Perturbations in kinetics of the thrombin generation assay identify women at risk of preeclampsia in the first trimester and provide the rationale for a preventive approach.

BACKGROUND: Activation of the coagulation system and increased thrombin generation have been implicated in the pathophysiology of preeclampsia, and this rationale supports the administration of low-molecular-weight heparin to prevent this syndrome in patients at risk. Yet, randomized trials of this prophylactic measure have yielded contradictory results. A possible explanation is that only a subset of patients with preeclampsia have excessive thrombin generation and would benefit from the administration of low-molecular-weight heparin. Therefore, the key questions are whether and when patients who subsequently develop preeclampsia present evidence of abnormal thrombin generation. OBJECTIVE: This study aimed to determine (1) the kinetics of thrombin generation throughout gestation in women with a normal pregnancy and in those with early and late preeclampsia, and (2) the diagnostic performance of in vivo thrombin generation parameters to predict the development of preeclampsia. STUDY DESIGN: This retrospective, nested case-control study was based on a prospective longitudinal cohort of singleton gestations. Cases comprised women who developed preeclampsia (n=49), and controls consisted of patients with a normal pregnancy (n=45). Preeclampsia was classified into early-onset (n=24) and late-onset (n=25). Longitudinal changes in the parameters of the thrombin generation assay (lag time, time to peak thrombin concentration, peak thrombin concentration, endogenous thrombin generation, and velocity index) throughout gestation were compared between the study groups, and normal pregnancy percentiles were derived from the control group. We tested whether a single parameter or a combination of parameters, derived from the kinetics of thrombin generation, could identify patients who subsequently developed preeclampsia. Time-related parameters <10th percentile were considered short, and concentration-related parameters >90th percentile were considered high. RESULTS: (1) Patients who developed preeclampsia (early- and late-onset) had abnormal thrombin generation kinetics as early as 8 to 16 weeks of pregnancy; (2) patients with a combination of a short lag time and high peak thrombin concentration at 8 to 16 weeks of pregnancy had an odds ratio of 43.87 for the subsequent development of preeclampsia (area under the curve, 0.79; sensitivity, 56.8%; specificity, 92.7%; positive likelihood ratio, 7.76); (3) at 16 to 22 weeks of gestation, patients with a combination of a short lag time and a high velocity index had an odds ratio of 16 for the subsequent development of preeclampsia (area under the curve, 0.78; sensitivity, 62.2%; specificity, 92.5%; positive likelihood ratio, 8.29). CONCLUSION: During early pregnancy, the thrombin generation assay can identify the subset of patients at a greater risk for the development of preeclampsia owing to accelerated and enhanced production of thrombin. This observation provides a rationale for testing the efficacy of low-molecular-weight heparin in this subset of patients. We propose that future research on the efficacy of low-molecular-weight heparin and other interventions targeting the coagulation system to prevent preeclampsia should be focused on patients with abnormal kinetics of thrombin generation.

Human Plasma Proteome During Normal Pregnancy.

The human plasma proteome is underexplored despite its potential value for monitoring health and disease. Herein, using a recently developed aptamer-based platform, we profiled 7288 proteins in 528 plasma samples from 91 normal pregnancies (Gene Expression Omnibus identifier GSE206454). The coefficient of variation was <20% for 93% of analytes (median 7%), and a cross-platform correlation for selected key angiogenic and anti-angiogenic proteins was significant. Gestational age was associated with changes in 953 proteins, including highly modulated placenta- and decidua-specific proteins, and they were enriched in biological processes including regulation of growth, angiogenesis, immunity, and inflammation. The abundance of proteins corresponding to RNAs specific to populations of cells previously described by single-cell RNA-Seq analysis of the placenta was highly modulated throughout gestation. Furthermore, machine learning-based prediction of gestational age and of time from sampling to term delivery compared favorably with transcriptomic models (mean absolute error of 2 weeks). These results suggested that the plasma proteome may provide a non-invasive readout of placental cellular dynamics and serve as a blueprint for investigating obstetrical disease.

Toward a new taxonomy of obstetrical disease: improved performance of maternal blood biomarkers for the great obstetrical syndromes when classified according to placental pathology.

BACKGROUND: The major challenge for obstetrics is the prediction and prevention of the great obstetrical syndromes. We propose that defining obstetrical diseases by the combination of clinical presentation and disease mechanisms as inferred by placental pathology will aid in the discovery of biomarkers and add specificity to those already known. OBJECTIVE: To describe the longitudinal profile of placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and the PlGF/sFlt-1 ratio throughout gestation, and to determine whether the association between abnormal biomarker profiles and obstetrical syndromes is strengthened by information derived from placental examination, eg, the presence or absence of placental lesions of maternal vascular malperfusion. STUDY DESIGN: This retrospective case cohort study was based on a parent cohort of 4006 pregnant women enrolled prospectively. The case cohort of 1499 pregnant women included 1000 randomly selected patients from the parent cohort and all additional patients with obstetrical syndromes from the parent cohort. Pregnant women were classified into six groups: 1) term delivery without pregnancy complications (n=540; control); 2) preterm labor and delivery (n=203); 3) preterm premature rupture of the membranes (n=112); 4) preeclampsia (n=230); 5) small-for-gestational-age neonate (n=334); and 6) other pregnancy complications (n=182). Maternal plasma concentrations of PlGF and sFlt-1 were determined by enzyme-linked immunosorbent assays in 7560 longitudinal samples. Placental pathologists, masked to clinical outcomes, diagnosed the presence or absence of placental lesions of maternal vascular malperfusion. Comparisons between mean biomarker concentrations in cases and controls were performed by utilizing longitudinal generalized additive models. Comparisons were made between controls and each obstetrical syndrome with and without subclassifying cases according to the presence or absence of placental lesions of maternal vascular malperfusion. RESULTS: 1) When obstetrical syndromes are classified based on the presence or absence of placental lesions of maternal vascular malperfusion, significant differences in the mean plasma concentrations of PlGF, sFlt-1, and the PlGF/sFlt-1 ratio between cases and controls emerge earlier in gestation; 2) the strength of association between an abnormal PlGF/sFlt-1 ratio and the occurrence of obstetrical syndromes increases when placental lesions of maternal vascular malperfusion are present (adjusted odds ratio [aOR], 13.6 vs 6.7 for preeclampsia; aOR, 8.1 vs 4.4 for small-for-gestational-age neonates; aOR, 5.5 vs 2.1 for preterm premature rupture of the membranes; and aOR, 3.3 vs 2.1 for preterm labor (all P<0.05); and 3) the PlGF/sFlt-1 ratio at 28 to 32 weeks of gestation is abnormal in patients who subsequently delivered due to preterm labor with intact membranes and in those with preterm premature rupture of the membranes if both groups have placental lesions of maternal vascular malperfusion. Such association is not significant in patients with these obstetrical syndromes who do not have placental lesions. CONCLUSION: Classification of obstetrical syndromes according to the presence or absence of placental lesions of maternal vascular malperfusion allows biomarkers to be informative earlier in gestation and enhances the strength of association between biomarkers and clinical outcomes. We propose that a new taxonomy of obstetrical disorders informed by placental pathology will facilitate the discovery and implementation of biomarkers as well as the prediction and prevention of such disorders.

