Integrated unbiased multiomics defines disease-independent placental clusters in common obstetrical syndromes.

BACKGROUND: Placental dysfunction, a root cause of common syndromes affecting human pregnancy, such as preeclampsia (PE), fetal growth restriction (FGR), and spontaneous preterm delivery (sPTD), remains poorly defined. These common, yet clinically disparate obstetrical syndromes share similar placental histopathologic patterns, while individuals within each syndrome present distinct molecular changes, challenging our understanding and hindering our ability to prevent and treat these syndromes. METHODS: Using our extensive biobank, we identified women with severe PE (n = 75), FGR (n = 40), FGR with a hypertensive disorder (FGR + HDP; n = 33), sPTD (n = 72), and two uncomplicated control groups, term (n = 113), and preterm without PE, FGR, or sPTD (n = 16). We used placental biopsies for transcriptomics, proteomics, metabolomics data, and histological evaluation. After conventional pairwise comparison, we deployed an unbiased, AI-based similarity network fusion (SNF) to integrate the datatypes and identify omics-defined placental clusters. We used Bayesian model selection to compare the association between the histopathological features and disease conditions vs SNF clusters. RESULTS: Pairwise, disease-based comparisons exhibited relatively few differences, likely reflecting the heterogeneity of the clinical syndromes. Therefore, we deployed the unbiased, omics-based SNF method. Our analysis resulted in four distinct clusters, which were mostly dominated by a specific syndrome. Notably, the cluster dominated by early-onset PE exhibited strong placental dysfunction patterns, with weaker injury patterns in the cluster dominated by sPTD. The SNF-defined clusters exhibited better correlation with the histopathology than the predefined disease groups. CONCLUSIONS: Our results demonstrate that integrated omics-based SNF distinctively reclassifies placental dysfunction patterns underlying the common obstetrical syndromes, improves our understanding of the pathological processes, and could promote a search for more personalized interventions.

Unc-13 homolog D mediates an antiviral effect of the chromosome 19 microRNA cluster miR-517a.

The function of microRNAs (miRNAs) can be cell autonomous or communicated to other cell types and has been implicated in diverse biological processes. We previously demonstrated that miR-517a-3p (miR-517a), a highly expressed member of the chromosome 19 miRNA cluster (C19MC) that is transcribed almost exclusively in human trophoblasts, attenuates viral replication via induction of autophagy in non-trophoblastic recipient cells. However, the molecular mechanisms underlying these effects remain unknown. Here, we identified unc-13 homolog D (UNC13D) as a direct, autophagy-related gene target of miR-517a, leading to repression of UNC13D. In line with the antiviral activity of miR-517a, silencing UNC13D suppressed replication of vesicular stomatitis virus (VSV), whereas overexpression of UNC13D increased VSV levels, suggesting a role for UNC13D silencing in the antiviral activity of miR-517a. We also found that miR-517a activated NF-κB signaling in HEK-293XL cells expressing TLR8, but the effect was not specific to C19MC miRNA. Taken together, our results define mechanistic pathways that link C19MC miRNA with inhibition of viral replication.

Expression and trafficking of placental microRNAs at the feto-maternal interface.

During pregnancy, placental trophoblasts at the feto-maternal interface produce a broad repertoire of microRNA (miRNA) species. These species include miRNA from the primate-specific chromosome 19 miRNA cluster (C19MC), which is expressed nearly exclusively in the placenta. Trafficking of these miRNAs among the maternal, placental, and fetal compartments is unknown. To determine miRNA expression and trafficking patterns during pregnancy, we sequenced miRNAs in triads of human placenta and of maternal and fetal blood and found large subject-to-subject variability, with C19MC exhibiting compartment-specific expression. We therefore created humanized mice that transgenically express the entire 160-kb human C19MC locus or lentivirally express C19MC miRNA members selectively in the placenta. C19MC transgenic mice expressed a low level of C19MC miRNAs in diverse organs. When pregnant, female C19MC mice exhibited a strikingly elevated (>40-fold) expression of C19MC miRNA in the placenta, compared with other organs, that resembled C19MC miRNAs patterns in humans. Our mouse models showed that placental miRNA traffic primarily to the maternal circulation and that maternal miRNA can traffic to the placenta and even into the fetal compartment. These findings define an extraordinary means of nonhormonal, miRNA-based communication between the placenta and feto-maternal compartments.-Chang, G., Mouillet, J.-F., Mishima, T., Chu, T., Sadovsky, E., Coyne, C. B., Parks, W. T., Surti, U., Sadovsky, Y. Expression and trafficking of placental microRNAs at the feto-maternal interface.

