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.

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 impact of opioids on the transcriptional landscape of human villous trophoblasts.

INTRODUCTION: Opioid use disorder (OUD) is implicated in major obstetrical diseases such as fetal growth restriction. Whether or not opioids directly impact placental trophoblast development and function remains unclear. We sought to examine the expression of opioid receptors (OPRs) in villous trophoblasts and the effect of opioids on placental transcriptomics. METHODS: Trophoblast stem (TS) cells and primary human trophoblast (PHT) cells from healthy term placentas were used to assess OPR expression in conditions that enhance trophoblast stemness vs differentiation. Placental RNAseq was conducted using our retrospective cohorts of pregnant people with OUD vs controls, both without major obstetrical complications. RT-qPCR was used to determine the effect of fentanyl on the expression of putative opioid targets and stemness or differentiation-associated genes in TS and PHT cells. RESULTS: Three main OPRs, including OPRM1, OPRD1, and OPRK1 were expressed in term PHT cells cultured in the stemness medium, whereas only OPRD1 and OPRK1 were expressed in TS cells. Interestingly, upon induction of differentiation, the expressed OPR mRNAs in TS or in PHT cells were downregulated. We found 286 differentially expressed long RNAs in placentas from the OUD participants vs controls. While three putative opioid targets differed their expression in stemness vs differentiation states of trophoblasts, fentanyl had no effect on their expression or the expression of major stemness or differentiation-relevant genes in TS and PHT cells. DISCUSSION: Trophoblastic expression of OPRs and opioid RNA targets is impacted by cell differentiation, suggesting differential susceptibility of villous trophoblasts to the effect of opioids.

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.

The lipase cofactor CGI58 controls placental lipolysis.

In eutherians, the placenta plays a critical role in the uptake, storage, and metabolism of lipids. These processes govern the availability of fatty acids to the developing fetus, where inadequate supply has been associated with substandard fetal growth. Whereas lipid droplets are essential for the storage of neutral lipids in the placenta and many other tissues, the processes that regulate placental lipid droplet lipolysis remain largely unknown. To assess the role of triglyceride lipases and their cofactors in determining placental lipid droplet and lipid accumulation, we assessed the role of patatin like phospholipase domain containing 2 (PNPLA2) and comparative gene identification-58 (CGI58) in lipid droplet dynamics in the human and mouse placenta. While both proteins are expressed in the placenta, the absence of CGI58, not PNPLA2, markedly increased placental lipid and lipid droplet accumulation. These changes were reversed upon restoration of CGI58 levels selectively in the CGI58-deficient mouse placenta. Using co-immunoprecipitation, we found that, in addition to PNPLA2, PNPLA9 interacts with CGI58. PNPLA9 was dispensable for lipolysis in the mouse placenta yet contributed to lipolysis in human placental trophoblasts. Our findings establish a crucial role for CGI58 in placental lipid droplet dynamics and, by extension, in nutrient supply to the developing fetus.

Term Human Placental Trophoblasts Express SARS-CoV-2 Entry Factors ACE2, TMPRSS2, and Furin.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had a massive impact on human lives worldwide. While the airborne SARS-CoV-2 primarily affects the lungs, viremia is not uncommon. As placental trophoblasts are directly bathed in maternal blood, they are vulnerable to SARS-CoV-2. Intriguingly, the human fetus is largely spared from SARS-CoV-2 infection. We tested whether the human placenta expresses the main SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin and showed that ACE2 and TMPRSS2 are expressed in the trophoblast rather than in other placental villous cells. While furin is expressed in the main placental villous cell types, we surveyed, trophoblasts exhibit the highest expression. In line with the expression of these entry factors, we demonstrated that a SARS-CoV-2 pseudovirus could enter primary human trophoblasts. Mechanisms underlying placental defense against SARS-CoV-2 infection likely involve postentry processing, which may be germane for mitigating interventions against SARS-CoV-2.IMPORTANCE Pregnant women worldwide have been affected by COVID-19. As the virus is commonly spread to various organs via the bloodstream and because human placental trophoblasts are directly bathed in maternal blood, feto-placental infection by SARS-CoV-2 seems likely. However, despite the heightened risk to pregnant women, thus far the transmission risk of COVID-19 to the feto-placental unit seems extremely low. This has been recently attributed to a negligible expression of SARS-CoV-2 entry factors in the human placenta. We therefore sought to explore the expression of the entry factors ACE2 and TMPRSS2 in the different cell types of human placental villi. Using a combination of transcriptome sequencing (RNA-seq), real-time quantitative PCR (RT-qPCR), in situ hybridization, and immunofluorescence, we found that trophoblasts, but not the other main villous cell types, express ACE2 and TMPRSS2, with a broad expression of furin. Correspondingly, we also showed that primary human trophoblasts are permissive to entry of SARS-CoV-2 pseudovirus particles.