Personalized assessment of cervical length improves prediction of spontaneous preterm birth: a standard and a percentile calculator.

BACKGROUND: A sonographic short cervix (length <25 mm during midgestation) is the most powerful predictor of preterm birth. Current clinical practice assumes that the same cervical length cutoff value should apply to all women when screening for spontaneous preterm birth, yet this approach may be suboptimal. OBJECTIVE: This study aimed to (1) create a customized cervical length standard that considers relevant maternal characteristics and gestational age at sonographic examination and (2) assess whether the customization of cervical length evaluation improves the prediction of spontaneous preterm birth. STUDY DESIGN: This retrospective analysis comprises a cohort of 7826 pregnant women enrolled in a longitudinal protocol between January 2006 and April 2017 at the Detroit Medical Center. Study participants met the following inclusion criteria: singleton pregnancy, ≥1 transvaginal sonographic measurements of the cervix, delivery after 20 weeks of gestation, and available relevant demographics and obstetrical history information. Data from women without a history of preterm birth or cervical surgery who delivered at term without progesterone treatment (N=5188) were used to create a customized standard of cervical length. The prediction of the primary outcome, spontaneous preterm birth at <37 weeks of gestation, was assessed in a subset of pregnancies (N=7336) that excluded cases with induced labor before 37 weeks of gestation. Area under the receiver operating characteristic curve and sensitivity at a fixed false-positive rate were calculated for screening at 20 to 23 6/7, 24 to 27 6/7, 28 to 31 6/7, and 32 to 35 6/7 weeks of gestation in asymptomatic patients. Survival analysis was used to determine which method is better at predicting imminent delivery among symptomatic women. RESULTS: The median cervical length remained fundamentally unchanged until 20 weeks of gestation and subsequently decreased nonlinearly with advancing gestational age among women who delivered at term. The effects of parity and maternal weight and height on the cervical length were dependent on the gestational age at ultrasound examination (interaction, P<.05 for all). Parous women had a longer cervix than nulliparous women, and the difference increased with advancing gestation after adjusting for maternal weight and height. Similarly, maternal weight was nonlinearly associated with a longer cervix, and the effect was greater later in gestation. The sensitivity at a 10% false-positive rate for prediction of spontaneous preterm birth at <37 weeks of gestation by a short cervix ranged from 29% to 40% throughout pregnancy, yet it increased to 50%, 50%, 53%, and 54% at 20 to 23 6/7, 24 to 27 6/7, 28 to 31 6/7, and 32 to 35 6/7 weeks of gestation, respectively, for a low, customized percentile (McNemar test, P<.001 for all). When a cervical length <25 mm was compared to the customized screening at 20 to 23 6/7 weeks of gestation by using a customized percentile cutoff value that ensured the same negative likelihood ratio for both screening methods, the customized approach had a significantly higher (about double) positive likelihood ratio in predicting spontaneous preterm birth at <33, <34, <35, <36, and <37 weeks of gestation. Among symptomatic women, the difference in survival between women with a customized cervical length percentile of ≥10th and those with a customized cervical length percentile of <10th was greater than the difference in survival between women with a cervical length ≥25 mm and those with a cervical length <25 mm. CONCLUSION: Compared to the use of a cervical length <25 mm, a customized cervical length assessment (1) identifies more women at risk of spontaneous preterm birth and (2) improves the distinction between patients at risk for impending preterm birth in those who have an episode of preterm labor.

Bacteria in the amniotic fluid without inflammation: early colonization vs. contamination.

OBJECTIVES: Intra-amniotic infection, defined by the presence of microorganisms in the amniotic cavity, is often accompanied by intra-amniotic inflammation. Occasionally, laboratories report the growth of bacteria or the presence of microbial nucleic acids in amniotic fluid in the absence of intra-amniotic inflammation. This study was conducted to determine the clinical significance of the presence of bacteria in amniotic fluid samples in the absence of intra-amniotic inflammation. METHODS: A retrospective cross-sectional study included 360 patients with preterm labor and intact membranes who underwent transabdominal amniocentesis for evaluation of the microbial state of the amniotic cavity as well as intra-amniotic inflammation. Cultivation techniques were used to isolate microorganisms, and broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was utilized to detect the nucleic acids of bacteria, viruses, and fungi. RESULTS: Patients whose amniotic fluid samples evinced microorganisms but did not indicate inflammation had a similar perinatal outcome to those without microorganisms or inflammation [amniocentesis-to-delivery interval (p=0.31), spontaneous preterm birth before 34 weeks (p=0.83), acute placental inflammatory lesions (p=1), and composite neonatal morbidity (p=0.8)]. CONCLUSIONS: The isolation of microorganisms from a sample of amniotic fluid in the absence of intra-amniotic inflammation is indicative of a benign condition, which most likely represents contamination of the specimen during the collection procedure or laboratory processing rather than early colonization or infection.

