Zika virus co-opts microRNA networks to persist in placental niches detected by spatial transcriptomics.

BACKGROUND: Zika virus congenital infection evades double-stranded RNA detection and may persist in the placenta for the duration of pregnancy without accompanying overt histopathologic inflammation. Understanding how viruses can persist and replicate in the placenta without causing overt cellular or tissue damage is fundamental to deciphering mechanisms of maternal-fetal vertical transmission. OBJECTIVE: Placenta-specific microRNAs are believed to be a tenet of viral resistance at the maternal-fetal interface. We aimed to test the hypothesis that the Zika virus functionally disrupts placental microRNAs, enabling viral persistence and fetal pathogenesis. STUDY DESIGN: To test this hypothesis, we used orthogonal approaches in human and murine experimental models. In primary human trophoblast cultures (n=5 donor placentae), we performed Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation to identify any significant alterations in the functional loading of microRNAs and their targets onto the RNA-induced silencing complex. Trophoblasts from same-donors were split and infected with a contemporary first-passage Zika virus strain HN16 (multiplicity of infection=1 plaque forming unit per cell) or mock infected. To functionally cross-validate microRNA-messenger RNA interactions, we compared our Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation results with an independent analysis of published bulk RNA-sequencing data from human placental disk specimens (n=3 subjects; Zika virus positive in first, second, or third trimester, CD45- cells sorted by flow cytometry) and compared it with uninfected controls (n=2 subjects). To investigate the importance of these microRNA and RNA interference networks in Zika virus pathogenesis, we used a gnotobiotic mouse model uniquely susceptible to the Zika virus. We evaluated if small-molecule enhancement of microRNA and RNA interference pathways with enoxacin influenced Zika virus pathogenesis (n=20 dams total yielding 187 fetal specimens). Lastly, placentae (n=14 total) from this mouse model were analyzed with Visium spatial transcriptomics (9743 spatial transcriptomes) to identify potential Zika virus-associated alterations in immune microenvironments. RESULTS: We found that Zika virus infection of primary human trophoblast cells led to an unexpected disruption of placental microRNA regulation networks. When compared with uninfected controls, Zika virus-infected placentae had significantly altered SLC12A8, SDK1, and VLDLR RNA-induced silencing complex loading and transcript levels (-22; adjusted P value <.05; Wald-test with false discovery rate correction q<0.05). In silico microRNA target analyses revealed that 26 of 119 transcripts (22%) in the transforming growth factor-ß signaling pathway were targeted by microRNAs that were found to be dysregulated following Zika virus infection in trophoblasts. In gnotobiotic mice, relative to mock controls, Zika virus-associated fetal pathogenesis included fetal growth restriction (P=.036) and viral persistence in placental tissue (P=.011). Moreover, spatial transcriptomics of murine placentae revealed that Zika virus-specific placental niches were defined by significant up-regulation of complement cascade components and coordinated changes in transforming growth factor-ß gene expression. Finally, treatment of Zika virus-infected mice with enoxacin abolished placental Zika virus persistence, rescued the associated fetal growth restriction, and the Zika virus-associated transcriptional changes in placental immune microenvironments were no longer observed. CONCLUSION: These results collectively suggest that (1) Zika virus infection and persistence is associated with functionally perturbed microRNA and RNA interference pathways specifically related to immune regulation in placental microenvironments and (2) enhancement of placental microRNA and RNA interference pathways in mice rescued Zika virus-associated pathogenesis, specifically persistence of viral transcripts in placental microenvironments and fetal growth restriction.

SARS-CoV-2 niches in human placenta revealed by spatial transcriptomics.

BACKGROUND: Functional placental niches are presumed to spatially separate maternal-fetal antigens and restrict the vertical transmission of pathogens. We hypothesized a high-resolution map of placental transcription could provide direct evidence for niche microenvironments with unique functions and transcription profiles. METHODS: We utilized Visium Spatial Transcriptomics paired with H&E staining to generate 17,927 spatial transcriptomes. By integrating these spatial transcriptomes with 273,944 placental single-cell and single-nuclei transcriptomes, we generated an atlas composed of at least 22 subpopulations in the maternal decidua, fetal chorionic villi, and chorioamniotic membranes. FINDINGS: Comparisons of placentae from uninfected healthy controls (n = 4) with COVID-19 asymptomatic (n = 4) and symptomatic (n = 5) infected participants demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in syncytiotrophoblasts occurred in both the presence and the absence of maternal clinical disease. With spatial transcriptomics, we found that the limit of detection for SARS-CoV-2 was 1/7,000 cells, and placental niches without detectable viral transcripts were unperturbed. In contrast, niches with high SARS-CoV-2 transcript levels were associated with significant upregulation in pro-inflammatory cytokines and interferon-stimulated genes, altered metallopeptidase signaling (TIMP1), with coordinated shifts in macrophage polarization, histiocytic intervillositis, and perivillous fibrin deposition. Fetal sex differences in gene expression responses to SARS-CoV-2 were limited, with confirmed mapping limited to the maternal decidua in males. CONCLUSIONS: High-resolution placental transcriptomics with spatial resolution revealed dynamic responses to SARS-CoV-2 in coordinate microenvironments in the absence and presence of clinically evident disease. FUNDING: This work was supported by the NIH (R01HD091731 and T32-HD098069), NSF (2208903), the Burroughs Welcome Fund and the March of Dimes Preterm Birth Research Initiatives, and a Career Development Award from the American Society of Gene and Cell Therapy.

