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The cell-free transcriptome in amniotic fluid (AF) has been shown to be informative of physiologic and pathologic processes in pregnancy; however, the change in AF proteome with gestational age has mostly been studied by targeted approaches. The objective of this study was to describe the gestational age-dependent changes in the AF proteome during normal pregnancy by using an omics platform. The abundance of 1310 proteins was measured on a high-throughput aptamer-based proteomics platform in AF samples collected from women during midtrimester (16-24 weeks of gestation, n = 15) and at term without labor (37-42 weeks of gestation, n = 13). Only pregnancies without obstetrical complications were included in the study. Almost 25% (320) of AF proteins significantly changed in abundance between the midtrimester and term gestation. Of these, 154 (48.1%) proteins increased, and 166 (51.9%) decreased in abundance at term compared to midtrimester. Tissue-specific signatures of the trachea, salivary glands, brain regions, and immune system were increased while those of the gestational tissues (uterus, placenta, and ovary), cardiac myocytes, and fetal liver were decreased at term compared to midtrimester. The changes in AF protein abundance were correlated with those previously reported in the cell-free AF transcriptome. Intersecting gestational age-modulated AF proteins and their corresponding mRNAs previously reported in the maternal blood identified neutrophil-related protein/mRNA pairs that were modulated in the same direction. The first study to utilize an aptamer-based assay to profile the AF proteome modulation with gestational age, it reveals that almost one-quarter of the proteins are modulated as gestation advances, which is more than twice the fraction of altered plasma proteins (~ 10%). The results reported herein have implications for future studies focused on discovering biomarkers to predict, monitor, and diagnose obstetrical diseases., (© 2022. The Author(s).)

Background: The amniotic fluid (AF) cell-free transcriptome is modulated by physiologic and pathologic processes during pregnancy. AF gene expression changes with advancing gestation reflect fetal development and organ maturation; yet, defining normal expression and splicing patterns for biomarker discovery in obstetrics requires larger heterogeneous cohorts, evaluation of potential confounding factors, and novel analytical approaches., Methods: Women with a normal pregnancy who had an AF sample collected during midtrimester (n = 30) or at term gestation (n = 68) were included. Expression profiling at exon level resolution was performed using Human Transcriptome Arrays. Differential expression was based on moderated t-test adjusted p < 0.05 and fold change > 1.25; for differential splicing, a splicing index > 2 and adjusted p < 0.05 were required. Functional profiling was used to interpret differentially expressed or spliced genes. The expression of tissue-specific and cell-type specific signatures defined by single-cell genomics was quantified and correlated with covariates. In-silico validation studies were performed using publicly available datasets., Results: 1) 64,071 genes were detected in AF, with 11% of the coding and 6% of the non-coding genes being differentially expressed between midtrimester and term gestation. Expression changes were highly correlated with those previously reported (R > 0.79, p < 0.001) and featured increased expression of genes specific to the trachea, salivary glands, and lung and decreased expression of genes specific to the cardiac myocytes, uterus, and fetal liver, among others. 2) Single-cell RNA-seq signatures of the cytotrophoblast, Hofbauer cells, erythrocytes, monocytes, T and B cells, among others, showed complex patterns of modulation with gestation (adjusted p < 0.05). 3) In 17% of the genes detected, we found differential splicing with advancing gestation in genes related to brain development processes and immunity pathways, including some that were missed based on differential expression analysis alone., Conclusions: This represents the largest AF transcriptomics study in normal pregnancy, reporting for the first time that single-cell genomic signatures can be tracked in the AF and display complex patterns of expression during gestation. We also demonstrate a role for alternative splicing in tissue-identity acquisition, organ development, and immune processes. The results herein may have implications for the development of fetal testing to assess placental function and fetal organ maturity.

Identification of pregnancies at risk of preterm birth (PTB), the leading cause of newborn deaths, remains challenging given the syndromic nature of the disease. We report a longitudinal multi-omics study coupled with a DREAM challenge to develop predictive models of PTB. The findings indicate that whole-blood gene expression predicts ultrasound-based gestational ages in normal and complicated pregnancies (r = 0.83) and, using data collected before 37 weeks of gestation, also predicts the delivery date in both normal pregnancies (r = 0.86) and those with spontaneous preterm birth (r = 0.75). Based on samples collected before 33 weeks in asymptomatic women, our analysis suggests that expression changes preceding preterm prelabor rupture of the membranes are consistent across time points and cohorts and involve leukocyte-mediated immunity. Models built from plasma proteomic data predict spontaneous preterm delivery with intact membranes with higher accuracy and earlier in pregnancy than transcriptomic models (AUROC = 0.76 versus AUROC = 0.6 at 27-33 weeks of gestation).

