Testing pulmonary physiology in ventilated non-human primates.

BACKGROUND: Animal models of respiratory viral infections are essential for investigating disease pathogenesis and the efficacy of antivirals and vaccine candidates. A major limitation in the research of respiratory diseases in animal models is correlating clinically relevant changes in pulmonary physiology with cellular and molecular mechanistic studies. Few animal models have captured and correlated physiologic changes in lung function and immune response within same experiment, which is critical given the heterogeneous nature of lung disease due to viral infections. In ventilated human patients, pulmonary physiology testing can be used to not only capture oxygenation, ventilation, but also pulmonary mechanics to yield quantitative measures of lung function and scalar tracings of flow-volume and pressure-volume loops. Application of this protocol during mechanical ventilation in non-human (NHP) models would represent a major advance in respiratory viral disease research. METHODS: We have applied and optimized a human pulmonary physiology testing protocol to ventilated pigtail macaques (Macaca nemestrina) at baseline and 5 days after influenza A (IAV) viral inoculation. RESULTS: The NHPs manifested clinical disease with hypothermia and loss of body weight. Declines in lung function were striking with a 66%-81% decline in P/F ratio, a measure of oxygenation reflecting the ratio of partial pressure of oxygen in arterial blood (PaO2 ) to the fraction of inspiratory oxygen concentration (FiO2 ). There was also a 16%-45% decline in lung compliance. CONCLUSION: We describe a new approach to performing pulmonary physiology testing protocol in non-human primates to better capture quantitative correlates of respiratory disease and demonstrate protection by therapeutics and vaccines.

Addressing a broken drug pipeline for preterm birth: why early preterm birth is an orphan disease.

Preterm birth remains one of the most urgent unresolved medical problems in obstetrics, yet only 2 therapeutics for preventing preterm birth have ever been approved by the United States Food and Drug Administration, and neither remains on the market. The recent withdrawal of 17-hydroxyprogesterone caproate (17-OHPC, Makena) marks a new but familiar era for obstetrics with no Food and Drug Administration-approved pharmaceuticals to address preterm birth. The lack of pharmaceuticals reflects a broad and ineffective pipeline hindered by extensive regulatory hurdles, soaring costs of performing drug research, and concerns regarding adverse effects among a particularly vulnerable population. The pharmaceutical industry has historically limited investments in research for diseases with similarly small markets, such as cystic fibrosis, given their rarity and diminished projected financial return. The Orphan Drug Act, however, incentivizes drug development for "orphan diseases", defined as affecting <200,000 people in the United States annually. Although the total number of preterm births in the United States exceeds this threshold annually, the early subset of preterm birth (<34 weeks' gestation) would qualify, which is predominantly caused by inflammation and infection. The scientific rationale for classifying preterm birth into early and late subsets is strong given that their etiologies differ, and therapeutics that may be efficacious for one subset may not work for the other. For example, antiinflammatory therapeutics would be expected to be highly effective for early but not late preterm birth. A robust therapeutic pipeline of antiinflammatory drugs already exists, which could be used to target spontaneous early preterm birth, in combination with antibiotics shown to sterilize the amniotic cavity. New applications for therapeutics targeting spontaneous early preterm birth could categorize as orphan disease drugs, which could revitalize the preterm birth therapeutic pipeline. Herein, we describe why drugs targeting early preterm birth should qualify for orphan status, which may increase pharmaceutical interest for this vitally important obstetrical condition.

Diminished antiviral innate immune gene expression in the placenta following a maternal SARS-CoV-2 infection.

BACKGROUND: COVID-19 is caused by the SARS-CoV-2 virus and is associated with critical illness requiring hospitalization, maternal mortality, stillbirth, and preterm birth. SARS-CoV-2 has been shown to induce placental pathology. However, substantial gaps exist in our understanding of the pathophysiology of COVID-19 disease in pregnancy and the long-term impact of SARS-CoV-2 on the placenta and fetus. To what extent a SARS-CoV-2 infection of the placenta alters the placental antiviral innate immune response is not well understood. A dysregulated innate immune response in the setting of maternal COVID-19 disease may increase the risk of inflammatory tissue injury or placental compromise and may contribute to deleterious pregnancy outcomes. OBJECTIVE: We sought to determine the impact of a maternal SARS-CoV-2 infection on placental immune response by evaluating gene expression of a panel of 6 antiviral innate immune mediators that act as biomarkers of the antiviral and interferon cytokine response. Our hypothesis was that a SARS-CoV-2 infection during pregnancy would result in an up-regulated placental antiviral innate immune response. STUDY DESIGN: We performed a case-control study on placental tissues (chorionic villous tissues and chorioamniotic membrane) collected from pregnant patients with (N=140) and without (N=24) COVID-19 disease. We performed real-time quantitative polymerase chain reaction and immunohistochemistry, and the placental histopathology was evaluated. Clinical data were abstracted. Fisher exact test, Pearson correlations, and linear regression models were used to examine proportions and continuous data between patients with active (<10 days since diagnosis) vs recovered COVID-19 (>10 days since diagnosis) at the time of delivery. Secondary regression models adjusted for labor status as a covariate and evaluated potential correlation between placental innate immune gene expression and other variables. RESULTS: SARS-CoV-2 viral RNA was detected in placental tissues from 5 women with COVID-19 and from no controls (0/24, 0%). Only 1 of 5 cases with detectable SARS-CoV-2 viral RNA in placental tissues was confirmed to express SARS-CoV-2 nucleocapsid and spike proteins in syncytiotrophoblast cells. We detected a considerably lower gene expression of 5 critical innate immune mediators (IFNB, IFIT1, MXA, IL6, IL1B) in the chorionic villi and chorioamniotic membranes from women with active or recovered COVID-19 than controls, which remained significant after adjustment for labor status. There were minimal correlations between placental gene expression and other studied variables including gestational age at diagnosis, time interval between COVID-19 diagnosis and delivery, prepregnancy body mass index, COVID-19 disease severity, or placental pathology. CONCLUSION: A maternal SARS-CoV-2 infection was associated with an impaired placental innate immune response in chorionic villous tissues and chorioamniotic membranes that was not correlated with gestational age at COVID-19 diagnosis, time interval from COVID-19 diagnosis to delivery, maternal obesity, disease severity, or placental pathology.

