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.

Radiological Society of North America/Quantitative Imaging Biomarker Alliance Shear Wave Speed Bias Quantification in Elastic and Viscoelastic Phantoms.

OBJECTIVES: To quantify the bias of shear wave speed (SWS) measurements between different commercial ultrasonic shear elasticity systems and a magnetic resonance elastography (MRE) system in elastic and viscoelastic phantoms. METHODS: Two elastic phantoms, representing healthy through fibrotic liver, were measured with 5 different ultrasound platforms, and 3 viscoelastic phantoms, representing healthy through fibrotic liver tissue, were measured with 12 different ultrasound platforms. Measurements were performed with different systems at different sites, at 3 focal depths, and with different appraisers. The SWS bias across the systems was quantified as a function of the system, site, focal depth, and appraiser. A single MRE research system was also used to characterize these phantoms using discrete frequencies from 60 to 500 Hz. RESULTS: The SWS from different systems had mean difference 95% confidence intervals of ±0.145 m/s (±9.6%) across both elastic phantoms and ± 0.340 m/s (±15.3%) across the viscoelastic phantoms. The focal depth and appraiser were less significant sources of SWS variability than the system and site. Magnetic resonance elastography best matched the ultrasonic SWS in the viscoelastic phantoms using a 140 Hz source but had a - 0.27 ± 0.027-m/s (-12.2% ± 1.2%) bias when using the clinically implemented 60-Hz vibration source. CONCLUSIONS: Shear wave speed reconstruction across different manufacturer systems is more consistent in elastic than viscoelastic phantoms, with a mean difference bias of < ±10% in all cases. Magnetic resonance elastographic measurements in the elastic and viscoelastic phantoms best match the ultrasound systems with a 140-Hz excitation but have a significant negative bias operating at 60 Hz. This study establishes a foundation for meaningful comparison of SWS measurements made with different platforms.

Optical coherence tomography of human fetal membrane sub-layers during loading.

Fetal membranes have important mechanical and antimicrobial roles in maintaining pregnancy. However, the small thickness (<800 µm) of fetal membranes places them outside the resolution limits of most ultrasound and magnetic resonance systems. Optical imaging methods like optical coherence tomography (OCT) have the potential to fill this resolution gap. Here, OCT and machine learning methods were developed to characterize the ex vivo properties of human fetal membranes under dynamic loading. A saline inflation test was incorporated into an OCT system, and tests were performed on n = 33 and n = 32 human samples obtained from labored and C-section donors, respectively. Fetal membranes were collected in near-cervical and near-placental locations. Histology, endogenous two photon fluorescence microscopy, and second harmonic generation microscopy were used to identify sources of contrast in OCT images of fetal membranes. A convolutional neural network was trained to automatically segment fetal membrane sub-layers with high accuracy (Dice coefficients >0.8). Intact amniochorion bilayer and separated amnion and chorion were individually loaded, and the amnion layer was identified as the load-bearing layer within intact fetal membranes for both labored and C-section samples, consistent with prior work. Additionally, the rupture pressure and thickness of the amniochorion bilayer from the near-placental region were greater than those of the near-cervical region for labored samples. This location-dependent change in fetal membrane thickness was not attributable to the load-bearing amnion layer. Finally, the initial phase of the loading curve indicates that amniochorion bilayer from the near-cervical region is strain-hardened compared to the near-placental region in labored samples. Overall, these studies fill a gap in our understanding of the structural and mechanical properties of human fetal membranes at high resolution under dynamic loading events.

Longitudinal ultrasonic dimensions and parametric solid models of the gravid uterus and cervix.

Tissue mechanics is central to pregnancy, during which maternal anatomic structures undergo continuous remodeling to serve a dual function to first protect the fetus in utero while it develops and then facilitate its passage out. In this study of normal pregnancy using biomechanical solid modeling, we used standard clinical ultrasound images to obtain measurements of structural dimensions of the gravid uterus and cervix throughout gestation. 2-dimensional ultrasound images were acquired from the uterus and cervix in 30 pregnant subjects in supine and standing positions at four time points during pregnancy (8-14, 14-16, 22-24, and 32-34 weeks). Offline, three observers independently measured from the images of multiple anatomic regions. Statistical analysis was performed to evaluate inter-observer variance, as well as effect of gestational age, gravity, and parity on maternal geometry. A parametric solid model developed in the Solidworks computer aided design (CAD) software was used to convert ultrasonic measurements to a 3-dimensional solid computer model, from which estimates of uterine and cervical volumes were made. This parametric model was compared against previous 3-dimensional solid models derived from magnetic resonance frequency images in pregnancy. In brief, we found several anatomic measurements easily derived from standard clinical imaging are reproducible and reliable, and provide sufficient information to allow biomechanical solid modeling. This structural dataset is the first, to our knowledge, to provide key variables to enable future computational calculations of tissue stress and stretch in pregnancy, making it possible to characterize the biomechanical milieu of normal pregnancy. This vital dataset will be the foundation to understand how the uterus and cervix malfunction in pregnancy leading to adverse perinatal outcomes.