Placental Vascular and Inflammatory Findings from Pregnancies Diagnosed with Coronavirus Disease 2019: A Systematic Review and Meta-analysis.

We aimed to perform a meta-analysis of the literature concerning histopathologic findings in the placentas of women with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection during pregnancy. Searches for articles in English included PubMed, Web of Science, Google Scholar, and reference lists (up to April 2021). Studies presenting data on placental histopathology according to the Amsterdam Consensus Group criteria in SARS-CoV-2 positive and negative pregnancies were identified. Lesions were categorized into: maternal and fetal vascular malperfusion (MVM and FVM, respectively), acute placental inflammation with maternal and fetal inflammatory response (MIR and FIR, respectively), chronic inflammatory lesions (CILs), and increased perivillous fibrin deposition (PVFD). A total of 15 studies reporting on 19,025 placentas, n = 699 of which were derived from women who were identified as being infected with SARS-CoV-2 and 18,326 as SARS-CoV-2-negative controls, were eligible for analysis. No significant difference in incidence of MVM (odds ratio [OR]: 1.18, 95% confidence interval [CI]: 0.73-1.90), FVM (OR: 1.23, 95% CI: 0.63-2.42), MIR (OR: 0.66, 95% CI: 0.29-1.52) or FIR (OR: 0.85, 95% CI: 0.44-1.63), and CILs (OR: 0.97, 95% CI: 0.55-1.72) was found between placentae from gravida identified as being SARS-CoV-2 infected. However, placenta from gravida identified as being infected with SARS-CoV-2 were associated with significantly increased occurrence of PVFD (OR: 2.77, 95% CI: 1.06-7.27). After subgroup analyses based on clinical severity of COVID-19 infection, no significant difference was observed in terms of reported placental pathology between symptomatic or asymptomatic SARS-CoV-2 gravidae placenta. Current evidence based on the available literature suggests that the only pathologic finding in the placentae of women who are pregnant identified as having been infected with SARS-CoV-2 was an increased prevalence of PVFD. KEY POINTS: · No association between SARS-CoV-2 and maternal or fetal placental malperfusion.. · No association between SARS-CoV-2 and maternal or fetal inflammatory response.. · SARS-CoV-2 is associated with increased perivillous fibrin deposition in placenta..

Perinatal COVID-19 outcomes: evaluating the strength of current evidence.

Purpose of the Study: Viral respiratory diseases, like those caused by novel strains of influenza and Coronaviridae, have historically disproportionately affected pregnant women and conferred increased risk of adverse perinatal outcomes. Initial reports published from Wuhan, China identified only limited symptoms in pregnant women and no cases of mortality, but more recent reports from other regions of the world have reported contrasting information. The purpose of the study was to evaluate initially published cases of SARS-CoV-2 infection in pregnant women in China and compare them to subsequently published studies from the remainder of the world.Materials and Methods: This review curates 199 maternal published cases of SARS-CoV-2 infection and COVID-19 initially reported in the literature from China and contrasts them to more recent literature reporting clinical findings and outcomes of 729 selected cases from the rest of the world, including the United States.Results: Overall, initial case reports and series from China reported no cases of maternal mortality, which contrasts with subsequent reports from other regions of the world demonstrating significant morbidity and mortality can and does occur in pregnant women infected with SARS-CoV-2.Conclusion: While initial reports suggest limited risks of infection in pregnancy with SARS-CoV-2, subsequent findings have demonstrated pregnant women are at risk for severe morbidity and mortality. Case studies and series that are imperative in the early stages of a pandemic to provide data on a novel pathogen cannot be used to provide generalizable information predicting group risks.

Complex species and strain ecology of the vaginal microbiome from pregnancy to postpartum and association with preterm birth.