Decidual Inflammation Drives Chemokine-Mediated Immune Infiltration Contributing to Term Labor.

Infiltration of maternal peripheral leukocytes into the uterine tissues is a critical event occurring before, during, and after term labor (TL). In this article, we investigate the contribution of uterine smooth muscle (myometrium) and pregnant endometrium (decidua) to the inflammatory process during human TL. We hypothesize that labor-related physiological inflammation is orchestrated by uterine-secreted cytokines, which dually activate the uterine vascular endothelium and maternal leukocytes to promote their adhesion and infiltration into the uterus. Using Luminex and ELISA assays, we examine a full range of cytokines (45 proteins) in media conditioned by primary decidual and myometrial cells from TL and term not in labor (TNL) women. The effect of conditioned media on the activation of human uterine microvascular endothelial cells was measured by qPCR and on peripheral leukocytes by flow cytometry. Transendothelial migration of calcein-labeled primary leukocytes toward media was assessed by fluorometry. Stromal decidual cells secrete significantly higher levels of multiple cytokines compared with myometrial cells (p < 0.05) and significantly more cytokines during TL than TNL. These cytokines activate uterine microvascular endothelial cells through the upregulation of cell adhesion molecule VCAM-1 and peripheral leukocytes by upregulation of CD11b. Furthermore, multiple cytokines secreted from the TL decidua and myometrium significantly increase migration of granulocytes, monocytes, and lymphocytes compared with TNL (p < 0.05), which was blocked by a broad-spectrum chemokine inhibitor (FX125L). These data reveal the critical role for decidual- and myometrial-secreted cytokines in the activation of inflammatory pathways leading to labor. We suggest that these pathways represent targets for therapeutic intervention during preterm labor.

One Vax Two Lives: a social media campaign and research program to address COVID-19 vaccine hesitancy in pregnancy.

The COVID-19 pandemic has disproportionately affected pregnant people by increasing health risks of maternal morbidity and mortality, stillbirth, and preterm birth. Although numerous studies have supported the safety and efficacy of COVID-19 vaccination in pregnancy in preventing or mitigating the risk for these adverse outcomes, many pregnant people remain hesitant. Approximately half of US adults regularly consume news from social media platforms, which are a fertile ground for the spread of vaccine disinformation. The lack of information regarding COVID-19 vaccine safety early in the pandemic fueled vaccine myths targeting the fears of pregnant people about vaccination risks. Saddened by the spike in maternal deaths of unvaccinated individuals during the COVID-19 Delta variant surge in the fall of 2021, we created a social media campaign to promote scientific communication regarding the risks of COVID-19 disease in pregnancy and the benefits of vaccination. We called the campaign "One Vax Two Lives," which refers to the ability of 1 maternal vaccine to benefit the health and lives of both the pregnant individual and their fetus. We present a blueprint of how we leveraged a large, interdisciplinary student workforce to create a social media campaign and research program studying vaccine hesitancy, which can be replicated by other groups. Community engagement and partnerships with key stakeholders, such as the Washington State Department of Health, were essential for amplifying the campaign and providing our team with feedback on content and approach. We present the analytics of our social media advertisements, web articles, and video content that helped inform the iterative design process of the multimedia content. Moving forward, we are launching collaborative research programs to study vaccine hesitancy and inform the development of new social media content designed for pregnant individuals who are: (1) Spanish-speaking Hispanic/Latina/x, (2) Black or Afro-Latinx, and (3) residents of rural communities in the State of Washington. Data from these mixed methods studies will inform new communication campaigns to reach vaccine-hesitant individuals. Finally, we discuss lessons learned and how the most impactful elements of the campaign can be translated to related areas of maternal public health.

Virulence, phenotype and genotype characteristics of invasive group B Streptococcus isolates obtained from Swedish pregnant women and neonates.