Quantitative assessment of cervical softening during pregnancy with shear wave elasticity imaging: an in vivo longitudinal study.

We report here the results of a longitudinal study of cervix stiffness during pregnancy. Thirty women, ages ranging from 19 to 37 years, were scanned with ultrasound at five time points beginning at their normal first-trimester screening (8-13 weeks) through term pregnancy (nominally 40 week) using a clinical ultrasound imaging system modified with a special ultrasound transducer and system software. The system estimated the shear wave speed (its square proportional to the shear modulus under idealized conditions) in the cervix. We found a constant fractional reduction (about 4% per week) in shear wave speed with increasing gestational age. We also demonstrated a spatial gradient in shear wave speed along the length of the cervix (softest at the distal end). Results were consistent with our previous ex vivo and in vivo work in women. Shear wave elasticity imaging may be a potentially useful clinical tool for objective assessment of cervical softening in pregnancy.

Quantitative Ultrasound Biomarkers Based on Backscattered Acoustic Power: Potential for Quantifying Remodeling of the Human Cervix during Pregnancy.

As pregnancy progresses, the cervix remodels from a rigid structure to one pliable enough to allow delivery of a fetus, a process that involves progressive disorganization of cervical microstructure. Quantitative ultrasound biomarkers that may detect this process include those derived from the backscattered echo signal, namely, acoustic attenuation and backscattered power loss. We recently reported that attenuation and backscattered power loss are affected by tissue anisotropy and heterogeneity in the ex vivo cervix. In this study, we compared attenuation and backscattered power difference in a group of women in early pregnancy (first trimester) with those in a group in late pregnancy (third trimester). We observed a significant decrease in the backscattered power difference in late as compared with early pregnancy, suggesting decreased microstructural organization in late pregnancy, a finding that is consistent with animal models of cervical remodeling. In contrast, we found no difference in attenuation between the time points. These results suggest that the backscattered power difference, but perhaps not attenuation, may be a useful clinical biomarker of cervical remodeling.

Quantitative Ultrasound Parameters Based on the Backscattered Echo Power Signal as Biomarkers of Cervical Remodeling: A Longitudinal Study in the Pregnant Rhesus Macaque.

Clinical prediction and especially prevention of abnormal birth timing, particularly pre-term, is poor. The cervix plays a key role in birth timing; it first serves as a rigid barrier to protect the developing fetus, then becomes the pathway to delivery of that fetus. Imaging biomarkers to define this remodeling process could provide insights to improve prediction of birth timing and elucidate novel targets for preventive therapies. Quantitative ultrasound (QUS) approaches that appear promising for this purpose include shear wave speed (SWS) estimation to quantify softness, as well as parameters based on backscattered power, such as the mean backscattered power difference (mBSPD) and specific attenuation coefficient (SAC), to quantify the organization of tissue microstructure. Invasive studies in rodents demonstrated that as pregnancy advances, cervical microstructure disorganizes as tissue softness and compliance increase. Our non-invasive studies in pregnant women and rhesus macaques suggested that QUS can detect these microstructural changes in vivo. Our previous study in the same cohort showed a progressive decline in SWS during pregnancy, consistent with increasing tissue softness, and we hypothesized that backscatter parameters would also decrease, consistent with increasing microstructural disorganization. In this study, we analyzed the mBSPD and SAC in the cervices of rhesus macaques (n = 18). We found that both mBSPD and SAC decreased throughout pregnancy (p < 0.001 for both parameters) and that the former appears to be a more reliable biomarker. In summary, biomarkers that can characterize tissue microstructural organization are promising for comprehensive characterization of cervical remodeling in pregnancy.

Detection of Changes in Cervical Softness Using Shear Wave Speed in Early versus Late Pregnancy: An in Vivo Cross-Sectional Study.