BACKGROUND: Lactobacillus was described as a keystone bacterial taxon in the human vagina over 100 years ago. Using metagenomics, we and others have characterized lactobacilli and other vaginal taxa across health and disease states, including pregnancy. While shifts in community membership have been resolved at the genus/species level, strain dynamics remain poorly characterized. METHODS: We performed a metagenomic analysis of the complex ecology of the vaginal econiche during and after pregnancy in a large U.S. based longitudinal cohort of women who were initially sampled in the third trimester of pregnancy, then validated key findings in a second cohort of women initially sampled in the second trimester of pregnancy. FINDINGS: First, we resolved microbial species and strains, interrogated their co-occurrence patterns, and probed the relationship between keystone species and preterm birth outcomes. Second, to determine the role of human heredity in shaping vaginal microbial ecology in relation to preterm birth, we performed a mtDNA-bacterial species association analysis. Finally, we explored the clinical utility of metagenomics in detection and co-occurrence patterns for the pathobiont Group B Streptococcus (causative bacterium of invasive neonatal sepsis). CONCLUSIONS: Our highly refined resolutions of the vaginal ecology during and post-pregnancy provide insights into not only structural and functional community dynamics, but highlight the capacity of metagenomics to reveal finer aspects of the vaginal microbial ecologic framework. FUNDING: NIH-NINR R01NR014792, NIH-NICHD R01HD091731, NIH National Children's Study Formative Research, Burroughs Wellcome Fund Preterm Birth Initiative, March of Dimes Preterm Birth Research Initiative, NIH-NIGMS (K12GM084897, T32GM007330, T32GM088129).

Placental Autophagy and Viral Replication Co-localize in Human and Non-human Primate Placentae Following Zika Virus Infection: Implications for Therapeutic Interventions.

Background: Multiple studies have shown both induction and inhibition of autophagy during Zika virus (ZIKV) infection. While some have proposed mechanisms by which autophagic dysregulation might facilitate ZIKV vertical transmission, there is a lack of in situ data in human and non-human primate models. This is an especially pertinent question as autophagy-inhibitors, such as hydroxychloroquine, have been proposed as potential therapeutic agents aimed at preventing vertical transmission of ZIKV and other RNA viruses. Objectives: Given the paucity of pre-clinical data in support of either autophagic enhancement or inhibition of placental ZIKV viral infection, we sought to assess cellular, spatial, and temporal associations between placental ZIKV infection and measures of autophagy in human primary cell culture and congenital infection cases, as well as an experimental non-human primate (marmoset, Callithrix jacchus) model. Study Design: Primary trophoblast cells were isolated from human placentae (n = 10) and infected in vitro with ZIKV. Autophagy-associated gene expression (ULK-1, BECN1, ATG5, ATG7, ATG12, ATG16L1, MAP1LC3A, MAP1LC3B, p62/SQSTM1) was then determined by TaqMan qPCR to determine fold-change with ZIKV-infection. In in vivo validation experiments, autophagy genes LC3B and p62/SQSTM1 were probed using in situ hybridization (ISH) in the placentae of human Congenital Zika Syndrome (CZS) cases (n = 3) and ZIKV-infected marmoset placenta (n = 1) and fetal tissue (n = 1). Infected and uninfected villi were compared for mean density and co-localization of autophagic protein markers. Results: Studies of primary cultured human trophoblasts revealed decreased expression of autophagy genes ATG5 and p62/SQSTM1 in ZIKV-infected trophoblasts [ATG5 fold change (±SD) 0.734-fold (±0.722), p = 0.036; p62/SQSTM1 0.661-fold (±0.666), p = 0.029]. Histologic examination by ISH and immunohistochemistry confirmed spatial association of autophagy and ZIKV infection in human congenital infection cases, as well as marmoset placental and fetal tissue samples. When quantified by densitometric data, autophagic protein LC3B, and p62/SQSTM1 expression in marmoset placenta were significantly decreased in in situ ZIKV-infected villi compared to less-infected areas [LC3B mean 0.951 (95% CI, 0.930-0.971), p = 0.018; p62/SQSTM1 mean 0.863 (95% CI, 0.810-0.916), p = 0.024]. Conclusion: In the current study, we observed that in the non-transformed human and non-human primate placenta, disruption (specifically down-regulation) of autophagy accompanies later ZIKV replication in vitro, in vivo, and in situ. The findings collectively suggest that dysregulated autophagy spatially and temporally accompanies placental ZIKV replication, providing the first in situ evidence in relevant primate pre-clinical and clinical models for the importance of timing of human therapeutic strategies aimed at agonizing/antagonizing autophagy. These studies have likely further implications for other congenitally transmitted viruses, particularly the RNA viruses, given the ubiquitous nature of autophagic disruption and dysregulation in host responses to viral infection during pregnancy.

Maternal diet alters human milk oligosaccharide composition with implications for the milk metagenome.

Human milk is the optimal nutrition source for infants, and oligosaccharides represent the third most abundant component in milk after lactose and fat. Human milk oligosaccharides (HMO) are favorable macromolecules which are, interestingly, indigestible by the infant but serve as substrates for bacteria. Hypothesizing that the maternal diet itself might influence HMO composition, we sought to directly determine the effect maternal diet on HMO and the milk bacteria. Employing a human cross-over study design, we demonstrate that distinct maternal dietary carbohydrate and energy sources preferentially alter milk concentrations of HMO, including fucosylated species. We find significant associations between the concentration of HMO-bound fucose and the abundance of fucosidase (a bacterial gene that digests fucose moieties) harbored by milk bacteria. These studies reveal a successive mechanism by which the maternal diet during lactation alters milk HMO composition, which in turn shapes the functional milk microbiome prior to infant ingestion.