Group B streptococci (GBS) are bacteria that can cause preterm birth and invasive neonatal disease. Heterogeneous expression of virulence factors enables GBS to exist as both commensal bacteria and to become highly invasive. A molecular epidemiological study comparing GBS bacterial traits, genotype and host characteristics may indicate whether it is possible to predict the risk of perinatal invasive GBS disease and more accurately target intrapartum antibiotic prophylaxis. A total of 229 invasive GBS isolates from Swedish pregnant women or neonates were assessed for virulence and phenotypic traits: hemolysis zone, hemolytic pigment (Granada agar), Streptococcus B Carrot Broth (SBCB) assay, CAMP factor, and hyaluronidase activity. Genes regulating hemolytic pigment synthesis (covR/covS, abx1, stk1, stp1) were sequenced. Of the virulence factors and phenotypes assessed, a Granada pigment or SBCB score ≥ 2 captured more than 90% of EOD isolates with excellent inter-rater reliability. High enzyme activity of hyaluronidase was observed in 16% (36/229) of the invasive GBS isolates and notably, in one case of stillbirth. Hyaluronidase activity was also significantly higher in GBS isolates obtained from pregnant/postpartum individuals versus the stillbirth or neonatal invasive isolates (p < 0.001). Sequencing analysis found that abx1 (g.T106I), stk1 (g.T211N), stp1 (g.K469R) and covS (g.V343M) variants were present significantly more often in the higher (Granada pigment score ≥ 2) versus lower pigmented isolates (p < 0.001, each variant). Among the 203 higher Granada pigment scoring isolates, 22 (10.8%) isolates had 3 of the four sequence variants and 10 (4.9%) had 2 of the four sequence variants. Although heterogeneity in GBS virulence factor expression was observed, the vast majority were more highly pigmented and contained several common sequence variants in genes regulating pigment synthesis. High activity of hyaluronidase may increase risk for stillbirth and invasive disease in pregnant or postpartum individuals. Our findings suggest that testing for GBS pigmentation and hyaluronidase may, albeit imperfectly, identify pregnant people at risk for invasive disease and represent a step towards a personalized medical approach for the administration of intrapartum antibiotic prophylaxis.

Low Prevalence of Severe Acute Respiratory Syndrome Coronavirus 2 Among Pregnant and Postpartum Patients With Universal Screening in Seattle, Washington.

We found low prevalence of SARS-CoV-2 (2.7% [5/188]) among pregnant and postpartum patients with universal testing. Prevalence among symptomatic patients was similar under initial targeted screening (22.2% [4/18]) and universal approaches (19.1% [8/42]). Among 170 asymptomatic patients, 2 were positive or inconclusive, respectively; repeat testing at 24 hours was negative.

Amniotic fluid interleukin 6 and interleukin 8 are superior predictors of fetal lung injury compared with maternal or fetal plasma cytokines or placental histopathology in a nonhuman primate model.

BACKGROUND: Intra-amniotic infection or inflammation is common in early preterm birth and associated with substantial neonatal lung morbidity owing to fetal exposure to proinflammatory cytokines and infectious organisms. Amniotic fluid interleukin 8, a proinflammatory cytokine, was previously correlated with the development of neonatal bronchopulmonary dysplasia, but whether amniotic fluid cytokines or placental pathology more accurately predicts neonatal lung pathology and morbidity is unknown. We have used a pregnant nonhuman primate model of group B Streptococcus infection to study the pathogenesis of intra-amniotic infection, bacterial invasion of the amniotic cavity and fetus, and microbial-host interactions. In this nonhuman primate model, we have studied the pathogenesis of group B Streptococcus strains with differing potential for virulence, which has resulted in a spectrum of intra-amniotic infection and fetal lung injury that affords the opportunity to study the inflammatory predictors of fetal lung pathology and injury. OBJECTIVE: This study aimed to determine whether fetal lung injury is best predicted by placental histopathology or the cytokine response in amniotic fluid or maternal plasma. STUDY DESIGN: Chronically catheterized pregnant monkeys (Macaca nemestrina, pigtail macaque) at 116 to 125 days gestation (term at 172 days) received a choriodecidual inoculation of saline (n=5), weakly hemolytic group B Streptococcus strain (n=5, low virulence), or hyperhemolytic group B Streptococcus strain (n=5, high virulence). Adverse pregnancy outcomes were defined as either preterm labor, microbial invasion of the amniotic cavity, or development of the fetal inflammatory response syndrome. Amniotic fluid and maternal and fetal plasma samples were collected after inoculation, and proinflammatory cytokines (tumor necrosis factor alpha, interleukin beta, interleukin 6, interleukin 8) were measured by a multiplex assay. Cesarean delivery was performed at the time of preterm labor or within 1 week of inoculation. Fetal necropsy was performed at the time of delivery. Placental pathology was scored in a blinded fashion by a pediatric pathologist, and fetal lung injury was determined by a semiquantitative score from histopathology evaluating inflammatory infiltrate, necrosis, tissue thickening, or collapse scored by a veterinary pathologist. RESULTS: The principal findings in our study are as follows: (1) adverse pregnancy outcomes occurred more frequently in animals receiving hyperhemolytic group B Streptococcus (80% with preterm labor, 80% with fetal inflammatory response syndrome) than in animals receiving weakly hemolytic group B Streptococcus (40% with preterm labor, 20% with fetal inflammatory response syndrome) and in controls (0% preterm labor, 0% fetal inflammatory response syndrome); (2) despite differences in the rate of adverse pregnancy outcomes and fetal inflammatory response syndrome, fetal lung injury scores were similar between animals receiving the weakly hemolytic group B Streptococcus strains and animals receiving the hyperhemolytic group B Streptococcus strains; (3) fetal lung injury score was significantly correlated with peak amniotic fluid cytokines interleukin 6 and interleukin 8 but not tumor necrosis factor alpha or interleukin 1 beta; and (4) fetal lung scores were poorly correlated with maternal and fetal plasma cytokine levels and placental pathology. CONCLUSION: Amniotic fluid interleukin 6 and interleukin 8 levels were superior predictors of fetal lung injury than placental histopathology or maternal plasma cytokines. This evidence supports a role for amniocentesis in the prediction of neonatal lung morbidity owing to intra-amniotic infection, which cannot be provided by cytokine analysis of maternal plasma or placental histopathology.