The aim of this study was to assess the ability of shear wave elasticity imaging (SWEI) to detect changes in cervical softness between early and late pregnancy. Using a cross-sectional study design, shear wave speed (SWS) measurements were obtained from women in the first trimester (5-14 wk of gestation) and compared with estimates from a previous study of women at term (37-41 wk). Two sets of five SWS measurements were made using commercial SWEI applications on an ultrasound system equipped with a prototype catheter transducer (128 elements, 3-mm diameter, 14-mm aperture). Average SWS estimates were 4.42 ± 0.32 m/s (n = 12) for the first trimester and 2.13 ± 0.66 m/s (n = 18) for the third trimester (p <0.0001). The area under the curve was 0.95 (95% confidence interval: 0.82-0.99) with a sensitivity and specificity of 83%. SWS estimates indicated that the third-trimester cervix is significantly softer than the first-trimester cervix. SWEI methods may be promising for assessing changes in cervical softness.

Anisotropy and Spatial Heterogeneity in Quantitative Ultrasound Parameters: Relevance to the Study of the Human Cervix.

Imaging biomarkers based on quantitative ultrasound can offer valuable information about properties that inform tissue function and behavior such as microstructural organization (e.g., collagen alignment) and viscoelasticity (i.e., compliance). For example, the cervix feels softer as its microstructure remodels during pregnancy, an increase in compliance that can be objectively quantified with shear wave speed and therefore shear wave speed estimation is a potential biomarker of cervical remodeling. Other proposed biomarkers include parameters derived from the backscattered echo signal, such as attenuation and backscattered power loss, because such parameters can provide insight into tissue microstructural alignment and organization. Of these, attenuation values for the pregnant cervix have been reported, but large estimate variance reduces their clinical value. That said, parameter estimates based on the backscattered echo signal may be incorrect if assumptions they rely on, such as tissue isotropy and homogeneity, are violated. For that reason, we explored backscatter and attenuation parameters as potential biomarkers of cervical remodeling via careful investigation of the assumptions of isotropy and homogeneity in cervical tissue. Specifically, we estimated the angle- and spatial-dependence of parameters of backscattered power and acoustic attenuation in the ex vivo human cervix, using the reference phantom method and electronic steering of the ultrasound beam. We found that estimates are anisotropic and spatially heterogeneous, presumably because the tissue itself is anisotropic and heterogeneous. We conclude that appropriate interpretation of imaging biomarkers of cervical remodeling must account for tissue anisotropy and heterogeneity.

Quantitative assessment of cervical softening during pregnancy in the Rhesus macaque with shear wave elasticity imaging.

Abnormal parturition, e.g. pre- or post-term birth, is associated with maternal and neonatal morbidity and increased economic burden. This could potentially be prevented by accurate detection of abnormal softening of the uterine cervix. Shear wave elasticity imaging (SWEI) techniques that quantify tissue softness, such as shear wave speed (SWS) measurement, are promising for evaluation of the cervix. Still, interpretation of results can be complicated by biological variability (i.e. spatial variations of cervix stiffness, parity), as well as by experimental factors (i.e. type of transducer, posture during scanning). Here we investigated the ability of SWEI to detect cervical softening, as well as sources of SWS variability that can affect this task, in the pregnant and nonpregnant Rhesus macaque. Specifically, we evaluated SWS differences when imaging the cervix transabdominally with a typical linear array abdominal transducer, and transrectally with a prototype intracavitary linear array transducer. Linear mixed effects (LME) models were used to model SWS as a function of menstrual cycle day (in nonpregnant animals) and gestational age (in pregnant animals). Other variables included parity, shear wave direction, and cervix side (anterior versus posterior). In the nonpregnant cervix, the LME model indicated that SWS increased by 2% (95% confidence interval 0-3%) per day, starting eight days before menstruation. During pregnancy, SWS significantly decreased at a rate of 6% (95% CI 5-7%) per week (intracavitary approach) and 3% (95% CI 2-4%) per week (transabdominal approach), and interactions between the scanning approach and other fixed effects were also significant. These results suggest that, while absolute SWS values are influenced by factors such as scanning approach and SWEI implementation, these sources of variability do not compromise the sensitivity of SWEI to cervical softening. Our results also highlight the importance of standardizing SWEI approaches to improve their accuracy for cervical assessment.

New techniques in evaluation of the cervix.

The process of parturition is poorly understood, but the cervix clearly plays a key role. Because of this, recent research efforts have been directed at objective quantification of cervical remodeling. Investigation has focused on two basic areas: (1) quantification of tissue deformability and (2) presence, orientation, and/or concentration of microstructural components (e.g. collagen). Methods to quantify tissue deformability include strain elastography and shear wave elasticity imaging (SWEI). Methods to describe tissue microstructure include attenuation and backscatter. A single parameter is unlikely to describe the complexities of cervical remodeling, but combining related parameters should improve accuracy of cervical evaluation. This chapter reviews options for cervical tissue characterization.