Maternal death due to COVID-19.

BACKGROUND: Despite 2.5 million infections and 169,000 deaths worldwide (as of April 20, 2020), no maternal deaths and only a few pregnant women afflicted with severe respiratory morbidity have been reported to be related to COVID-19 disease. Given the disproportionate burden of severe and fatal respiratory disease previously documented among pregnant women following other coronavirus-related outbreaks (SARS-CoV in 2003 and MERS-CoV in 2012) and influenza pandemics over the last century, the absence of reported maternal morbidity and mortality with COVID-19 disease is unexpected. OBJECTIVE: To describe maternal and perinatal outcomes and death in a case series of pregnant women with COVID-19 disease. STUDY DESIGN: We describe here a multiinstitution adjudicated case series from Iran that includes 9 pregnant women diagnosed with severe COVID-19 disease in their second or third trimester. All 9 pregnant women received a diagnosis of SARS-CoV-2 infection by reverse transcription polymerase chain reaction nucleic acid testing. Outcomes of these women were compared with their familial/household members with contact to the affected patient on or after their symptom onset. All data were reported at death or after a minimum of 14 days from date of admission with COVID-19 disease. RESULTS: Among 9 pregnant women with severe COVID-19 disease, at the time of reporting, 7 of 9 died, 1 of 9 remains critically ill and ventilator dependent, and 1 of 9 recovered after prolonged hospitalization. We obtained self-verified familial/household cohort data in all 9 cases, and in each and every instance, maternal outcomes were more severe compared with outcomes of other high- and low-risk familial/household members (n=33 members for comparison). CONCLUSION: We report herein maternal deaths owing to COVID-19 disease. Until rigorously collected surveillance data emerge, it is prudent to be aware of the potential for maternal death among pregnant women diagnosed as having COVID-19 disease in their second or third trimester.

The Development of the Human Microbiome: Why Moms Matter.

The human body is cohabitated with trillions of commensal bacteria that are essential for our health. However, certain bacteria can also cause diseases in the human host. Before the microbiome can be attributed to disease risk and pathogenesis, normal acquisition and development of the microbiome must be understood. Here, we explore the evidence surrounding in utero microbial exposures and the significant of this exposure in the proper development of the fetal and neonatal microbiome. We further explore the development of the fetal and neonatal microbiome and its relationship to preterm birth, feeding practices, and mode of delivery, and maternal diet.

Visualization of microbes by 16S in situ hybridization in term and preterm placentas without intraamniotic infection.

BACKGROUND: Numerous reports have documented bacteria in the placental membranes and basal plate decidua in the absence of immunopathology using histologic techniques. Similarly, independent metagenomic characterizations have identified an altered taxonomic makeup in association with spontaneous preterm birth. Here we sought to corroborate these findings by localizing presumptive intact bacteria using molecular histology within the placental microanatomy. OBJECTIVE: Here we examined for microbes in term and preterm gestations using a signal-amplified 16S universal in situ hybridization probe set for bacterial rRNA, alongside traditional histologic methods of Warthin-Starry and Gram stains, as well as clinical culture methodologies. We further sought to differentiate accompanying 16S gene sequencing taxonomic profiles from germ-free (gnotobiotic) mouse and extraction and amplicon contamination controls. STUDY DESIGN: Placentas were collected from a total of 53 subjects, composed of term labored (n = 4) and unlabored cesarean deliveries (n = 22) and preterm vaginal (n = 18) and cesarean deliveries (n = 8); a placenta from a single subject with clinical and histologic evident choriomanionitis was employed as a positive control (n = 1). The preterm cohort included spontaneous preterm birth with (n = 6) and without (n = 10) preterm premature rupture of membranes, as well as medically indicated preterm births (n = 10). Placental microbes were visualized using an in situ hybridization probe set designed against highly conserved regions of the bacterial 16S ribosome, which produces an amplified stable signal using branched DNA probes. Extracted bacterial nucleic acids from these same samples were subjected to 16S rRNA metagenomic sequencing (Illumina, V4) for course taxonomic analysis, alongside environmental and kit contaminant controls. A subset of unlabored, cesarean-delivered term pregnancies were also assessed with clinical culture for readily cultivatable pathogenic microbes. RESULTS: Molecular in situ hybridization of bacterial rRNA enabled visualization and localization of low-abundance microbes after systematic high-power scanning. Despite the absence of clinical or histologic chorioamnionitis in 52 of 53 subjects, instances of 16S rRNA signal were confidently observed in 13 of 16 spontaneous preterm birth placentas, which was not significantly different from term unlabored cesarean specimens (18 of 22; P > .05). 16S rRNA signal was largely localized to the villous parenchyma and/or syncytiotrophoblast, and less commonly the chorion and the maternal intervillous space. In all term and unlabored cesarean deliveries, visualization of evident placental microbes by in situ hybridization occurred in the absence of clinical or histologic detection using conventional clinical cultivation, hematoxylin-eosin, and Gram staining. In 1 subject, appreciable villous bacteria localized to an infarction, where 16S microbial detection was confirmed by Warthin-Starry stain. In all instances, parallel sample principle coordinate analysis using Bray-Cutis distances of 16S rRNA gene sequencing data demonstrated consistent taxonomic distinction from all negative or potential contamination controls (P = .024, PERMANOVA). Classification from contaminant filtered data identified a distinct taxonomic makeup among term and preterm cohorts when compared with contaminant controls (false discovery rate <0.05). CONCLUSION: Presumptively intact placental microbes are visualized as low-abundance, low-biomass and sparse populations within the placenta regardless of gestational age and mode of delivery. Their taxonomic makeup is distinct from contamination controls. These findings further support several previously published findings, including our own, which have used metagenomics to characterize low-abundance and low-biomass microbial communities in the placenta.