A new customized fetal growth standard for African American women: the PRB/NICHD Detroit study.

BACKGROUND: The assessment of fetal growth disorders requires a standard. Current nomograms for the assessment of fetal growth in African American women have been derived either from neonatal (rather than fetal) biometry data or have not been customized for maternal ethnicity, weight, height, and parity and fetal sex. OBJECTIVE: We sought to (1) develop a new customized fetal growth standard for African American mothers; and (2) compare such a standard to 3 existing standards for the classification of fetuses as small (SGA) or large (LGA) for gestational age. STUDY DESIGN: A retrospective cohort study included 4183 women (4001 African American and 182 Caucasian) from the Detroit metropolitan area who underwent ultrasound examinations between 14-40 weeks of gestation (the median number of scans per pregnancy was 5, interquartile range 3-7) and for whom relevant covariate data were available. Longitudinal quantile regression was used to build models defining the "normal" estimated fetal weight (EFW) centiles for gestational age in African American women, adjusted for maternal height, weight, and parity and fetal sex, and excluding pathologic factors with a significant effect on fetal weight. The resulting Perinatology Research Branch/Eunice Kennedy Shriver National Institute of Child Health and Human Development (hereinafter, PRB/NICHD) growth standard was compared to 3 other existing standards--the customized gestation-related optimal weight (GROW) standard; the Eunice Kennedy Shriver National Institute of Child Health and Human Development (hereinafter, NICHD) African American standard; and the multinational World Health Organization (WHO) standard--utilized to screen fetuses for SGA (<10th centile) or LGA (>90th centile) based on the last available ultrasound examination for each pregnancy. RESULTS: First, the mean birthweight at 40 weeks was 133 g higher for neonates born to Caucasian than to African American mothers and 150 g higher for male than female neonates; maternal weight, height, and parity had a positive effect on birthweight. Second, analysis of longitudinal EFW revealed the following features of fetal growth: (1) all weight centiles were about 2% higher for male than for female fetuses; (2) maternal height had a positive effect on EFW, with larger fetuses being affected more (2% increase in the 95th centile of weight for each 10-cm increase in height); and (3) maternal weight and parity had a positive effect on EFW that increased with gestation and varied among the weight centiles. Third, the screen-positive rate for SGA was 7.2% for the NICHD African American standard, 12.3% for the GROW standard, 13% for the WHO standard customized by fetal sex, and 14.4% for the PRB/NICHD customized standard. For all standards, the screen-positive rate for SGA was at least 2-fold higher among fetuses delivered preterm than at term. Fourth, the screen-positive rate for LGA was 8.7% for the GROW standard, 9.2% for the PRB/NICHD customized standard, 10.8% for the WHO standard customized by fetal sex, and 12.3% for the NICHD African American standard. Finally, the highest overall agreement among standards was between the GROW and PRB/NICHD customized standards (Cohen's interrater agreement, kappa = 0.85). CONCLUSION: We developed a novel customized PRB/NICHD fetal growth standard from fetal data in an African American population without assuming proportionality of the effects of covariates, and without assuming that these effects are equal on all centiles of weight; we also provide an easy-to-use centile calculator. This standard classified more fetuses as being at risk for SGA compared to existing standards, especially among fetuses delivered preterm, but classified about the same number of LGA. The comparison among the 4 growth standards also revealed that the most important factor determining agreement among standards is whether they account for the same factors known to affect fetal growth.

Statistical power of likelihood ratio and Wald tests in latent class models with covariates.

This paper discusses power and sample-size computation for likelihood ratio and Wald testing of the significance of covariate effects in latent class models. For both tests, asymptotic distributions can be used; that is, the test statistic can be assumed to follow a central Chi-square under the null hypothesis and a non-central Chi-square under the alternative hypothesis. Power or sample-size computation using these asymptotic distributions requires specification of the non-centrality parameter, which in practice is rarely known. We show how to calculate this non-centrality parameter using a large simulated data set from the model under the alternative hypothesis. A simulation study is conducted evaluating the adequacy of the proposed power analysis methods, determining the key study design factor affecting the power level, and comparing the performance of the likelihood ratio and Wald test. The proposed power analysis methods turn out to perform very well for a broad range of conditions. Moreover, apart from effect size and sample size, an important factor affecting the power is the class separation, implying that when class separation is low, rather large sample sizes are needed to achieve a reasonable power level.

Power Analysis for the Likelihood-Ratio Test in Latent Markov Models: Shortcutting the Bootstrap p-Value-Based Method.

The latent Markov (LM) model is a popular method for identifying distinct unobserved states and transitions between these states over time in longitudinally observed responses. The bootstrap likelihood-ratio (BLR) test yields the most rigorous test for determining the number of latent states, yet little is known about power analysis for this test. Power could be computed as the proportion of the bootstrap p values (PBP) for which the null hypothesis is rejected. This requires performing the full bootstrap procedure for a large number of samples generated from the model under the alternative hypothesis, which is computationally infeasible in most situations. This article presents a computationally feasible shortcut method for power computation for the BLR test. The shortcut method involves the following simple steps: (1) obtaining the parameters of the model under the null hypothesis, (2) constructing the empirical distributions of the likelihood ratio under the null and alternative hypotheses via Monte Carlo simulations, and (3) using these empirical distributions to compute the power. We evaluate the performance of the shortcut method by comparing it to the PBP method and, moreover, show how the shortcut method can be used for sample-size determination.

A mitochondrial regulator protein, MNRR1, is elevated in the maternal blood of women with preeclampsia.