Higher severe acute respiratory syndrome coronavirus 2 infection rate in pregnant patients.

BACKGROUND: During the early months of the coronavirus disease 2019 pandemic, risks associated with severe acute respiratory syndrome coronavirus 2 in pregnancy were uncertain. Pregnant patients can serve as a model for the success of clinical and public health responses during public health emergencies as they are typically in frequent contact with the medical system. Population-based estimates of severe acute respiratory syndrome coronavirus 2 infections in pregnancy are unknown because of incomplete ascertainment of pregnancy status or inclusion of only single centers or hospitalized cases. Whether pregnant women were protected by the public health response or through their interactions with obstetrical providers in the early months of pandemic is not clearly understood. OBJECTIVE: This study aimed to estimate the severe acute respiratory syndrome coronavirus 2 infection rate in pregnancy and to examine the disparities by race and ethnicity and English language proficiency in Washington State. STUDY DESIGN: Pregnant patients with a polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 infection diagnosed between March 1, 2020, and June 30, 2020 were identified within 35 hospitals and clinics, capturing 61% of annual deliveries in Washington State. Infection rates in pregnancy were estimated overall and by Washington State Accountable Community of Health region and cross-sectionally compared with severe acute respiratory syndrome coronavirus 2 infection rates in similarly aged adults in Washington State. Race and ethnicity and language used for medical care of pregnant patients were compared with recent data from Washington State. RESULTS: A total of 240 pregnant patients with severe acute respiratory syndrome coronavirus 2 infections were identified during the study period with 70.7% from minority racial and ethnic groups. The principal findings in our study were as follows: (1) the severe acute respiratory syndrome coronavirus 2 infection rate was 13.9 per 1000 deliveries in pregnant patients (95% confidence interval, 8.3-23.2) compared with 7.3 per 1000 (95% confidence interval, 7.2-7.4) in adults aged 20 to 39 years in Washington State (rate ratio, 1.7; 95% confidence interval, 1.3-2.3); (2) the severe acute respiratory syndrome coronavirus 2 infection rate reduced to 11.3 per 1000 deliveries (95% confidence interval, 6.3-20.3) when excluding 45 cases of severe acute respiratory syndrome coronavirus disease 2 detected through asymptomatic screening (rate ratio, 1.3; 95% confidence interval, 0.96-1.9); (3) the proportion of pregnant patients in non-White racial and ethnic groups with severe acute respiratory syndrome coronavirus disease 2 infection was 2- to 4-fold higher than the race and ethnicity distribution of women in Washington State who delivered live births in 2018; and (4) the proportion of pregnant patients with severe acute respiratory syndrome coronavirus 2 infection receiving medical care in a non-English language was higher than estimates of pregnant patients receiving care with limited English proficiency in Washington State (30.4% vs 7.6%). CONCLUSION: The severe acute respiratory syndrome coronavirus 2 infection rate in pregnant people was 70% higher than similarly aged adults in Washington State, which could not be completely explained by universal screening at delivery. Pregnant patients from nearly all racial and ethnic minority groups and patients receiving medical care in a non-English language were overrepresented. Pregnant women were not protected from severe acute respiratory syndrome coronavirus 2 infection in the early months of the pandemic. Moreover, the greatest burden of infections occurred in nearly all racial and ethnic minority groups. These data coupled with a broader recognition that pregnancy is a risk factor for severe illness and maternal mortality strongly suggested that pregnant people should be broadly prioritized for coronavirus disease 2019 vaccine allocation in the United States similar to some states.

Disease severity, pregnancy outcomes, and maternal deaths among pregnant patients with severe acute respiratory syndrome coronavirus 2 infection in Washington State.