The assembly of miRNA-mRNA-protein regulatory networks using high-throughput expression data.

MOTIVATION: Inference of gene regulatory networks from high throughput measurement of gene and protein expression is particularly attractive because it allows the simultaneous discovery of interactive molecular signals for numerous genes and proteins at a relatively low cost. RESULTS: We developed two score-based local causal learning algorithms that utilized the Markov blanket search to identify direct regulators of target mRNAs and proteins. These two algorithms were specifically designed for integrated high throughput RNA and protein data. Simulation study showed that these algorithms outperformed other state-of-the-art gene regulatory network learning algorithms. We also generated integrated miRNA, mRNA, and protein expression data based on high throughput analysis of primary trophoblasts, derived from term human placenta and cultured under standard or hypoxic conditions. We applied the new algorithms to these data and identified gene regulatory networks for a set of trophoblastic proteins found to be differentially expressed under the specified culture conditions.

Human placental trophoblasts confer viral resistance to recipient cells.

Placental trophoblasts form the interface between the fetal and maternal environments and serve to limit the maternal-fetal spread of viruses. Here we show that cultured primary human placental trophoblasts are highly resistant to infection by a number of viruses and, importantly, confer this resistance to nonplacental recipient cells by exosome-mediated delivery of specific microRNAs (miRNAs). We show that miRNA members of the chromosome 19 miRNA cluster, which are almost exclusively expressed in the human placenta, are packaged within trophoblast-derived exosomes and attenuate viral replication in recipient cells by the induction of autophagy. Together, our findings identify an unprecedented paracrine and/or systemic function of placental trophoblasts that uses exosome-mediated transfer of a unique set of placental-specific effector miRNAs to directly communicate with placental or maternal target cells and regulate their immunity to viral infections.

MicroRNAs in placental health and disease.

MicroRNAs (miRNAs) constitute a large family of small noncoding RNAs that are encoded by the genomes of most organisms. They regulate gene expression through posttranscriptional mechanisms to attenuate protein output in various genetic networks. The discovery of miRNAs has transformed our understanding of gene regulation and sparked intense efforts intended to harness their potential as diagnostic markers and therapeutic tools. Over the last decade, a flurry of studies has shed light on placental miRNAs but has also raised many questions regarding the scope of their biologic action. Moreover, the recognition that miRNAs of placental origin are released continually in the maternal circulation throughout pregnancy suggested that circulating miRNAs might serve as biomarkers for placental function during pregnancy. Although this generated much enthusiasm, recently recognized challenges have delayed the application of miRNA-based biomarkers and therapeutics in clinical practice. In this review, we summarize key findings in the field and discuss current knowledge related to miRNAs in the context of placental biology.

The unique expression and function of miR-424 in human placental trophoblasts.

Placental hypoperfusion causes cellular hypoxia and is associated with fetal growth restriction and preeclampsia. In response to hypoxia, the repertoire of genes expressed in placental trophoblasts changes, which influences key cellular processes such as differentiation and fusion. Diverse miRNAs were recently found to modulate the cellular response to hypoxia. Here we show that miR-424, which was previously shown to be upregulated by hypoxia in nontrophoblastic cell types, is uniquely downregulated in primary human trophoblasts by hypoxia or chemicals known to hinder cell differentiation. We also identify FGFR1 as a direct target of miR-424 in human trophoblasts. This effect is unique to miR-424 and is not seen with other members of this miRNA family that are expressed in trophoblasts, such as miR-15 and miR-16. Our findings establish a unique role for miR-424 during differentiation of human trophoblasts.