OBJECTIVE: Preeclampsia, one of the most serious obstetric complications, is a heterogenous disorder resulting from different pathologic processes. However, placental oxidative stress and an anti-angiogenic state play a crucial role. Mitochondria are a major source of cellular reactive oxygen species. Abnormalities in mitochondrial structures, proteins, and functions have been observed in the placentae of patients with preeclampsia, thus mitochondrial dysfunction has been implicated in the mechanism of the disease. Mitochondrial nuclear retrograde regulator 1 (MNRR1) is a newly characterized bi-organellar protein with pleiotropic functions. In the mitochondria, this protein regulates cytochrome c oxidase activity and reactive oxygen species production, whereas in the nucleus, it regulates the transcription of a number of genes including response to tissue hypoxia and inflammatory signals. Since MNRR1 expression changes in response to hypoxia and to an inflammatory signal, MNRR1 could be a part of mitochondrial dysfunction and involved in the pathologic process of preeclampsia. This study aimed to determine whether the plasma MNRR1 concentration of women with preeclampsia differed from that of normal pregnant women. METHODS: This retrospective case-control study included 97 women with preeclampsia, stratified by gestational age at delivery into early (<34 weeks, n = 40) and late (≥34 weeks, n = 57) preeclampsia and by the presence or absence of placental lesions consistent with maternal vascular malperfusion (MVM), the histologic counterpart of an anti-angiogenic state. Women with an uncomplicated pregnancy at various gestational ages who delivered at term served as controls (n = 80) and were further stratified into early (n = 25) and late (n = 55) controls according to gestational age at venipuncture. Maternal plasma MNRR1 concentrations were determined by an enzyme-linked immunosorbent assay. RESULTS: 1) Women with preeclampsia at the time of diagnosis (either early or late disease) had a significantly higher median (interquartile range, IQR) plasma MNRR1 concentration than the controls [early preeclampsia: 1632 (924-2926) pg/mL vs. 630 (448-4002) pg/mL, p = .026, and late preeclampsia: 1833 (1441-5534) pg/mL vs. 910 (526-6178) pg/mL, p = .021]. Among women with early preeclampsia, those with MVM lesions in the placenta had the highest median (IQR) plasma MNRR1 concentration among the three groups [with MVM: 2066 (1070-3188) pg/mL vs. without MVM: 888 (812-1781) pg/mL, p = .03; and with MVM vs. control: 630 (448-4002) pg/mL, p = .04]. There was no significant difference in the median plasma MNRR1 concentration between women with early preeclampsia without MVM lesions and those with an uncomplicated pregnancy (p = .3). By contrast, women with late preeclampsia, regardless of MVM lesions, had a significantly higher median (IQR) plasma MNRR1 concentration than women in the control group [with MVM: 1609 (1392-3135) pg/mL vs. control: 910 (526-6178), p = .045; and without MVM: 2023 (1578-8936) pg/mL vs. control, p = .01]. CONCLUSIONS: MNRR1, a mitochondrial regulator protein, is elevated in the maternal plasma of women with preeclampsia (both early and late) at the time of diagnosis. These findings may reflect some degree of mitochondrial dysfunction, intravascular inflammation, or other unknown pathologic processes that characterize this obstetrical syndrome.

The vaginal immunoproteome for the prediction of spontaneous preterm birth: A retrospective longitudinal study.

Background: Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Most cases of preterm birth occur spontaneously and result from preterm labor with intact (spontaneous preterm labor [sPTL]) or ruptured (preterm prelabor rupture of membranes [PPROM]) membranes. The prediction of spontaneous preterm birth (sPTB) remains underpowered due to its syndromic nature and the dearth of independent analyses of the vaginal host immune response. Thus, we conducted the largest longitudinal investigation targeting vaginal immune mediators, referred to herein as the immunoproteome, in a population at high risk for sPTB. Methods: Vaginal swabs were collected across gestation from pregnant women who ultimately underwent term birth, sPTL, or PPROM. Cytokines, chemokines, growth factors, and antimicrobial peptides in the samples were quantified via specific and sensitive immunoassays. Predictive models were constructed from immune mediator concentrations. Results: Throughout uncomplicated gestation, the vaginal immunoproteome harbors a cytokine network with a homeostatic profile. Yet, the vaginal immunoproteome is skewed toward a pro-inflammatory state in pregnant women who ultimately experience sPTL and PPROM. Such an inflammatory profile includes increased monocyte chemoattractants, cytokines indicative of macrophage and T-cell activation, and reduced antimicrobial proteins/peptides. The vaginal immunoproteome has improved predictive value over maternal characteristics alone for identifying women at risk for early (<34 weeks) sPTB. Conclusions: The vaginal immunoproteome undergoes homeostatic changes throughout gestation and deviations from this shift are associated with sPTB. Furthermore, the vaginal immunoproteome can be leveraged as a potential biomarker for early sPTB, a subset of sPTB associated with extremely adverse neonatal outcomes. Funding: This research was conducted by the Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health and Human Services (NICHD/NIH/DHHS) under contract HHSN275201300006C. ALT, KRT, and NGL were supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health.