BACKGROUND: Evidence is accumulating that coronavirus disease 2019 increases the risk of hospitalization and mechanical ventilation in pregnant patients and for preterm delivery. However, the impact on maternal mortality and whether morbidity is differentially affected by disease severity at delivery and trimester of infection are unknown. OBJECTIVE: This study aimed to describe disease severity and outcomes of severe acute respiratory syndrome coronavirus 2 infections in pregnancy across the Washington State, including pregnancy complications and outcomes, hospitalization, and case fatality. STUDY DESIGN: Pregnant patients with a polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 infection between March 1, 2020, and June 30, 2020, were identified in a multicenter retrospective cohort study from 35 sites in Washington State. Sites captured 61% of annual state deliveries. Case-fatality rates in pregnancy were compared with coronavirus disease 2019 fatality rates in similarly aged adults in Washington State using rate ratios and rate differences. Maternal and neonatal outcomes were compared by trimester of infection and disease severity at the time of delivery. RESULTS: The principal study findings were as follows: (1) among 240 pregnant patients in Washington State with severe acute respiratory syndrome coronavirus 2 infections, 1 in 11 developed severe or critical disease, 1 in 10 were hospitalized for coronavirus disease 2019, and 1 in 80 died; (2) the coronavirus disease 2019-associated hospitalization rate was 3.5-fold higher than in similarly aged adults in Washington State (10.0% vs 2.8%; rate ratio, 3.5; 95% confidence interval, 2.3-5.3); (3) pregnant patients hospitalized for a respiratory concern were more likely to have a comorbidity or underlying conditions including asthma, hypertension, type 2 diabetes mellitus, autoimmune disease, and class III obesity; (4) 3 maternal deaths (1.3%) were attributed to coronavirus disease 2019 for a maternal mortality rate of 1250 of 100,000 pregnancies (95% confidence interval, 257-3653); (5) the coronavirus disease 2019 case fatality in pregnancy was a significant 13.6-fold (95% confidence interval, 2.7-43.6) higher in pregnant patients than in similarly aged individuals in Washington State with an absolute difference in mortality rate of 1.2% (95% confidence interval, -0.3 to 2.6); and (6) preterm birth was significantly higher among women with severe or critical coronavirus disease 2019 at delivery than for women who had recovered from coronavirus disease 2019 (45.4% severe or critical coronavirus disease 2019 vs 5.2% mild coronavirus disease 2019; P<.001). CONCLUSION: Coronavirus disease 2019 hospitalization and case-fatality rates in pregnant patients were significantly higher than in similarly aged adults in Washington State. These data indicate that pregnant patients are at risk of severe or critical disease and mortality compared to nonpregnant adults, and also at risk for preterm birth.

MicroRNA Signature of Epithelial-Mesenchymal Transition in Group B Streptococcal Infection of the Placental Chorioamniotic Membranes.

BACKGROUND: Infection-induced preterm birth is a major cause of neonatal mortality and morbidity and leads to preterm premature rupture of placental chorioamniotic membranes. The loss of amniotic epithelial cells and tensile strength preceding membrane rupture is poorly understood. We hypothesized that intrauterine bacterial infection induces changes in microRNA (miRNA) expression, leading to amniotic epithelial cell loss and membrane weakening. METHODS: Ten pregnant pigtail macaques received choriodecidual inoculation of either group B Streptococcus (GBS) or saline (n = 5/group). Placental chorioamniotic membranes were studied using RNA microarray and immunohistochemistry. Chorioamniotic membranes from women with preterm premature rupture of membranes (pPROM) and normal term pregnancies were studied using transmission electron microscopy. RESULTS: In our model, an experimental GBS infection was associated with changes in the miRNA profile in the chorioamniotic membranes consistent with epithelial to mesenchymal transition (EMT) with loss of epithelial (E-cadherin) and gain of mesenchymal (vimentin) markers. Similarly, loss of desmosomes (intercellular junctions) was seen in placental tissues from women with pPROM. CONCLUSIONS: We describe EMT as a novel mechanism for infection-associated chorioamniotic membrane weakening, which may be a common pathway for many etiologies of pPROM. Therapy based on anti-miRNA targeting of EMT may prevent pPROM due to perinatal infection.

Importance of Inclusion of Pregnant and Breastfeeding Women in COVID-19 Therapeutic Trials.

Investigators are employing unprecedented innovation in the design of clinical trials to rapidly and rigorously assess potentially promising therapies for coronavirus disease 2019 (COVID-19); this is in stark contrast to the continued near-universal regressive practice of exclusion of pregnant and breastfeeding women from these trials. The few trials that allow their inclusion focus on postexposure prophylaxis or outpatient treatment of milder disease, limiting the options available to pregnant women with severe COVID-19 to compassionate use of remdesivir, or off-label drug use of hydroxychloroquine or other therapies. These restrictions were put in place despite experience with these drugs in pregnant women. In this Viewpoint, we call attention to the need and urgency to engage pregnant women in COVID-19 treatment trials now in order to develop data-driven recommendations regarding the risks and benefits of therapies in this unique but not uncommon population.

Clinical characteristics of 46 pregnant women with a severe acute respiratory syndrome coronavirus 2 infection in Washington State.

BACKGROUND: The impact of coronavirus disease 2019 on pregnant women is incompletely understood, but early data from case series suggest a variable course of illness from asymptomatic or mild disease to maternal death. It is unclear whether pregnant women manifest enhanced disease similar to influenza viral infection or whether specific risk factors might predispose to severe disease. OBJECTIVE: To describe maternal disease and obstetrical outcomes associated with coronavirus disease 2019 in pregnancy to rapidly inform clinical care. STUDY DESIGN: This is a retrospective study of pregnant patients with a laboratory-confirmed severe acute respiratory syndrome coronavirus 2 infection from 6 hospital systems in Washington State between Jan. 21, 2020, and April 17, 2020. Demographics, medical and obstetrical history, and coronavirus disease 2019 encounter data were abstracted from medical records. RESULTS: A total of 46 pregnant patients with a severe acute respiratory syndrome coronavirus 2 infection were identified from hospital systems capturing 40% of births in Washington State. Nearly all pregnant individuals with a severe acute respiratory syndrome coronavirus 2 infection were symptomatic (93.5%, n=43) and the majority were in their second or third trimester (43.5% [n=20] and 50.0% [n=23], respectively). Symptoms resolved in a median of 24 days (interquartile range, 13-37). Notably, 7 women were hospitalized (16%) including 1 admitted to the intensive care unit. A total of 6 cases (15%) were categorized as severe coronavirus disease 2019 with nearly all patients being either overweight or obese before pregnancy or with asthma or other comorbidities. Of the 8 deliveries that occurred during the study period, there was 1 preterm birth at 33 weeks' gestation to improve pulmonary status in a woman with class III obesity, and 1 stillbirth of unknown etiology. CONCLUSION: Severe coronavirus disease 2019 developed in approximately 15% of pregnant patients and occurred primarily in overweight or obese women with underlying conditions. Obesity and coronavirus disease 2019 may synergistically increase risk for a medically indicated preterm birth to improve maternal pulmonary status in late pregnancy. These findings support categorizing pregnant patients as a higher-risk group, particularly those with chronic comorbidities.

