on pregnancy is still unclear. The presence of angiotensin-converting enzyme (ACE) 2 (ACE2), the main receptor for SARS-CoV-2, in human placentas indicates that this organ can be vulnerable for viral infection during pregnancy. However, for this to happen, additional molecular processes are critical to allow viral entry in cells, its replication and disease manifestation, particularly in the placenta and/or feto-maternal circulation. Beyond the risk of vertical transmission, COVID-19 is also proposed to deplete ACE2 protein and its biological actions in the placenta. It is postulated that such effects may impair essential processes during placentation and maternal hemodynamic adaptations in COVID-19 pregnancy, features also observed in several disorders of pregnancy. This review gathers information indicating risks and protective features related to ACE2 changes in COVID-19 pregnancies. First, we describe the mechanisms of SARS-CoV-2 infection having ACE2 as a main entry door and current evidence of viral infection in the placenta. Further, we discuss the central role of ACE2 in physiological systems such as the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS), both active during placentation and hemodynamic adaptations of pregnancy. Significant knowledge gaps are also identified and should be urgently filled to better understand the fate of ACE2 in COVID-19 pregnancies and the potential associated risks. Emerging knowledge will be able to improve the early stratification of high-risk pregnancies with COVID-19 exposure as well as to guide better management and follow-up of these mothers and their children.

Placental response to maternal SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic affected people at all ages. Whereas pregnant women seemed to have a worse course of disease than age-matched non-pregnant women, the risk of feto-placental infection is low. Using a cohort of 66 COVID-19-positive women in late pregnancy, we correlated clinical parameters with disease severity, placental histopathology, and the expression of viral entry and Interferon-induced transmembrane (IFITM) antiviral transcripts. All newborns were negative for SARS-CoV-2. None of the demographic parameters or placental histopathological characteristics were associated with disease severity. The fetal-maternal transfer ratio for IgG against the N or S viral proteins was commonly less than one, as recently reported. We found that the expression level of placental ACE2, but not TMPRSS2 or Furin, was higher in women with severe COVID-19. Placental expression of IFITM1 and IFITM3, which have been implicated in antiviral response, was higher in participants with severe disease. We also showed that IFITM3 protein expression, which localized to early and late endosomes, was enhanced in severe COVID-19. Our data suggest an association between disease severity and placental SARS-CoV-2 processing and antiviral pathways, implying a role for these proteins in placental response to SARS-CoV-2.

Pregnancy and pandemics: Interaction of viral surface proteins and placenta cells.

Throughout history, pandemics of infectious diseases caused by emerging viruses have spread worldwide. Evidence from previous outbreaks demonstrated that pregnant women are at high risk of contracting the diseases and suffering from adverse outcomes. However, while some viruses can cause major health complications for the mother and her fetus, others do not appear to affect pregnancy. Viral surface proteins bind to specific receptors on the cellular membrane of host cells and begin therewith the infection process. During pregnancy, the molecular features of these proteins may determine specific target cells in the placenta, which may explain the different outcomes. In this review, we display information on Variola, Influenza, Zika and Corona viruses focused on their surface proteins, effects on pregnancy, and possible target placental cells. This will contribute to understanding viral entry during pregnancy, as well as to develop strategies to decrease the incidence of obstetrical problems in current and future infections.

Maternal nutrients and effects of gestational COVID-19 infection on fetal brain development.

BACKGROUND & AIMS: Maternal gestational infection is a well-characterized risk factor for offsprings' development of mental disorders including schizophrenia, autism, and attention deficit disorder. The inflammatory response elicited by the infection is partly directed against the placenta and fetus and is the putative pathogenic mechanism for fetal brain developmental abnormalities. Fetal brain abnormalities are generally irreversible after birth and increase risk for later mental disorders. Maternal immune activation in animals models this pathophysiology. SARS-CoV-2 produces maternal inflammatory responses during pregnancy similar to previously studied common respiratory viruses. METHOD: Choline, folic acid, Vitamin D, and n-3 polyunsaturated fatty acids are among the nutrients that have been studied as possible mitigating factors for effects of maternal infection and inflammation on fetal development. Clinical and animal studies relevant to their use in pregnant women who have been infected are reviewed. RESULTS: Higher maternal choline levels have positive effects on the development of brain function for infants of mothers who experienced viral infections in early pregnancy. No other nutrient has been studied in the context of viral inflammation. Vitamin D reduces pro-inflammatory cytokines in some, but not all, studies. Active folic acid metabolites decrease anti-inflammatory cytokines. N-3 polyunsaturated fatty acids have no effect. CONCLUSIONS: Vitamin D and folic acid are already supplemented in food additives and in prenatal vitamins. Despite recommendations by several public health agencies and medical societies, choline intake is often inadequate in early gestation when the brain is forming. A public health initiative for choline supplements during the pandemic could be helpful for women planning or already pregnant who also become exposed or infected with SARS-CoV-2.