Human pregnancies last 40 weeks on average. Preterm births, defined as live births before 37 weeks, occur in about one in ten pregnancies. Being born too early is the main cause of a number of diseases and death in newborn babies. Preterm births are further divided into those that happen early ­ before 34 weeks ­ and those that happen late ­ between 34 and 37 weeks. There are also differences between preterm births in which the amniotic sac ruptures before or after the start of labor. Although several factors can lead to spontaneous preterm birth, bacteria getting into the amniotic fluid around the fetus are a well-known trigger. These bacteria usually come from the vagina. In the past, researchers have studied the number and types of bacteria in the vagina of people who had a normal pregnancy and those that had a preterm birth to predict who is more at risk of preterm birth. However, predictions based only on data about bacteria have been less useful so far. Instead, it might be better to investigate a person's immune response during pregnancy. Shaffer et al. addressed this gap by asking whether measuring the levels of proteins involved in the immune response could help predict preterm births. Shaffer et al. collected vaginal fluids from 739 individuals of predominately African American ethnicity with an average BMI of 28.7 ­ representing a population at high risk for spontaneous preterm birth. The swabs were taken at multiple points during their pregnancy, and 31 different immune-related proteins in those fluids were measured. The researchers further noted whether these individuals had a normal or a preterm birth. The data showed that, compared to normal births, preterm births are associated with higher levels of proteins that attract white blood cells and promote inflammation, such as IL-6 and IL-1ß. Vaginal fluids from individuals who went on to have an early preterm birth where the amniotic sac ruptured before labor, contained lower levels of proteins known as defensins, which defend the body from bacteria. With these new data from vaginal swabs, Shaffer et al. could make better predictions about the likelihood of preterm birth in general and early preterm birth with the amniotic sac ruptured before labor. For the latter scenario, the predictions were not improved when combining immune protein data with other characteristics of the pregnant person, such as age. These findings suggest that clinicians may be able to use measurements of immune-related proteins to help predict preterm births, so that pregnant individuals at high risk can receive extra care. Further research will have to validate the data and determine whether the findings apply more widely.

COVID-19 is associated with early emergence of preeclampsia: results from a large regional collaborative.

Objective: To investigate the relationship between preeclampsia and SARS-CoV-2 infection during pregnancy. Methods: This was a retrospective cohort study of pregnant women between March and October 2020. Pregnant patients admitted to 14 obstetrical centers in Michigan, USA formed the study population. Of the N = 1458 participants, 369 had SARS-CoV-2 infection (cases). Controls were uninfected pregnancies that were delivered in the same obstetric unit within 30 days of the index case. Robust Poisson regression was used to estimate relative risk (RR) of preterm and term preeclampsia and preeclampsia involving placental lesions. The analysis included adjustment for relevant clinical and demographic risk factors.Results: SARS-CoV-2 infection during pregnancy increased the risk of preeclampsia [adjusted aRR = 1.69 (1.26-2.26)], preeclampsia involving placental lesions [aRR = 1.97(1.14-3.4)] and preterm preeclampsia 2.48(1.48-4.17). Although the highest rate of preeclampsia was observed in patients infected with SARS-CoV-2 who were symptomatic (18.4%), there was increased risk even in asymptomatic SARS-CoV-2 infected patients (14.2%) relative to non-infected controls (8.7%) (p < 0.05). This association with symptomatology was also noted with preterm preeclampsia for which the rate doubled from 2.7% in controls to 5.2% in asymptomatic cases and reached 11.8% among symptomatic cases (p < 0.05). The rate of preterm preeclampsia among cases of pregnant people self-identified as Black reached 10.1% and was almost double the rate of the reminder of the group of infected pregnancies (5.3%), although the rate among uninfected was almost the same (2.7%) for both Black and non-Black groups (interaction p = 0.05).Conclusions: Infection with SARS-CoV-2 increases the risk of preeclampsia even in the absence of symptoms, although symptomatic persons are at even higher risk. Racial disparities in the development of preterm preeclampsia after SARS-CoV-2 infection may explain discrepancies in prematurity between different populations.

Proteomic profile of extracellular vesicles in maternal plasma of women with fetal death.

OBJECTIVES: Fetal death is a complication of pregnancy caused by multiple etiologies rather than being the end-result of a single disease process. Many soluble analytes in the maternal circulation, such as hormones and cytokines, have been implicated in its pathophysiology. However, changes in the protein content of extracellular vesicles (EVs), which could provide additional insight into the disease pathways of this obstetrical syndrome, have not been examined. This study aimed to characterize the proteomic profile of EVs in the plasma of pregnant women who experienced fetal death and to evaluate whether such a profile reflected the pathophysiological mechanisms of this obstetrical complication. Moreover, the proteomic results were compared to and integrated with those obtained from the soluble fraction of maternal plasma. METHODS: This retrospective case-control study included 47 women who experienced fetal death and 94 matched, healthy, pregnant controls. Proteomic analysis of 82 proteins in the EVs and the soluble fractions of maternal plasma samples was conducted by using a bead-based, multiplexed immunoassay platform. Quantile regression analysis and random forest models were implemented to assess differences in the concentration of proteins in the EV and soluble fractions and to evaluate their combined discriminatory power between clinical groups. Hierarchical cluster analysis was applied to identify subgroups of fetal death cases with similar proteomic profiles. A p-value of <.05 was used to infer significance, unless multiple testing was involved, with the false discovery rate controlled at the 10% level (q < 0.1). All statistical analyses were performed by using the R statistical language and environment-and specialized packages. RESULTS: Nineteen proteins (placental growth factor, macrophage migration inhibitory factor, endoglin, regulated upon activation normal T cell expressed and presumably secreted (RANTES), interleukin (IL)-6, macrophage inflammatory protein 1-alpha, urokinase plasminogen activator surface receptor, tissue factor pathway inhibitor, IL-8, E-Selectin, vascular endothelial growth factor receptor 2, pentraxin 3, IL-16, galectin-1, monocyte chemotactic protein 1, disintegrin and metalloproteinase domain-containing protein 12, insulin-like growth factor-binding protein 1, matrix metalloproteinase-1(MMP1), and CD163) were found to have different plasma concentrations (of an EV or a soluble fraction) in women with fetal death compared to controls. There was a similar pattern of change for the dysregulated proteins in the EV and soluble fractions and a positive correlation between the log2-fold changes of proteins significant in either the EV or the soluble fraction (ρ = 0.89, p < .001). The combination of EV and soluble fraction proteins resulted in a good discriminatory model (area under the ROC curve, 82%; sensitivity, 57.5% at a 10% false-positive rate). Unsupervised clustering based on the proteins differentially expressed in either the EV or the soluble fraction of patients with fetal death relative to controls revealed three major clusters of patients. CONCLUSION: Pregnant women with fetal death have different concentrations of 19 proteins in the EV and soluble fractions compared to controls, and the direction of changes in concentration was similar between fractions. The combination of EV and soluble protein concentrations revealed three different clusters of fetal death cases with distinct clinical and placental histopathological characteristics.