Zika virus and the nonmicrocephalic fetus: why we should still worry.

Zika virus is a mosquito-transmitted flavivirus and was first linked to congenital microcephaly caused by a large outbreak in northeastern Brazil. Although the Zika virus epidemic is now in decline, pregnancies in large parts of the Americas remain at risk because of ongoing transmission and the potential for new outbreaks. This review presents why Zika virus is still a complex and worrisome public health problem with an expanding spectrum of birth defects and how Zika virus and related viruses evade the immune response to injure the fetus. Recent reports indicate that the spectrum of fetal brain and other anomalies associated with Zika virus exposure is broader and more complex than microcephaly alone and includes subtle fetal brain and ocular injuries; thus, the ability to prenatally diagnose fetal injury associated with Zika virus infection remains limited. New studies indicate that Zika virus imparts disproportionate effects on fetal growth with an unusual femur-sparing profile, potentially providing a new approach to identify viral injury to the fetus. Studies to determine the limitations of prenatal and postnatal testing for detection of Zika virus-associated birth defects and long-term neurocognitive deficits are needed to better guide women with a possible infectious exposure. It is also imperative that we investigate why the Zika virus is so adept at infecting the placenta and the fetal brain to better predict other viruses with similar capabilities that may give rise to new epidemics. The efficiency with which the Zika virus evades the early immune response to enable infection of the mother, placenta, and fetus is likely critical for understanding why the infection may either be fulminant or limited. Furthermore, studies suggest that several emerging and related viruses may also cause birth defects, including West Nile virus, which is endemic in many parts of the United States. With mosquito-borne diseases increasing worldwide, there remains an urgent need to better understand the pathogenesis of the Zika virus and related viruses to protect pregnancies and child health.

The fetal origins of mental illness.

The impact of infections and inflammation during pregnancy on the developing fetal brain remains incompletely defined, with important clinical and research gaps. Although the classic infectious TORCH pathogens (ie, Toxoplasma gondii, rubella virus, cytomegalovirus [CMV], herpes simplex virus) are known to be directly teratogenic, emerging evidence suggests that these infections represent the most extreme end of a much larger spectrum of injury. We present the accumulating evidence that prenatal exposure to a wide variety of viral and bacterial infections-or simply inflammation-may subtly alter fetal brain development, leading to neuropsychiatric consequences for the child later in life. The link between influenza infections in pregnant women and an increased risk for development of schizophrenia in their children was first described more than 30 years ago. Since then, evidence suggests that a range of infections during pregnancy may also increase risk for autism spectrum disorder and depression in the child. Subsequent studies in animal models demonstrated that both pregnancy infections and inflammation can result in direct injury to neurons and neural progenitor cells or indirect injury through activation of microglia and astrocytes, which can trigger cytokine production and oxidative stress. Infectious exposures can also alter placental serotonin production, which can perturb neurotransmitter signaling in the developing brain. Clinically, detection of these subtle injuries to the fetal brain is difficult. As the neuropsychiatric impact of perinatal infections or inflammation may not be known for decades after birth, our construct for defining teratogenic infections in pregnancy (eg, TORCH) based on congenital anomalies is insufficient to capture the full adverse impact on the child. We discuss the clinical implications of this body of evidence and how we might place greater emphasis on prevention of prenatal infections. For example, increasing uptake of the seasonal influenza vaccine is a key strategy to reduce perinatal infections and the risk for fetal brain injury. An important research gap exists in understanding how antibiotic therapy during pregnancy affects the fetal inflammatory load and how to avoid inflammation-mediated injury to the fetal brain. In summary, we discuss the current evidence and mechanisms linking infections and inflammation with the increased lifelong risk of neuropsychiatric disorders in the child, and how we might improve prenatal care to protect the fetal brain.

Human Cervical Mucus Plugs Exhibit Insufficiencies in Antimicrobial Activity Towards Group B Streptococcus.

Preterm birth is a leading cause of neonatal mortality and lacks an effective therapy. Ascending microbial infections from the lower genital tract lead to infection of the placenta, amniotic fluid, and fetus causing preterm birth or stillbirth. Directly in the path of an ascending infection is the cervical mucus plug (CMP), a dense mucoid structure in the cervical canal with potential antimicrobial properties. In this study, we aimed to define the components of CMP responsible for antimicrobial activity against a common lower genital tract organism associated with preterm birth and stillbirths, namely, group B streptococcus (GBS). Using a quantitative proteomic approach, we identified antimicrobial factors in CMPs that were collected from healthy human pregnancies. However, we noted that the concentration of antimicrobial peptides present in the human CMPs were insufficient to directly kill GBS, and antimicrobial activity, when observed, was due to antibiotics retained in the CMPs. Despite this insufficiency, CMP proteins were able to activate leukocytes in whole blood resulting in increased rates of bacterial killing, suggesting a role for the CMP in enhancing complement-mediated killing or leukocyte activation. This study provides new insight into how the human CMP may limit ascending bacterial infection.