Maternal respiratory SARS-CoV-2 infection in pregnancy is associated with a robust inflammatory response at the maternal-fetal interface.

BACKGROUND: Pregnant women are at increased risk for severe outcomes from coronavirus disease 2019 (COVID-19), but the pathophysiology underlying this increased morbidity and its potential effect on the developing fetus is not well understood. METHODS: We assessed placental histology, ACE2 expression, and viral and immune dynamics at the term placenta in pregnant women with and without respiratory severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. FINDINGS: The majority (13 of 15) of placentas analyzed had no detectable viral RNA. ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term, suggesting that low ACE2 expression may protect the term placenta from viral infection. Using immortalized cell lines and primary isolated placental cells, we found that cytotrophoblasts, the trophoblast stem cells and precursors to syncytiotrophoblasts, rather than syncytiotrophoblasts or Hofbauer cells, are most vulnerable to SARS-CoV-2 infection in vitro. To better understand potential immune mechanisms shielding placental cells from infection in vivo, we performed bulk and single-cell transcriptomics analyses and found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited robust immune responses, including increased activation of natural killer (NK) and T cells, increased expression of interferon-related genes, as well as markers associated with pregnancy complications such as preeclampsia. CONCLUSIONS: SARS-CoV-2 infection in late pregnancy is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. FUNDING: NIH (T32GM007205, F30HD093350, K23MH118999, R01AI157488, U01DA040588) and Fast Grant funding support from Emergent Ventures at the Mercatus Center.

SARS-CoV-2 colonization of maternal and fetal cells of the human placenta promotes alteration of local renin-angiotensin system.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection appears to increase the risk of adverse pregnancy outcomes, such as pre-eclampsia in pregnant women. The mechanism(s) by which this occurs remains unclear. METHODS: We investigated the pathophysiology of SARS-CoV-2 at maternal-fetal interface in pregnant women who tested positive for the virus using RNA in situ hybridization (viral RNA), immunohistochemistry, and hematoxylin and eosin staining. To investigate whether viral infection alters the renin angiotensin system (RAS) in placenta, which controls blood pressure, we treated human trophoblasts with recombinant spike protein or a live modified virus with a vesicular stomatitis viral backbone expressing spike protein (VSV-S). FINDINGS: Viral colonization was highest in maternal decidua, fetal trophoblasts, Hofbauer cells, and in placentas delivered prematurely. We localized SARS-CoV-2 to cells expressing angiotensin-converting enzyme 2 (ACE2) and demonstrate that infected placentas had significantly reduced ACE2. In response to both spike protein and VSV-S, cellular ACE2 decreased although angiotensin II receptor type 1 (AT1R) increased with concomitant increase in soluble fms-like tyrosine kinase-1 (sFlt1). Viral infection decreased pro-angiogenic factors, AT2R, and placental growth factor, which competitively binds to sFlt1. Sera from infected pregnant women had elevated levels of sFlt1 and angiotensin II type 1-receptor autoantibodies prior to delivery, both signatory markers of pre-eclampsia. CONCLUSIONS: SARS-CoV-2 colonizes ACE2-expressing maternal and fetal cells in the placenta. Infection in pregnant women correlates with alteration of placental RAS. As RAS regulates blood pressure, SARS-CoV-2 infection may thus increase adverse hemodynamic outcomes, such as pre-eclampsia in pregnant women. FUNDING: NIH/NICHD grants R01 HD091218 and 3R01HD091218-04S1 (RADx-UP Supplement).