Maternal SARS-COV-2 infection and prematurity: the Southern Michigan COVID-19 collaborative.

OBJECTIVE: COVID-19 has been reported to increase the risk of prematurity, however, due to the frequent absence of unaffected controls as well as inadequate accounting for confounders in many studies, the question requires further investigation. We sought to determine the impact of COVID-19 disease on preterm birth (PTB) overall, as well as related subcategories such as early prematurity, spontaneous, medically indicated preterm birth, and preterm labor (PTL). We assessed the impact of confounders such as COVID-19 risk factors, a-priori risk factors for PTB, symptomatology, and disease severity on rates of prematurity. METHODS: This was a retrospective cohort study of pregnant women from March 2020 till October 1st, 2020. The study included patients from 14 obstetric centers in Michigan, USA. Cases were defined as women diagnosed with COVID-19 at any point during their pregnancy. Cases were matched with uninfected women who delivered in the same unit, within 30 d of the delivery of the index case. Outcomes of interest were frequencies of prematurity overall and subcategories of preterm birth (early, spontaneous/medically indicated, preterm labor, and premature preterm rupture of membranes) in cases compared to controls. The impact of modifiers of these outcomes was documented with extensive control for potential confounders. A p value <.05 was used to infer significance. RESULTS: The rate of prematurity was 8.9% in controls, 9.4% in asymptomatic cases, 26.5% in symptomatic COVID-19 cases, and 58.8% among cases admitted to the ICU. Gestational age at delivery was noted to decrease with disease severity. Cases were at an increased risk of prematurity overall [adjusted relative risk (aRR) = 1.62 (1.2-2.18)] and of early prematurity (<34 weeks) [aRR = 1.8 (1.02-3.16)] when compared to controls. Medically indicated prematurity related to preeclampsia [aRR = 2.46 (1.47-4.12)] or other indications [aRR = 2.32 (1.12-4.79)], were the primary drivers of overall prematurity risk. Symptomatic cases were at an increased risk of preterm labor [aRR = 1.74 (1.04-2.8)] and spontaneous preterm birth due to premature preterm rupture of membranes [aRR = 2.2(1.05-4.55)] when compared to controls and asymptomatic cases combined. The gestational age at delivery followed a dose-response relation with disease severity, as more severe cases tended to deliver earlier (Wilcoxon p < .05). CONCLUSIONS: COVID-19 is an independent risk factor for preterm birth. The increased preterm birth rate in COVID-19 was primarily driven by medically indicated delivery, with preeclampsia as the principal risk factor. Symptomatic status and disease severity were significant drivers of preterm birth.

The amniotic fluid proteome predicts imminent preterm delivery in asymptomatic women with a short cervix.

Preterm birth, the leading cause of perinatal morbidity and mortality, is associated with increased risk of short- and long-term adverse outcomes. For women identified as at risk for preterm birth attributable to a sonographic short cervix, the determination of imminent delivery is crucial for patient management. The current study aimed to identify amniotic fluid (AF) proteins that could predict imminent delivery in asymptomatic patients with a short cervix. This retrospective cohort study included women enrolled between May 2002 and September 2015 who were diagnosed with a sonographic short cervix (< 25 mm) at 16-32 weeks of gestation. Amniocenteses were performed to exclude intra-amniotic infection; none of the women included had clinical signs of infection or labor at the time of amniocentesis. An aptamer-based multiplex platform was used to profile 1310 AF proteins, and the differential protein abundance between women who delivered within two weeks from amniocentesis, and those who did not, was determined. The analysis included adjustment for quantitative cervical length and control of the false-positive rate at 10%. The area under the receiver operating characteristic curve was calculated to determine whether protein abundance in combination with cervical length improved the prediction of imminent preterm delivery as compared to cervical length alone. Of the 1,310 proteins profiled in AF, 17 were differentially abundant in women destined to deliver within two weeks of amniocentesis independently of the cervical length (adjusted p-value < 0.10). The decreased abundance of SNAP25 and the increased abundance of GPI, PTPN11, OLR1, ENO1, GAPDH, CHI3L1, RETN, CSF3, LCN2, CXCL1, CXCL8, PGLYRP1, LDHB, IL6, MMP8, and PRTN3 were associated with an increased risk of imminent delivery (odds ratio > 1.5 for each). The sensitivity at a 10% false-positive rate for the prediction of imminent delivery by a quantitative cervical length alone was 38%, yet it increased to 79% when combined with the abundance of four AF proteins (CXCL8, SNAP25, PTPN11, and MMP8). Neutrophil-mediated immunity, neutrophil activation, granulocyte activation, myeloid leukocyte activation, and myeloid leukocyte-mediated immunity were biological processes impacted by protein dysregulation in women destined to deliver within two weeks of diagnosis. The combination of AF protein abundance and quantitative cervical length improves prediction of the timing of delivery compared to cervical length alone, among women with a sonographic short cervix.

Maternal whole blood mRNA signatures identify women at risk of early preeclampsia: a longitudinal study.