Femur-sparing pattern of abnormal fetal growth in pregnant women from New York City after maternal Zika virus infection.

BACKGROUND: Zika virus is a mosquito-transmitted flavivirus, which can induce fetal brain injury and growth restriction following maternal infection during pregnancy. Prenatal diagnosis of Zika virus-associated fetal injury in the absence of microcephaly is challenging due to an incomplete understanding of how maternal Zika virus infection affects fetal growth and the use of different sonographic reference standards around the world. We hypothesized that skeletal growth is unaffected by Zika virus infection and that the femur length can represent an internal standard to detect growth deceleration of the fetal head and/or abdomen by ultrasound. OBJECTIVE: We sought to determine if maternal Zika virus infection is associated with a femur-sparing pattern of intrauterine growth restriction through analysis of fetal biometric measures and/or body ratios using the 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project and World Health Organization Fetal Growth Chart sonographic references. STUDY DESIGN: Pregnant women diagnosed with a possible recent Zika virus infection at Columbia University Medical Center after traveling to an endemic area were retrospectively identified and included if a fetal ultrasound was performed. Data were collected regarding Zika virus testing, fetal biometry, pregnancy, and neonatal outcomes. The 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project and World Health Organization Fetal Growth Chart sonographic standards were applied to obtain Z-scores and/or percentiles for fetal head circumference, abdominal circumference, and femur length specific for each gestational week. A novel 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project standard was also developed to generate Z-scores for fetal body ratios with respect to femur length (head circumference:femur length, abdominal circumference:femur length). Data were then grouped within clinically relevant gestational age strata (<24, 24-27 6/7, 28-33 6/7, >34 weeks) to analyze time-dependent effects of Zika virus infection on fetal size. Statistical analysis was performed using Wilcoxon signed-rank test on paired data, comparing either abdominal circumference or head circumference to femur length. RESULTS: A total of 56 pregnant women were included in the study with laboratory evidence of a confirmed or possible recent Zika virus infection. Based on the Centers for Disease Control and Prevention definition for microcephaly after congenital Zika virus exposure, microcephaly was diagnosed in 5% (3/56) by both the 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project and World Health Organization Fetal Growth Chart standards (head circumference Z-score ≤-2 or ≤2.3%). Using 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project, intrauterine fetal growth restriction was diagnosed in 18% of pregnancies (10/56; abdominal circumference Z-score ≤-1.3, <10%). Analysis of fetal size using the last ultrasound scan for all subjects revealed a significantly abnormal skewing of fetal biometrics with a smaller abdominal circumference vs femur length by either 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project or World Health Organization Fetal Growth Chart (P < .001 for both). A difference in distribution of fetal abdominal circumference compared to femur length was first apparent in the 24-27 6/7 week strata (2014 International Fetal and Newborn Growth Consortium for the 21st Century Project, P = .002; World Health Organization Fetal Growth Chart, P = .001). A significantly smaller head circumference compared to femur length was also observed by 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project as early as the 28-33 6/7 week strata (2014 International Fetal and Newborn Growth Consortium for the 21st Century Project, P = .007). Overall, a femur-sparing pattern of growth restriction was detected in 52% of pregnancies with either head circumference:femur length or abdominal circumference:femur length fetal body ratio <10th percentile (2014 International Fetal and Newborn Growth Consortium for the 21st Century Project Z-score ≤-1.3). CONCLUSION: An unusual femur-sparing pattern of fetal growth restriction was detected in the majority of fetuses with congenital Zika virus exposure. Fetal body ratios may represent a more sensitive ultrasound biomarker to detect viral injury in nonmicrocephalic fetuses that could impart long-term risk for complications of congenital Zika virus infection.

Evidence of cardiac involvement in the fetal inflammatory response syndrome: disruption of gene networks programming cardiac development in nonhuman primates.