Clinical Importance of Placental Testing among Suspected Cases of Congenital Zika Syndrome.

Contemporaneous Zika virus (ZIKV) strains can cause congenital Zika syndrome (CZS). Current ZIKV clinical laboratory testing strategies are limited and include IgM serology (which may wane 12 weeks after initial exposure) and nucleic acid testing (NAT) of maternal serum, urine, and placenta for (+) strand ZIKV RNA (which is often transient). The objectives of this study were to determine if use of additional molecular tools, such as quantitative PCR and microscopy, would add to the diagnostic value of current standard placental ZIKV testing in cases with maternal endemic exposure and indeterminate testing. ZIKV RNA was quantified from dissected sections of placental villi, chorioamnion sections, and full cross-sections of umbilical cord in all cases examined. Quantitation with high-resolution automated electrophoresis determined relative amounts of precisely verified ZIKV (74-nt amplicons). In order to localize and visualize stable and actively replicating placental ZIKV in situ, labeling of flaviviridae glycoprotein, RNA ISH against both (+) and (⁻) ZIKV-specific ssRNA strands, and independent histologic examination for significant pathologic changes were employed. We demonstrate that the use of these molecular tools added to the diagnostic value of placental ZIKV testing among suspected cases of congenital Zika syndrome with poorly ascribed maternal endemic exposure.

Increases in bioactive lipids accompany early metabolic changes associated with ß-cell expansion in response to short-term high-fat diet.

Pancreatic ß-cell expansion is a highly regulated metabolic adaptation to increased somatic demands, including obesity and pregnancy; adult ß cells otherwise rarely proliferate. We previously showed that high-fat diet (HFD) feeding induces mouse ß-cell proliferation in less than 1 wk in the absence of insulin resistance. Here we metabolically profiled tissues from a short-term HFD ß-cell expansion mouse model to identify pathways and metabolite changes associated with ß-cell proliferation. Mice fed HFD vs. chow diet (CD) showed a 14.3% increase in body weight after 7 days; ß-cell proliferation increased 1.75-fold without insulin resistance. Plasma from 1-wk HFD-fed mice induced ß-cell proliferation ex vivo. The plasma, as well as liver, skeletal muscle, and bone, were assessed by LC and GC mass-spectrometry for global metabolite changes. Of the 1,283 metabolites detected, 159 showed significant changes [false discovery rate (FDR) < 0.1]. The majority of changes were in liver and muscle. Pathway enrichment analysis revealed key metabolic changes in steroid synthesis and lipid metabolism, including free fatty acids and other bioactive lipids. Other important enrichments included changes in the citric acid cycle and 1-carbon metabolism pathways implicated in DNA methylation. Although the minority of changes were observed in bone and plasma (<20), increased p-cresol sulfate was increased >4 fold in plasma (the largest increase in all tissues), and pantothenate (vitamin B5) decreased >2-fold. The results suggest that HFD-mediated ß-cell expansion is associated with complex, global metabolite changes. The finding could be a significant insight into Type 2 diabetes pathogenesis and potential novel drug targets.

Timing of gestational exposure to Zika virus is associated with postnatal growth restriction in a murine model.