PURPOSE: To determine whether previously established mRNA signatures are predictive of early preeclampsia when evaluated by maternal cellular transcriptome analysis in samples collected before clinical manifestation. MATERIALS AND METHODS: We profiled gene expression at exon-level resolution in whole blood samples collected longitudinally from 49 women with normal pregnancy (controls) and 13 with early preeclampsia (delivery <34 weeks of gestation). After preprocessing and removal of gestational age-related trends in gene expression, data were converted into Z-scores based on the mean and standard deviation among controls for six gestational-age intervals. The average Z-scores of mRNAs in each previously established signature considered herein were compared between cases and controls at 9-11, 11-17, 17-22, 22-28, 28-32, and 32-34 weeks of gestation.Results: (1) Average expression of the 16-gene untargeted cellular mRNA signature was higher in women diagnosed with early preeclampsia at 32-34 weeks of gestation, yet more importantly, also prior to diagnosis at 28-32 weeks and 22-28 weeks of gestation, compared to controls (all, p < .05). (2) A combination of four genes from this signature, including a long non-protein coding RNA [H19 imprinted maternally expressed transcript (H19)], fibronectin 1 (FN1), tubulin beta-6 class V (TUBB6), and formyl peptide receptor 3 (FPR3) had a sensitivity of 0.85 (0.55-0.98) and a specificity of 0.92 (0.8-0.98) for prediction of early preeclampsia at 22-28 weeks of gestation. (3) H19, FN1, and TUBB6 were increased in women with early preeclampsia as early as 11-17 weeks of gestation (all, p < .05). (4) After diagnosis at 32-34 weeks, but also prior to diagnosis at 11-17 weeks, women destined to have early preeclampsia showed a coordinated increase in whole blood expression of several single-cell placental signatures, including the 20-gene signature of extravillous trophoblast (all, p < .05). (5) A combination of three mRNAs from the extravillous trophoblast signature (MMP11, SLC6A2, and IL18BP) predicted early preeclampsia at 11-17 weeks of gestation with a sensitivity of 0.83 (0.52-0.98) and specificity of 0.94 (0.79-0.99). CONCLUSIONS: Circulating early transcriptomic markers for preeclampsia can be found either by untargeted profiling of the cellular transcriptome or by focusing on placental cell-specific mRNAs. The untargeted cellular mRNA signature was consistently increased in early preeclampsia after 22 weeks of gestation, and individual mRNAs of this signature were significantly increased as early as 11-17 weeks of gestation. Several single-cell placental signatures predicted future development of the disease at 11-17 weeks and were also increased in women already diagnosed at 32-34 weeks of gestation.

Gasdermin D: in vivo evidence of pyroptosis in spontaneous labor at term.

Objective: Pyroptosis is an inflammatory form of programmed cell death that is mediated by the activation of the inflammasome and depends on the pore-forming function of gasdermin D. Therefore, the detection of gasdermin D represents in vivo evidence of pyroptosis. We recently showed that there is intra-amniotic inflammasome activation in spontaneous labor at term; however, evidence of pyroptosis is lacking. The objectives of this study were to investigate (1) whether gasdermin D is detectable in the amniotic fluid of women who delivered at term; (2) whether amniotic fluid gasdermin D concentrations are associated with the process of spontaneous labor at term; and (3) whether gasdermin D is expressed in the chorioamniotic membranes from these patients.Methods: This retrospective cross-sectional study included amniotic fluid samples from 41 women who underwent spontaneous labor at term (n = 17) or delivered at term without labor (n = 24). As a readout of pyroptosis, gasdermin D was determined in amniotic fluid samples using a specific and sensitive ELISA kit. The 90th percentile of amniotic fluid gasdermin D concentrations was calculated among women without spontaneous labor at term (reference group). The association between high amniotic fluid gasdermin D concentrations (≥90th percentile in the reference group) and spontaneous labor at term was tested using the Fisher's exact test. A p value <.05 was considered significant. Multiplex immunofluorescence staining and phenoptics (multispectral imaging) were performed to determine gasdermin D expression in the chorioamniotic membranes and to colocalize this protein with the inflammasome-related molecules caspase-1 and interleukin-1ß.Results: (1) Gasdermin D is present in the amniotic fluid of women who delivered at term; (2) the 90th percentile of amniotic fluid gasdermin D concentrations in women who delivered at term without spontaneous labor was 3.4 ng/mL; (3) the proportion of women with amniotic fluid gasdermin D concentrations above the threshold was higher in those who underwent term labor than in those who delivered at term without labor; (4) amniotic fluid concentrations of gasdermin D > 3.4 ng/mL were significantly associated with the presence of spontaneous labor in women who delivered at term (odds ratio 6.0, p-value .048); and (5) the protein expression of gasdermin D is increased in the chorioamniotic membranes of women who underwent spontaneous labor at term and is colocalized with caspase-1 and IL-1ß.Conclusions: Gasdermin D is increased in the amniotic fluid and chorioamniotic membranes of women who underwent spontaneous labor at term compared to those without labor. These data provide evidence implicating pyroptosis in the mechanisms that lead to the sterile inflammatory process of term parturition.

Preterm labor is characterized by a high abundance of amniotic fluid prostaglandins in patients with intra-amniotic infection or sterile intra-amniotic inflammation.

OBJECTIVE: To distinguish between prostaglandin and prostamide concentrations in the amniotic fluid of women who had an episode of preterm labor with intact membranes through the utilisation of liquid chromatography-tandem mass spectrometry. STUDY DESIGN: Liquid chromatography-tandem mass spectrometry analysis of amniotic fluid of women with preterm labor and (1) subsequent delivery at term (2) preterm delivery without intra-amniotic inflammation; (3) preterm delivery with sterile intra-amniotic inflammation (interleukin (IL)-6>2.6 ng/mL without detectable microorganisms); and (4) preterm delivery with intra-amniotic infection [IL-6>2.6 ng/mL with detectable microorganisms]. RESULTS: (1) amniotic fluid concentrations of PGE2, PGF2α, and PGFM were higher in patients with intra-amniotic infection than in those without intra-amniotic inflammation; (2) PGE2 and PGF2α concentrations were also greater in patients with intra-amniotic infection than in those with sterile intra-amniotic inflammation; (3) patients with sterile intra-amniotic inflammation had higher amniotic fluid concentrations of PGE2 and PGFM than those without intra-amniotic inflammation who delivered at term; (4) PGFM concentrations were also greater in women with sterile intra-amniotic inflammation than in those without intra-amniotic inflammation who delivered preterm; (5) amniotic fluid concentrations of prostamides (PGE2-EA and PGF2α-EA) were not different among patients with preterm labor; (6) amniotic fluid concentrations of prostaglandins, but no prostamides, were higher in cases with intra-amniotic inflammation; and (7) the PGE2:PGE2-EA and PGF2α:PGF2α-EA ratios were higher in patients with intra-amniotic infection compared to those without inflammation. CONCLUSIONS: Mass spectrometric analysis of amniotic fluid indicated that amniotic fluid concentrations of prostaglandins, but no prostamides, were higher in women with preterm labor and intra-amniotic infection than in other patients with an episode of preterm labor. Yet, women with intra-amniotic infection had greater amniotic fluid concentrations of PGE2 and PGF2α than those with sterile intra-amniotic inflammation, suggesting that these two clinical conditions may be differentiated by using mass spectrometric analysis of amniotic fluid.