Defining trophoblast injury patterns in the transcriptomes of dysfunctional placentas.

Trophoblast injury is central to clinically relevant placenta dysfunction. We hypothesized that the mRNA of primary human trophoblasts, exposed to distinct injuries in vitro, capture transcriptome patterns of placental biopsies obtained from common obstetrical syndromes. We deployed a CIBERSORTx deconvolution method to correlate trophoblastic RNAseq-based expression matrices with the transcriptome of omics-defined placental dysfunction patterns in vivo. We found distinct trophoblast injury patterns in placental biopsies from women with fetal growth restriction and a hypertensive disorder, or in biopsies clustered by their omics analysis. Our RNAseq data are useful for defining the contribution of trophoblast injuries to placental dysfunction syndromes.

MiR-205 silences MED1 in hypoxic primary human trophoblasts.

Acting through degradation of target mRNA or inhibition of translation, microRNAs (miRNAs) regulate development, differentiation, and cellular response to diverse cues. We analyzed changes in miRNA expression in human placental trophoblasts exposed to hypoxia, which may result from hypoperfusion and placental injury. Using an miRNA microarray screen, confirmed by Northern blot analysis, we defined a set of seven miRNAs (miR-93, miR-205, miR-224, miR-335, miR-424, miR-451, and miR-491) that are differentially regulated in primary trophoblasts exposed to hypoxia. We combined in silico prediction of miRNA targets with gene expression profiling data to identify a series of potential targets for the miRNAs, which were further analyzed using luciferase reporter assays. Among experimentally confirmed targets, we found that the transcriptional coactivator MED1, which plays an important role in placental development, is a target for miR-205. Using gain- and loss-of-function assays, we confirmed that miR-205 interacts with a specific target in the 3'-UTR sequence of MED1 and silences MED1 expression in human trophoblasts exposed to hypoxia, suggesting that miR-205 plays a role in trophoblast injury.

Expression patterns of placental microRNAs.

Among different types of small RNA molecules, distinct types of microRNAs (miRNAs) are expressed in many cell types, where they modulate RNA stability and translation, thus controlling virtually every aspect of tissue development, proliferation, differentiation, and function. Aberrant miRNA expression has been linked to discrete pathologic processes. As the placenta plays a pivotal role in governing fetal development, it is not surprising that the placenta expresses numerous types of miRNAs. Whereas many of these miRNAs are ubiquitously expressed, certain miRNA species are largely unique to the placenta. Research in the field of placental miRNAs is in its early phase, with most studies centering on cataloging placental miRNA species or examining differences in placental miRNA expression between placentas from normal pregnancies and those from pregnancies complicated by pathologies that are associated with placental dysfunction. Recent research endeavors ventured to assess the function of miRNAs in cultured placental trophoblasts, using in vitro conditions that model relevant pathophysiological processes. The impact of miRNA-mediated repression on the trophoblast transcriptome, particularly in response to genetic and environmental perturbations, remains largely unknown. Further in-depth studies are required to unravel the functional significance of miRNAs in molding placental robustness, which must constantly adapt to altered maternal physiologic status to sustain optimal support to the developing embryo. In this review, we summarize the current information about placental miRNAs expression, and the lingering challenges in this field.

Trophoblastic extracellular vesicles and viruses: Friends or foes?

Cells produce cytoplasmic vesicles to facilitate the processing and transport of RNAs, proteins, and other signaling molecules among intracellular organelles. Moreover, most cells release a range of extracellular vesicles (EVs) that mediate intercellular communication in both physiological and pathological settings. In addition to a better understanding of their biological functions, the diagnostic and therapeutic prospects of EVs, particularly the nano-sized small EVs (sEVs, exosomes), are currently being rigorously pursued. While EVs and viruses such as retroviruses might have evolved independently, they share a number of similar characteristics, including biogenesis pathways, size distribution, cargo, and cell-targeting mechanisms. The interplay of EVs with viruses has profound effects on viral replication and infectivity. Our research indicates that sEVs, produced by primary human trophoblasts, can endow other non-placental cell types with antiviral response. Better insights into the interaction of EVs with viruses may illuminate new ways to attenuate viral infections during pregnancy, and perhaps develop new antiviral therapeutics to protect the feto-placental unit during critical times of human development.