It is increasingly evident that cytokines and growth factors produced in the decidua play a pivotal role in the regulation of the local immune microenvironment and the establishment of pregnancy. One of the major growth factors produced in the decidua is vascular endothelial growth factor (VEGF), which acts not only on endothelial cells, but also on multiple other cell types, including macrophages. We sought to determine whether decidua-derived VEGF affects macrophage recruitment and polarization using human endometrial/decidual tissue samples, primary human endometrial stromal cells (ESCs), and the human monocyte cell line THP1. In situ hybridization was used for assessment of local VEGF expression and immunohistochemistry was used for identification and localization of CD68-positive endometrial macrophages. Macrophage migration in culture was assessed using a transwell migration assay, and the various M1/M2 phenotypic markers and VEGF expression were assessed using quantitative real-time PCR (qRT-PCR). We found dramatic increases in both VEGF levels and macrophage numbers in the decidua during early pregnancy compared to the secretory phase endometrium (non-pregnant), with a significant increase in M2 macrophage markers, suggesting that M2 is the predominant macrophage phenotype in the decidua. However, decidual samples from preeclamptic pregnancies showed a significant shift in macrophage phenotype markers, with upregulation of M1 and downregulation of M2 markers. In THP1 cultures, VEGF treatment significantly enhanced macrophage migration and induced M1 macrophages to shift to an M2 phenotype. Moreover, treatment with conditioned media from decidualized ESCs induced changes in macrophage migration and polarization similar to that of VEGF treatment. These effects were abrogated by the addition of a potent VEGF inhibitor. Together these results suggest that decidual VEGF plays a significant role in macrophage recruitment and M2 polarization, and that inhibition of VEGF signaling may contribute to the shift in macrophage polarity observed in different pregnancy disorders, including preeclampsia. [ABSTRACT FROM AUTHOR]

• Vaginal fluid immune mediators negatively correlated with CST IV vaginal bacteria. • CXCL10, in particular, was negatively correlated with 15 CST IV bacteria. • Specific cytokines were negatively correlated with CST IV bacteria in preterm births. • Sneathia sanguinegens was negatively correlated with cytokines in preterm births. Recent studies suggest that alterations in the vaginal microbiome allow for the assessment of the risk for spontaneous preterm birth (PTB), the leading cause of neonatal morbidity and mortality worldwide. However, the associations between the local immune response and the vaginal microbiome are still poorly understood. Herein, we characterize the vaginal host immune-microbiome interactions in women who ultimately underwent PTB and in those who delivered at term. Vaginal fluid samples from 52 pregnant women (of whom 18 underwent PTB and 34 delivered at term) were collected between 10 and 32 weeks of gestation in a case-control study. Concentrations of 33 immune mediators were determined using sensitive and specific immunoassays. The previously published 16S rRNA gene sequence and bacterial phylotype data of these subjects were utilized in this study. Linear mixed effects models were utilized to test associations between vaginal immune mediator concentrations and bacterial phylotype relative abundances. 1) In the overall study population, vaginal concentrations of CXCL10, CCL2, CCL3, SLP1 and VEGF negatively correlated with non- Lactobacillus , Community State Type IV (CST IV) members of the vaginal microbiome; 2) CXCL10, in particular, negatively correlated with 15 bacterial phylotypes, most of which are typical members of CST IV, such as Gardnerella vaginalis , Megasphaera spp., and Atopobium vaginae ; 3) Gemella spp., also members of CST IV, negatively correlated with vaginal concentrations of VEGF, CCL2, CCL3, SLPI, and CXCL10; 4) when comparing PTB cases to term controls, five soluble immune mediators (CCL26, CCL22, CCL2, CXCL10, and IL-16), especially CCL26, were negatively correlated with five typical members of CST IV: Sneathia sanguinegens , Parvimonas micra , Veillonellaceae, BVAB2, and Gemella spp.; and 5) Sneathia sanguinegens had stronger negative associations with all five soluble immune mediators (CCL26, CCL22, CCL2, CXCL10, and IL-16) in PTB cases than in term controls. The assessment of vaginal host immune-microbiome interactions revealed that specific soluble immune mediators, mainly CXCL10, negatively correlated with typical members of CST IV of the vaginal microbiome. Sneathia sanguinegens , in particular, had stronger negative associations with different immune mediators, including CXCL10 and CCL26, in women who ultimately underwent PTB compared to those who delivered at term. These findings provide insight into the vaginal host immune-microbiome interactions in normal and complicated pregnancies. [ABSTRACT FROM AUTHOR]