Prostaglandin and prostamide concentrations in amniotic fluid of women with spontaneous labor at term with and without clinical chorioamnionitis.

OBJECTIVE: Prostaglandins (PGs) are considered universal mediators for the process of physiological parturition. This is based on observations that amniotic fluid concentrations of PGs are elevated prior to and during the onset of labor (mostly utilizing immunoassays). Distinguishing PGs from similarly structured molecules (i.e. prostamides; PG-EA) is difficult given the cross-reactivity of available antibodies and the chemical similarity between these compounds. Herein, this limitation was overcome by utilizing mass spectrometry to determine PG and PG-EA concentrations in amniotic fluid of women with spontaneous labor at term and in those with clinical chorioamnionitis (CHAM), the most common infection-related diagnosis made in labor and delivery units worldwide. STUDY DESIGN: Liquid chromatography-tandem mass spectrometry (LC MS/MS) was used to determine the PG and PG-EA content in amniotic fluid samples of women with spontaneous labor at term with (n = 14) or without (n = 28) CHAM. Controls included women who delivered at term without labor (n = 10). RESULTS: PGE2, PGF2α, and 13,14-dihydro-15-keto-PGF2α (PGFM) were higher in amniotic fluid of women with spontaneous labor at term than in those without labor. PGE2, PGF2α, and PGFM were also higher in amniotic fluid of women with CHAM than in those without labor. However, PGE2-EA and PGF2α-EA were lower in amniotic fluid of women with CHAM than in those without CHAM. The ratios of PGE2 to PGE2-EA and PGF2α to PGF2α-EA were higher in amniotic fluid of women with spontaneous labor at term with or without CHAM than in those without labor; yet, the ratio of PGF2α to PGF2α-EA was greater in women with CHAM than in those without this clinical condition. CONCLUSIONS: Spontaneous labor at term with or without CHAM is characterized by elevated amniotic fluid concentrations of prostaglandins (PGE2, PGF2α, and PGFM) but not prostamides. Quantification of these products by LC MS/MSlc==may potentially be of utility in identifying their physiological functions relevant to parturition. SUMMARY: Prostaglandins (PGs) are critical for the onset and progression of labor. Structural similarities of PGs and prostamides (PG-EA) prevents their specific identification by immunoassay. We utilized LC MS/MS to determine PG and PG-EA content in amniotic fluid (AF) of women with spontaneous labor at term with or without CHAM and women who delivered at term without labor. Higher aamniotic ffluid PG levels were observed in women with spontaneous labor with and without CHAM compared to women delivering without labor. PG-EA levels in amniotic fluid of women with spontaneous labor and CHAM were lower than in women with spontaneous labor without CHAM but not those without labor. Ratios of PGs to PG-EAs were higher in AF of women with labor and CHAM compared to those without labor. Delineation of these products by LC MS/MS may potentially be of utility in identifying their physiological functions relevant to parturition.

Gasdermin D: Evidence of pyroptosis in spontaneous preterm labor with sterile intra-amniotic inflammation or intra-amniotic infection.

PROBLEM: Pyroptosis, inflammatory programmed cell death, is initiated through the inflammasome and relies on the pore-forming actions of the effector molecule gasdermin D. Herein, we investigated whether gasdermin D is detectable in women with spontaneous preterm labor and sterile intra-amniotic inflammation or intra-amniotic infection. METHOD OF STUDY: Amniotic fluid samples (n = 124) from women with spontaneous preterm labor were subdivided into the following groups: (a) those who delivered at term (n = 32); and those who delivered preterm (b) without intra-amniotic inflammation (n = 41), (c) with sterile intra-amniotic inflammation (n = 32), or (d) with intra-amniotic infection (n = 19), based on amniotic fluid IL-6 concentrations and the microbiological status of amniotic fluid (culture and PCR/ESI-MS). Gasdermin D concentrations were measured using an ELISA kit. Multiplex immunofluorescence staining was also performed to determine the expression of gasdermin D, caspase-1, and interleukin-1ß in the chorioamniotic membranes. Flow cytometry was used to detect pyroptosis (active caspase-1) in decidual cells from women with preterm labor and birth. RESULTS: (a) Gasdermin D was detected in the amniotic fluid and chorioamniotic membranes from women who underwent spontaneous preterm labor/birth with either sterile intra-amniotic inflammation or intra-amniotic infection, but was rarely detected in those without intra-amniotic inflammation. (b) Amniotic fluid concentrations of gasdermin D were higher in women with intra-amniotic infection than in those with sterile intra-amniotic inflammation, and its expression in the chorioamniotic membranes was associated with caspase-1 and IL-1ß (inflammasome mediators). (c) Decidual stromal cells and leukocytes isolated from women with preterm labor and birth are capable of undergoing pyroptosis given their expression of active caspase-1. CONCLUSION: Pyroptosis can occur in the context of sterile intra-amniotic inflammation and intra-amniotic infection in patients with spontaneous preterm labor and birth.