RNA Network Interactions During Differentiation of Human Trophoblasts.

In the human placenta, two trophoblast cell layers separate the maternal blood from the villous basement membrane and fetal capillary endothelial cells. The inner layer, which is complete early in pregnancy and later becomes discontinuous, comprises the proliferative mononuclear cytotrophoblasts, which fuse together and differentiate to form the outer layer of multinucleated syncytiotrophoblasts. Because the syncytiotrophoblasts are responsible for key maternal-fetal exchange functions, tight regulation of this differentiation process is critical for the proper development and the functional role of the placenta. The molecular mechanisms regulating the fusion and differentiation of trophoblasts during human pregnancy remain poorly understood. To decipher the interactions of non-coding RNAs (ncRNAs) in this process, we exposed cultured primary human trophoblasts to standard in vitro differentiation conditions or to conditions known to hinder this differentiation process, namely exposure to hypoxia (O2 < 1%) or to the addition of dimethyl sulfoxide (DMSO, 1.5%) to the culture medium. Using next generation sequencing technology, we analyzed the differential expression of trophoblastic lncRNAs, miRNAs, and mRNAs that are concordantly modulated by both hypoxia and DMSO. Additionally, we developed a model to construct a lncRNA-miRNA-mRNA co-expression network and inferred the functions of lncRNAs and miRNAs via indirect gene ontology analysis. This study improves our knowledge of the interactions between ncRNAs and mRNAs during trophoblast differentiation and identifies key biological processes that may be impaired in common gestational diseases, such as fetal growth restriction or preeclampsia.

Transgenic expression of human C19MC miRNAs impacts placental morphogenesis.

INTRODUCTION: The chromosome 19 miRNA cluster (C19MC) encodes a large family of microRNAs (miRNAs) that are abundantly expressed in the placenta of higher primates and also in certain cancers. In the placenta, miRNAs from this cluster account for nearly 40% of all miRNAs present in trophoblasts. However, the function of these miRNAs in the placenta remains poorly understood. Recent observations reveal a role for these miRNAs in cell migration, and suggest that they are involved in the development and function of the human placenta. Here, we examine the placenta in transgenic mice expressing the human C19MC miRNAs. METHODS: We produced transgenic mice using pronuclear microinjection of a bacterial artificial chromosome plasmid carrying the entire human C19MC locus and derived a homozygous line using crossbreeding. We performed morphological characterization and profiled gene expression changes in the placentas of the transgenic mice. RESULTS: C19MC transgenic mice delivered on time with no gross malformations. The placentas of transgenic mice expressed C19MC miRNAs and were larger than wild type placentas. Histologically, we found that the transgenic placenta exhibited projections of spongiotrophoblasts that penetrated deep into the labyrinth. Gene expression analysis revealed alterations in the expression of several genes involved in cell migration, with evidence of enhanced cell proliferation. DISCUSSION: Mice that were humanized for transgenically overexpressed C19MC miRNAs exhibit enlarged placentas with aberrant delineation of cell layers. The observed phenotype and the related gene expression changes suggest disrupted migration of placental cell subpopulations.

Placental small extracellular vesicles: Current questions and investigative opportunities.

The discovery of regulated trafficking of extracellular vesicles (EVs) has added a new dimension to our understanding of local and distant communication among cells and tissues. Notwithstanding the expanded landscape of EV subtypes, the majority of research in the field centers on small and large EVs that are commonly termed exosomes, microvesicles and apoptotic cell-derived vesicles. In the context of pregnancy, EV-based communication has a special role in the crosstalk among the placenta, maternal and fetal compartments, with most studies focusing on trophoblastic EVs and their effect on other placental cell types, endothelial cells, and distant tissues. Many unanswered questions in the field of EV biology center on the mechanisms of vesicle biogenesis, loading of cargo molecules, EV release and trafficking, the interaction of EVs with target cells and the endocytic pathways underlying their uptake, and the intracellular processing of EVs inside target cells. These questions are directly relevant to EV-based placental-maternal-fetal communication and have unique implications in the context of interaction between two organisms. Despite rapid progress in the field, the number of speculative, unsubstantiated assumptions about placental EVs is concerning. Here we attempt to delineate existing knowledge in the field, focusing primarily on placental small EVs (exosomes). We define central questions that require investigative attention in order to advance the field.