BACKGROUND: Vertical transmission of Zika virus leads to infection of neuroprogenitor cells and destruction of brain parenchyma. Recent evidence suggests that the timing of infection as well as host factors may affect vertical transmission. As a result, congenital Zika virus infection may only become clinically apparent in the postnatal period. OBJECTIVE: We sought to develop an outbred mouse model of Zika virus vertical transmission to determine if the timing of gestational Zika virus exposure yields phenotypic differences at birth and through adolescence. We hypothesized that later gestational inoculations would only become apparent in adolescence. STUDY DESIGN: To better recapitulate human exposures, timed pregnant Swiss-Webster dams (n = 15) were subcutaneously inoculated with 1 × 104 plaque-forming units of first passage contemporary Zika virus HN16 strain or a mock injection on embryonic day 4, 8, or 12 with bioactive antiinterferon alpha receptor antibody administered in days preceding and proceeding inoculation. The antibody was given to prevent the robust type I interferon signaling cascade that make mice inherently resistant to Zika virus infection. At birth and adolescence (6 weeks of age) offspring were assessed for growth, brain weight, and biparietal head diameters, and Zika virus viral levels by reverse transcription-polymerase chain reaction or in situ hybridization. RESULTS: Pups of Zika virus-infected dams infected at embryonic days 4 and 8 but not 12 were growth restricted (P < .003). Brain weights were significantly smaller at birth (P = .01) for embryonic day 8 Zika virus-exposed offspring. At 6 weeks of age, biparietal diameters were smaller for all Zika virus-exposed males and females (P < .05), with embryonic day 8-exposed males smallest by biparietal diameter and growth-restriction measurements (weight >2 SD, P = .0007). All pups and adolescent mice were assessed for Zika virus infection by reverse transcription-polymerase chain reaction. Analysis of all underweight pups reveled 1 to be positive for neuronal Zika virus infection by in situ hybridization, while a second moribund animal was diffusely positive at 8 days of age by Zika virus infectivity throughout the brain, kidneys, and intestine. CONCLUSION: These findings demonstrate that postnatal effects of infection occurring at single time points continue to be detrimental to offspring in the postnatal period in a subset of littermates and subject to a window of gestational susceptibility coinciding with placentation. This model recapitulates frequently encountered clinical scenarios in nonendemic regions, including the majority of the United States, where travel-related exposure occurs in short and well-defined windows of gestation. Our low rate of infection and relatively rare evidence of congenital Zika syndrome parallels human population-based data.

Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication.

Zika virus (ZIKV) is an emerging mosquito-borne (Aedes genus) arbovirus of the Flaviviridae family. Although ZIKV has been predominately associated with a mild or asymptomatic dengue-like disease, its appearance in the Americas has been accompanied by a multi-fold increase in reported incidence of fetal microcephaly and brain malformations. The source and mode of vertical transmission from mother to fetus is presumptively transplacental, although a causal link explaining the interval delay between maternal symptoms and observed fetal malformations following infection has been missing. In this study, we show that primary human placental trophoblasts from non-exposed donors (n = 20) can be infected by primary passage ZIKV-FLR isolate, and uniquely allowed for ZIKV viral RNA replication when compared to dengue virus (DENV). Consistent with their being permissive for ZIKV infection, primary trophoblasts expressed multiple putative ZIKV cell entry receptors, and cellular function and differentiation were preserved. These findings suggest that ZIKV-FLR strain can replicate in human placental trophoblasts without host cell destruction, thereby serving as a likely permissive reservoir and portal of fetal transmission with risk of latent microcephaly and malformations.

Testing for Zika virus infection in pregnancy: key concepts to deal with an emerging epidemic.

Zika virus is an emerging mosquito-borne (Aedes genus) arbovirus of the Flaviviridae family. Following epidemics in Micronesia and French Polynesia during the past decade, more recent Zika virus infection outbreaks were first reported in South America as early as May 2013 and spread to now 50 countries throughout the Americas. Although no other flavivirus has previously been known to cause major fetal malformations following perinatal infection, reports of a causal link between Zika virus and microcephaly, brain and ocular malformations, and fetal loss emerged from hard-hit regions of Brazil by October 2015. Among the minority of infected women with symptoms, clinical manifestations of Zika virus infection may include fever, headache, arthralgia, myalgia, and maculopapular rash; however, only 1 of every 4-5 people who are infected have any symptoms. Thus, clinical symptom reporting is an ineffective screening tool for the relative risk assessment of Zika virus infection in the majority of patients. As previously occurred with other largely asymptomatic viral infections posing perinatal transmission risk (such as HIV or cytomegalovirus), we must develop and implement rapid, sensitive, and specific screening and diagnostic testing for both viral detection and estimation of timing of exposure. Unfortunately, despite an unprecedented surge in attempts to rapidly advance perinatal clinical testing for a previously obscure arbovirus, there are several ongoing hindrances to molecular- and sonographic-based screening and diagnosis of congenital Zika virus infection. These include the following: (1) difficulty in estimating the timing of exposure for women living in endemic areas and thus limited interpretability of immunoglobulin M serologies; (2) cross-reaction of immunoglobulin serologies with other endemic flaviruses, such as dengue; (3) persistent viremia and viruria in pregnancy weeks to months after primary exposure; and (4) fetal brain malformations and anomalies preceding the sonographic detection of microcephaly. In this commentary, we discuss screening and diagnostic considerations that are grounded not only in the realities of current obstetrical practice in a largely global population but also in basic immunology and virology. We review recent epidemiological data pertaining to the risk of congenital Zika virus malformations based on trimester of exposure and consider side by side with emerging data demonstrating replication of Zika virus in placental and fetal tissue throughout gestation. We discuss limitations to ultrasound based strategies that rely largely or solely on the detection of microcephaly and provide alternative neurosonographic approaches for the detection of malformations that may precede or occur independent of a small head circumference. This expert review provides information that is of value for the following: (1) obstetrician, maternal-fetal medicine specialist, midwife, patient, and family in cases of suspected Zika virus infection; (2) review of the methodology for laboratory testing to explore the presence of the virus and the immune response; (3) ultrasound-based assessment of the fetus suspected to be exposed to Zika virus with particular emphasis on the central nervous system; and (4) identification of areas ready for development.