Statistical analysis of shear wave speed in the uterine cervix.

Although cervical softening is critical in pregnancy, there currently is no objective method for assessing the softness of the cervix. Shear wave speed (SWS) estimation is a noninvasive tool used to measure tissue mechanical properties such as stiffness. The goal of this study was to determine the spatial variability and assess the ability of SWS to classify ripened versus unripened tissue samples. Ex vivo human hysterectomy samples (n = 22) were collected; a subset (n = 13) were ripened. SWS estimates were made at 4 to 5 locations along the length of the canal on both anterior and posterior halves. A linear mixed model was used for a robust multivariate analysis. Receiver operating characteristic (ROC) analysis and the area under the ROC curve (AUC) were calculated to describe the utility of SWS to classify ripened versus unripened tissue samples. Results showed that all variables used in the linear mixed model were significant ( p < 0.05). Estimates at the mid location for the unripened group were 3.45 ± 0.95 m/s (anterior) and 3.56 ± 0.92 m/s (posterior), and 2.11 ± 0.45 m/s (anterior) and 2.68 ± 0.57 m/s (posterior) for the ripened ( p < 0.001). The AUCs were 0.91 and 0.84 for anterior and posterior, respectively, suggesting that SWS estimates may be useful for quantifying cervical softening.

Evaluating the feasibility of acoustic radiation force impulse shear wave elasticity imaging of the uterine cervix with an intracavity array: a simulation study.

The uterine cervix softens, shortens, and dilates throughout pregnancy in response to progressive disorganization of its layered collagen microstructure. This process is an essential part of normal pregnancy, but premature changes are associated with preterm birth. Clinically, there are no reliable noninvasive methods to objectively measure cervical softening or assess cervical microstructure. The goal of these preliminary studies was to evaluate the feasibility of using an intracavity ultrasound array to generate acoustic radiation force impulse (ARFI) excitations in the uterine cervix through simulation, and to optimize the acoustic radiation force (ARF) excitation for shear wave elasticity imaging (SWEI) of the tissue stiffness. The cervix is a unique soft tissue target for SWEI because it has significantly greater acoustic attenuation (α = 1.3 to 2.0 dB·cm(-1)·MHz(-)1) than other soft tissues, and the pathology being studied tends to lead to an increase in tissue compliance, with healthy cervix being relatively stiff compared with other soft tissues (E ≈ 25 kPa). Additionally, the cervix can only be accessed in vivo using a transvaginal or catheter-based array, which places additional constraints on the excitation focal characteristics that can be used during SWEI. Finite element method (FEM) models of SWEI show that larger-aperture, catheter-based arrays can utilize excitation frequencies up to 7 MHz to generate adequate focal gain up to focal depths 10 to 15 mm deep, with higher frequencies suffering from excessive amounts of near-field acoustic attenuation. Using full-aperture excitations can yield ~40% increases in ARFI-induced displacements, but also restricts the depth of field of the excitation to ~0.5 mm, compared with 2 to 6 mm, which limits the range that can be used for shear wave characterization of the tissue. The center-frequency content of the shear wave particle velocity profiles ranges from 1.5 to 2.5 kHz, depending on the focal configuration and the stiffness of the material being imaged. Overall, SWEI is possible using catheter-based imaging arrays to generate adequate displacements in cervical tissue for shear wave imaging, although specific considerations must be made when optimizing these arrays for this shear wave imaging application.

Nonlinear optical microscopy and ultrasound imaging of human cervical structure.

The cervix softens and shortens as its collagen microstructure rearranges in preparation for birth, but premature change may lead to premature birth. The global preterm birth rate has not decreased despite decades of research, likely because cervical microstructure is poorly understood. Our group has developed a multilevel approach to evaluating the human cervix. We are developing quantitative ultrasound (QUS) techniques for noninvasive interrogation of cervical microstructure and corroborating those results with high-resolution images of microstructure from second harmonic generation imaging (SHG) microscopy. We obtain ultrasound measurements from hysterectomy specimens, prepare the tissue for SHG, and stitch together several hundred images to create a comprehensive view of large areas of cervix. The images are analyzed for collagen orientation and alignment with curvelet transform, and registered with QUS data, facilitating multiscale analysis in which the micron-scale SHG images and millimeter-scale ultrasound data interpretation inform each other. This novel combination of modalities allows comprehensive characterization of cervical microstructure in high resolution. Through a detailed comparative study, we demonstrate that SHG imaging both corroborates the quantitative ultrasound measurements and provides further insight. Ultimately, a comprehensive understanding of specific microstructural cervical change in pregnancy should lead to novel approaches to the prevention of preterm birth.