DEAD-box protein-103 (DP103, Ddx20) is essential for early embryonic development and modulates ovarian morphology and function.

The DEAD-box helicase DP103 (Ddx20, Gemin3) is a multifunctional protein that interacts with Epstein-Barr virus nuclear proteins (EBNA2/EBNA3) and is a part of the spliceosomal small nuclear ribonucleoproteins complex. DP103 also aggregates with the micro-RNA machinery complex. We have previously shown that DP103 interacts with the nuclear receptor steroidogenic factor-1 (SF-1, NR5A1), a key regulator of reproductive development, and represses its transcriptional activity. To further explore the physiological function of DP103, we disrupted the corresponding gene in mice. Homozygous Dp103-null mice die early in embryonic development before a four-cell stage. Although heterozygous mice are healthy and fertile, analysis of steroidogenic tissues revealed minor abnormalities in mutant females, including larger ovaries, altered estrous cycle, and reduced basal secretion of ACTH. Our data point to diverse functions of murine DP103, with an obligatory role during early embryonic development and also in modulation of steroidogenesis.

A novel domain within the DEAD-box protein DP103 is essential for transcriptional repression and helicase activity.

Members of the DEAD-box family of helicases, distinguished by a core characteristic sequence of Asp-Glu-Ala-Asp, are expressed in a wide range of prokaryotes and eukaryotes and exhibit diverse cellular functions, including DNA transcription, recombination and repair, RNA processing, translation, and posttranslational regulation. Although ubiquitous, the function of most DEAD-box proteins is unknown. We and others have recently cloned DP103, which harbors conserved DEAD-box, helicase, and ATPase domains in its N terminus. DP103 (also termed Gemin3 and DDX20) interacts with SF-1, SMN, EBNA2, and EBNA3C in mammalian cells. Here we demonstrate that a discrete domain within the nonconserved C-terminal region of DP103 directly interacts with SF-1. This domain exhibits an autonomous repression function and is necessary and sufficient for repressing the transcriptional activity of SF-1. Furthermore, intact DP103 exhibits helicase activity. Importantly, the C-terminal domain is obligatory but not sufficient for this unwinding activity of DP103. Together, our results support a novel paradigm for transcriptional repression and demonstrate the bifunctional role of the C-terminal domain of DP103.

The expression level of C19MC miRNAs in early pregnancy and in response to viral infection.

INTRODUCTION: We have previously shown that miRNAs produced from the Chromosome 19 MiRNA Cluster (C19MC), which are expressed almost exclusively in primate trophoblasts and are released into the maternal circulation, reduce viral replication in non-placental cells and can modulate migratory behavior of extravillous trophoblast. We sought to define the expression pattern of C19MC miRNA in early pregnancy and in response to viral infection in vitro and in vivo. METHODS: We prospectively followed women undergoing in vitro fertilization (IVF) and determined their blood level of C19MC miRNA using RT-qPCR. To examine the effect of viral exposure on C19MC miRNAs expression, we used three systems: (1) a transgenic mouse overexpressing the C19MC cluster and exposed to Togaviridae during pregnancy, (2) cultured primary human trophoblasts exposed to Vesicular Stomatitis Virus in vitro, and (3) amniotic fluid from women exposed to cytomegalovirus during pregnancy. RESULTS: In 27 IVF pregnancies, C19MC miRNAs were detected as early as 2 weeks after implantation, and their levels increased thereafter. There was no change in C19MC miRNA expression levels in the mouse placenta in response to viral exposure. Similarly, Vesicular Stomatitis Virus infection of primary human trophoblast did not selectively increase C19MC miRNA expression. C19MC miRNA expression in the amniotic fluid was not affected by vertical transmission of cytomegalovirus. DISCUSSION: The expression of C19MC miRNAs in maternal circulation very early in pregnancy suggests a role in the establishment of the maternal-fetal interface. The levels of C19MC miRNA are not influenced by diverse types of viral infection.