Maturation of the infant microbiome community structure and function across multiple body sites and in relation to mode of delivery.

Human microbial communities are characterized by their taxonomic, metagenomic and metabolic diversity, which varies by distinct body sites and influences human physiology. However, when and how microbial communities within each body niche acquire unique taxonomical and functional signatures in early life remains underexplored. We thus sought to determine the taxonomic composition and potential metabolic function of the neonatal and early infant microbiota across multiple body sites and assess the effect of the mode of delivery and its potential confounders or modifiers. A cohort of pregnant women in their early third trimester (n = 81) were prospectively enrolled for longitudinal sampling through 6 weeks after delivery, and a second matched cross-sectional cohort (n = 81) was additionally recruited for sampling once at the time of delivery. Samples across multiple body sites, including stool, oral gingiva, nares, skin and vagina were collected for each maternal-infant dyad. Whole-genome shotgun sequencing and sequencing analysis of the gene encoding the 16S rRNA were performed to interrogate the composition and function of the neonatal and maternal microbiota. We found that the neonatal microbiota and its associated functional pathways were relatively homogeneous across all body sites at delivery, with the notable exception of the neonatal meconium. However, by 6 weeks after delivery, the infant microbiota structure and function had substantially expanded and diversified, with the body site serving as the primary determinant of the composition of the bacterial community and its functional capacity. Although minor variations in the neonatal (immediately at birth) microbiota community structure were associated with the cesarean mode of delivery in some body sites (oral gingiva, nares and skin; R2 = 0.038), this was not true for neonatal stool (meconium; Mann-Whitney P > 0.05), and there was no observable difference in community function regardless of delivery mode. For infants at 6 weeks of age, the microbiota structure and function had expanded and diversified with demonstrable body site specificity (P < 0.001, R2 = 0.189) but without discernable differences in community structure or function between infants delivered vaginally or by cesarean surgery (P = 0.057, R2 = 0.007). We conclude that within the first 6 weeks of life, the infant microbiota undergoes substantial reorganization, which is primarily driven by body site and not by mode of delivery.

Increased Umbilical Cord PAI-1 Levels in Placental Insufficiency Are Associated with Fetal Hypoxia and Angiogenesis.

In intrauterine growth restriction (IUGR), a subset of pregnancies undergoes placental vascular dysregulation resulting in restricted blood flow and fetal hypoxemia. Altered transcription of hypoxic regulated plasminogen activator inhibitor 1 (PAI-1) has been associated with pregnancy complications and angiogenic regulation. Here we assessed circulating PAI-1 as an indicator of placental insufficiency. Venous umbilical PAI-1 of hypoxemic (VpO2 20 versus 35 mmHg, p < 0.0001) placental insufficient pregnancies (resistance index 0.9 versus 0.63, p < 0.05) (n = 18) was compared to controls (n = 12). PAI-1 was increased (~10-fold, p < 0.001) and had a positive predictive ratio of 6.7. Further, PAI-1 levels correlated to blood oxygen (r = -0.68, p < 0.0001). The plasma's angiogenic potency measured in vitro was associated with umbilical cord blood PAI-1 levels (r = 0.65, p < 0.01). This association was attenuated by PAI-1 inhibiting antibody (p < 0.001). The results demonstrate PAI-1 as a potential marker of placental insufficiency and identify its close association with pathological hypoxia and angiogenesis in a subset of growth restricted pregnancies.

Alterations in expression of imprinted genes from the H19/IGF2 loci in a multigenerational model of intrauterine growth restriction (IUGR).