BACKGROUND: Most early preterm births are associated with intraamniotic infection and inflammation, which can lead to systemic inflammation in the fetus. The fetal inflammatory response syndrome describes elevations in the fetal interleukin-6 level, which is a marker for inflammation and fetal organ injury. An understanding of the effects of inflammation on fetal cardiac development may lead to insight into the fetal origins of adult cardiovascular disease. OBJECTIVE: The purpose of this study was to determine whether the fetal inflammatory response syndrome is associated with disruptions in gene networks that program fetal cardiac development. STUDY DESIGN: We obtained fetal cardiac tissue after necropsy from a well-described pregnant nonhuman primate model (pigtail macaque, Macaca nemestrina) of intrauterine infection (n=5) and controls (n=5). Cases with the fetal inflammatory response syndrome (fetal plasma interleukin-6 >11 pg/mL) were induced by either choriodecidual inoculation of a hypervirulent group B streptococcus strain (n=4) or intraamniotic inoculation of Escherichia coli (n=1). RNA and protein were extracted from fetal hearts and profiled by microarray and Luminex (Millipore, Billerica, MA) for cytokine analysis, respectively. Results were validated by quantitative reverse transcriptase polymerase chain reaction. Statistical and bioinformatics analyses included single gene analysis, gene set analysis, Ingenuity Pathway Analysis (Qiagen, Valencia, CA), and Wilcoxon rank sum. RESULTS: Severe fetal inflammation developed in the context of intraamniotic infection and a disseminated bacterial infection in the fetus. Interleukin-6 and -8 in fetal cardiac tissues were elevated significantly in fetal inflammatory response syndrome cases vs controls (P<.05). A total of 609 probe sets were expressed differentially (>1.5-fold change, P<.05) in the fetal heart (analysis of variance). Altered expression of select genes was validated by quantitative reverse transcriptase polymerase chain reaction that included several with known functions in cardiac injury, morphogenesis, angiogenesis, and tissue remodeling (eg, angiotensin I converting enzyme 2, STEAP family member 4, natriuretic peptide A, and secreted frizzled-related protein 4; all P<.05). Multiple gene sets and pathways that are involved in cardiac morphogenesis and vasculogenesis were downregulated significantly by gene set and Ingenuity Pathway Analysis (hallmark transforming growth factor beta signaling, cellular morphogenesis during differentiation, morphology of cardiovascular system; all P<.05). CONCLUSION: Disruption of gene networks for cardiac morphogenesis and vasculogenesis occurred in the preterm fetal heart of nonhuman primates with preterm labor, intraamniotic infection, and severe fetal inflammation. Inflammatory injury to the fetal heart in utero may contribute to the development of heart disease later in life. Development of preterm labor therapeutics must also target fetal inflammation to lessen organ injury and potential long-term effects on cardiac function.

Group B streptococcal infection of the choriodecidua induces dysfunction of the cytokeratin network in amniotic epithelium: a pathway to membrane weakening.

Early events leading to intrauterine infection remain poorly defined, but may hold the key to preventing preterm delivery. To determine molecular pathways within fetal membranes (chorioamnion) associated with early choriodecidual infection that may progress to preterm premature rupture of membranes (PPROM), we examined the effects of a Group B Streptococcus (GBS) choriodecidual infection on chorioamnion in a nonhuman primate model. Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118-125 days gestation (term = 172 days) received choriodecidual inoculation of either GBS (n = 5) or saline (n = 5). Cesarean section was performed in the first week after GBS or saline inoculation. RNA extracted from chorioamnion (inoculation site) was profiled by microarray. Single gene, Gene Set, and Ingenuity Pathway Analysis results were validated using qRT-PCR (chorioamnion), Luminex (amniotic fluid, AF), immunohistochemistry, and transmission electron microscopy (TEM). Despite uterine quiescence in most cases, significant elevations of AF cytokines (TNF-α, IL-8, IL-1ß, IL-6) were detected in GBS versus controls (p<0.05). Choriodecidual infection resolved by the time of cesarean section in 3 of 5 cases and GBS was undetectable by culture and PCR in the AF. A total of 331 genes were differentially expressed (>2-fold change, p<0.05). Remarkably, GBS exposure was associated with significantly downregulated expression of multiple cytokeratin (CK) and other cytoskeletal genes critical for maintenance of tissue tensile strength. Immunofluorescence revealed highly significant changes in the CK network within amniocytes with dense CK aggregates and retraction from the cell periphery (all p = 0.006). In human pregnancies affected by PPROM, there was further evidence of CK network retraction with significantly shorter amniocyte foot processes (p = 0.002). These results suggest early choriodecidual infection results in decreased cellular membrane integrity and tensile strength via dysfunction of CK networks. Downregulation of CK expression and perturbations in the amniotic epithelial cell intermediate filament network occur after GBS choriodecidual infection, which may contribute to PPROM.

Choriodecidual Group B Streptococcal Infection Induces miR-155-5p in the Fetal Lung in Macaca nemestrina.

The mechanisms underlying fetal lung injury remain poorly defined. MicroRNAs (miRNAs) are small noncoding, endogenous RNAs that regulate gene expression and have been implicated in the pathogenesis of lung disease. Using a nonhuman primate model of choriodecidual infection, we sought to determine if differentially expressed miRNAs were associated with acute fetal lung injury. After inoculating 10 chronically catheterized pregnant monkeys (Macaca nemestrina) with either group B streptococcus (GBS) at 1 × 10(6) CFU (n = 5) or saline (n = 5) in the choriodecidual space, we extracted fetal lung mRNA and miRNA and profiled the changes in expression by microarray analysis. We identified 9 differentially expressed miRNAs in GBS-exposed fetal lungs, but of these, only miR-155-5p was validated by quantitative reverse transcription-PCR (P = 0.02). Significantly elevated miR-155-5p expression was also observed when immortalized human fetal airway epithelial (FeAE) cells were exposed to proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]). Overexpression of miR-155-5p in FeAE cells in turn increased the production of IL-6 and CXCL10/gamma interferon-induced protein 10, which are implicated in leukocyte recruitment but also in protection from lung injury. Interestingly, while miR-155-5p decreased fibroblast growth factor 9 (FGF9) expression in a luciferase reporter assay, FGF9 levels were actually increased in GBS-exposed fetal lungs in vivo. FGF9 overexpression is associated with abnormal lung development. Thus, upregulation of miR-155-5p may serve as a compensatory mechanism to lessen the increase in FGF9 and prevent aberrant lung development. Understanding the complicated networks regulating lung development in the setting of infection is a key step in identifying how to prevent fetal lung injury leading to bronchopulmonary dysplasia.