The Function of TrophomiRs and Other MicroRNAs in the Human Placenta.

In eutherian organisms, the placenta interfaces the fetal and maternal environments. Located at the placental villous surface, in direct contact with maternal blood, is the trophoblast layer, which mediates the crucial maternal-fetal exchange of gases, nutrients, and waste products, produces hormones that support the pregnancy, and provides immunologic defense. Discovery of microRNAs (miRNAs) and their role in development, differentiation, and homeostatic resilience has increased our understanding of genomic and epigenomic networks that regulate placental function. Moreover, unique miRNA species, which are expressed by human trophoblasts and are termed "trophomiRs," may show specialized functions during normal and pathological pregnancies. Placental miRNAs, packaged within exosomes and other vesicles or bound in protein complexes, are capable of communicating distinctive signals to maternal and/or fetal tissues. Additional research may usher in the use of circulating miRNAs as pregnancy-related disease biomarkers, providing new diagnostic and therapeutic options during pregnancy.

The expression and post-transcriptional regulation of FSTL1 transcripts in placental trophoblasts.

INTRODUCTION: Follistatin-like-1 (FSTL1) is a widely expressed secreted protein with diverse but poorly understood functions. Originally described as a pro-inflammatory molecule, it has recently been reported to play a role in signaling pathways that regulate development and homeostasis. Distinctively, FSTL1 harbors within its 3'-UTR the sequence encoding microRNA-198 (miR-198), shown to be inversely regulated relative to FSTL1 expression and to exhibit opposite actions on cellular processes such as cell migration. We sought to investigate the expression of FSTL1 and to assess its interplay with miR-198 in human trophoblasts. METHODS: We used a combination of northern blot analyses, quantitative PCR, small RNA sequencing, western blot and immunohistochemistry to characterize FSTL1 and miR-198 expression in placental trophoblasts. We also used reporter assays to examine the post-transcriptional regulation of FSTL1 and assess its putative regulation by miR-198. RESULTS: We detected the expression of FSTL1 transcript in both the human extravillous trophoblast line HTR-8/SVneo and in primary term human villous trophoblasts. We also found that the expression of FSTL1 was largely restricted to extravillous trophoblasts. Hypoxia enhanced the expression of FSTL1 protein in cultured primary villous trophoblasts. Interestingly, we did not detect any evidence for expression or function of mature miR-198 in human trophoblasts. DISCUSSION: Our data indicate that placental FSTL1 is expressed particularly in extravillous trophoblasts. We also found no evidence for placental expression of miR-198, or for its regulation of FSTL1, implying that the post-transcriptional regulation of FSTL1 by miR-198 is tissue specific.

The role of trophoblastic microRNAs in placental viral infection.

During the past decade, various types of small non-coding RNAs were found to be expressed in all kingdoms and phyla of life. Intense research efforts have begun to shed light on their biological functions, although much remains to be determined in order to fully characterize their scope of biological action. Typically, small RNAs provide sequence specificity to a protein complex that is driven to silence a long target RNA. MicroRNAs (miRNAs) are small RNAs that are coded in the genome of most eukaryotes, and contribute to the cellular identity by regulating cell-specific gene networks by translational repression or degradation of mRNA. These effects commonly fine-tune gene expression associated with developmental or environmental cues. Different cell types can be characterized by their distinctive cellular miRNA landscape. The human placenta expresses a unique set of miRNAs, a high proportion of which is derived from a large cluster located on chromosome 19, (termed chromosome 19 miRNA cluster, or C19MC). Interestingly, a fraction of these placenta-enriched miRNAs are released to the extracellular environment through exosomes that were recently found to induce an antiviral immunity. In this review, we explore relevant placental viral infections and discuss the antiviral role of exosome-packaged placental C19MC miRNAs in this context.