BACKGROUND: The H19/IGF2 imprinted loci have attracted recent attention because of their role in cellular differentiation and proliferation, heritable gene regulation, and in utero or early postnatal growth and development. Expression from the imprinted H19/IGF2 locus involves a complex interplay of 3 means of epigenetic regulation: proper establishment of DNA methylation, promoter occupancy of CTCF, and expression of microRNA-675. We have demonstrated previously in a multigenerational rat model of intrauterine growth restriction the epigenetic heritability of adult metabolic syndrome in a F2 generation. We have further demonstrated abrogation of the F2 adult metabolic syndrome phenotype with essential nutrient supplementation of intermediates along the 1-carbon pathway and shown that alterations in the metabolome precede the adult onset of metabolic syndrome. The upstream molecular and epigenomic mediators underlying these observations, however, have yet to be elucidated fully. OBJECTIVE: In the current study, we sought to characterize the impact of the intrauterine growth-restricted lineage and essential nutrient supplementation on both levels and molecular mediators of H19 and IGF2 gene expression in the F2 generation. STUDY DESIGN: F2 intrauterine growth-restricted and sham lineages were obtained by exposing P1 (grandmaternal) pregnant dams to bilateral uterine artery ligation or sham surgery at gestational day 19.5. F1 pups were allocated to the essential nutrient supplemented or control diet at postnatal day 21, and bred at 6-7 weeks of age. Hepatic tissues from the resultant F2 offspring at birth and at weaning (day 21) were obtained. Bisulfite modification and sequencing was employed for methylation analysis. H19 and IGF2 expression was measured by quantitative polymerase chain reaction. Promoter occupancy was quantified by the use of chromatin immunoprecipitation, or ChIP, against CTCF insulator proteins. RESULTS: Growth-restricted F2 on control diet demonstrated significant down-regulation in H19 expression compared with sham lineage (0.7831 vs 1.287; P < .05); however, essential nutrient supplementation diet abrogates this difference (4.995 vs 5.100; P > .05). Conversely, Igf2 was up-regulated by essential nutrient supplemented diet on the sham lineage (2.0 fold, P = .01), an effect that was not observed in the growth restricted offspring. A significant differential methylation was observed in the promoter region of region H19 among the intrauterine growth-restricted lineage (18% vs 25%; P < .05) on a control diet, whereas the essential nutrient supplemented diet was alternately associated with hypermethylation in both lineages (sham: 50%; intrauterine growth restriction: 84%, P < .05). Consistent with essential nutrient supplementation impacting the epigenome, a decrease of CTCF promoter occupancy was observed in CTCF4 of the growth restricted lineage (2.45% vs 0.56%; P < .05) on the control diet, an effect that was repressed with essential nutrient supplementation. CONCLUSION: Heritable growth restriction is associated with changes in H19 gene expression; these changes are reversible with diet supplementation to favorably impact adult metabolic syndrome.

The perinatal microbiome and pregnancy: moving beyond the vaginal microbiome.

The human microbiome, the collective genome of the microbial community that is on and within us, has recently been mapped. The initial characterization of healthy subjects has provided investigators with a reference population for interrogating the microbiome in metabolic, intestinal, and reproductive health and disease states. Although it is known that bacteria can colonize the vagina, recent metagenomic studies have shown that the vaginal microbiome varies among reproductive age women. Similarly, the richness and diversity of intestinal microbiota also naturally fluctuate among gravidae in both human and nonhuman primates, as well as mice. Moreover, recent evidence suggests that microbiome niches in pregnancy are not limited to maternal body sites, as the placenta appears to harbor a low biomass microbiome that is presumptively established in early pregnancy and varies in association with a remote history of maternal antenatal infection as well as preterm birth. In this article, we will provide a brief overview on metagenomics science as a means to investigate the microbiome, observations pertaining to both variation and the presumptive potential role of a varied microbiome during pregnancy, and how future studies of the microbiome in pregnancy may lend to a better understanding of human biology, reproductive health, and parturition.

Heritable IUGR and adult metabolic syndrome are reversible and associated with alterations in the metabolome following dietary supplementation of 1-carbon intermediates.

Metabolic syndrome (MetS), following intrauterine growth restriction (IUGR), is epigenetically heritable. Recently, we abrogated the F2 adult phenotype with essential nutrient supplementation (ENS) of intermediates along the 1-carbon pathway. With the use of the same grandparental uterine artery ligation model, we profiled the F2 serum metabolome at weaning [postnatal day (d)21; n = 76] and adulthood (d160; n = 12) to test if MetS is preceded by alterations in the metabolome. Indicative of developmentally programmed MetS, adult F2, formerly IUGR rats, were obese (621 vs. 461 g; P < 0.0001), dyslipidemic (133 vs. 67 mg/dl; P < 0.001), and glucose intolerant (26 vs. 15 mg/kg/min; P < 0.01). Unbiased gas chromatography-mass spectrometry (GC-MS) profiling revealed 34 peaks corresponding to 12 nonredundant metabolites and 9 unknowns to be changing at weaning [false discovery rate (FDR) < 0.05]. Markers of later-in-life MetS included citric acid, glucosamine, myoinositol, and proline (P < 0.03). Hierarchical clustering revealed grouping by IUGR lineage and supplementation at d21 and d160. Weanlings grouped distinctly for ENS and IUGR by partial least-squares discriminate analysis (PLS-DA; P < 0.01), whereas paternal and maternal IUGR (IUGR(pat)/IUGR(mat), respectively) control-fed rats, destined for MetS, had a distinct metabolome at weaning (randomForest analysis; class error < 0.1) and adulthood (PLS-DA; P < 0.05). In sum, we have found that alterations in the metabolome accompany heritable IUGR, precede adult-onset MetS, and are partially amenable to dietary intervention.