Placental response to maternal SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic affected people at all ages. Whereas pregnant women seemed to have a worse course of disease than age-matched non-pregnant women, the risk of feto-placental infection is low. Using a cohort of 66 COVID-19-positive women in late pregnancy, we correlated clinical parameters with disease severity, placental histopathology, and the expression of viral entry and Interferon-induced transmembrane (IFITM) antiviral transcripts. All newborns were negative for SARS-CoV-2. None of the demographic parameters or placental histopathological characteristics were associated with disease severity. The fetal-maternal transfer ratio for IgG against the N or S viral proteins was commonly less than one, as recently reported. We found that the expression level of placental ACE2, but not TMPRSS2 or Furin, was higher in women with severe COVID-19. Placental expression of IFITM1 and IFITM3, which have been implicated in antiviral response, was higher in participants with severe disease. We also showed that IFITM3 protein expression, which localized to early and late endosomes, was enhanced in severe COVID-19. Our data suggest an association between disease severity and placental SARS-CoV-2 processing and antiviral pathways, implying a role for these proteins in placental response to SARS-CoV-2.

Unique microRNA Signals in Plasma Exosomes from Pregnancies Complicated by Preeclampsia.

Although preeclampsia is a common and serious complication of pregnancy, insight into its pathobiology and diagnosis is lacking. Circulating plasma exosomes, which contain RNA and other molecules and have recently become accessible for diagnostics, may be informative in this regard. We tested the hypothesis that preeclampsia may affect the miRNA cargo within circulating maternal blood exosomes. We collected plasma from 60 pregnant women at term, including 20 women with pregnancy complicated by preeclampsia, and 20 women with fetal growth restriction and 20 with healthy pregnancy, serving as controls. We isolated exosomes from the maternal plasma by continuous density gradient ultracentrifugation. Our main outcome variable was exosomal miRNA cargo, analyzed by quantitative polymerase chain reaction-based TaqMan advanced miRNA assay in a card format and the expression of differentially expressed exosomal miRNA in whole plasma from the same participants. We found that 7 miRNA species were differentially expressed in exosomes from women with preeclampsia and those from controls. In contrast, there was no significant difference in exosomal miRNA expression between women with fetal growth restriction and controls. The results were not affected by fetal sex. Only one of the preeclampsia-related, differentially expressed exosomal miRNAs was significantly different in whole plasma miRNA analysis. We concluded that unlike whole plasma miRNA, exosomes extracted from the plasma of women with preeclampsia exhibit a unique miRNA profile, suggesting that plasma exosomal miRNA could provide insight into the pathophysiology of preeclampsia, and may play a role in disease diagnostics.

Internalization of trophoblastic small extracellular vesicles and detection of their miRNA cargo in P-bodies.

Pregnancy is a unique situation, in which placenta-derived small extracellular vesicles (sEVs) may communicate with maternal and foetal tissues. While relevant to homoeostatic and pathological functions, the mechanisms underlying sEV entry and cargo handling in target cells remain largely unknown. Using fluorescently or luminescently labelled sEVs, derived from primary human placental trophoblasts or from a placental cell line, we interrogated the endocytic pathways used by these sEVs to enter relevant target cells, including the neighbouring primary placental fibroblasts and human uterine microvascular endothelial cells. We found that trophoblastic sEVs can enter target cells, where they retain biological activity. Importantly, using a broad series of pharmacological inhibitors and siRNA-dependent silencing approaches, we showed that trophoblastic sEVs enter target cells using macropinocytosis and clathrin-mediated endocytosis pathways, but not caveolin-dependent endocytosis. Tracking their intracellular course, we localized the sEVs to early endosomes, late endosomes, and lysosomes. Finally, we used coimmunoprecipitation to demonstrate the association of the sEV microRNA (miRNA) with the P-body proteins AGO2 and GW182. Together, our data systematically detail endocytic pathways used by placental sEVs to enter relevant fibroblastic and endothelial target cells, and provide support for "endocytic escape" of sEV miRNA to P-bodies, a key site for cytoplasmic RNA regulation.

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.

Klf14 is an imprinted transcription factor that regulates placental growth.

INTRODUCTION: Imprinted genes are preferentially expressed from one parentally inherited allele, and many are crucial to the regulation of placental function and fetal growth. Murine Krüppel-like factor 14 (Klf14) is a maternally expressed imprinted transcription factor that is a component of the Mest imprinted gene cluster on mouse chromosome 6. We sought to determine if loss of Klf14 expression alters the course of normal mouse extraembryonic development. We also used high-throughput RNA sequencing (RNAseq) to identify a set of differentially expressed genes (DEGs) in placentas with loss of Klf14. METHODS: We generated a Klf14 knockout (Klf14null) mouse using recombineering and transgenic approaches. To identify DEGs in the mouse placenta we compared mRNA transcriptomes derived from 17.5dpc Klf14matKO and wild-type littermate placentas by RNAseq. Candidate DEGs were confirmed with quantitative reverse transcription PCR (qPCR) on an independent cohort of male and female gestational age matched Klf14matKO placentas. RESULTS: We found that 17.5dpc placentas inheriting a maternal null allele (Klf14matKO) had a modest overgrowth phenotype and a near complete ablation of Klf14 expression. However, there was no effect on fetal growth. We identified 20 DEGs differentially expressed in Klf14matKO placentas by RNAseq, and subsequently validated five that are highly upregulated (Begain, Col26a1, Fbln5, Gdf10, and Nell1) by qPCR. The most enriched functional gene-networks included those classified as regulating cellular development and metabolism. CONCLUSION: These results suggest that loss of the maternal Klf14 locus in the mouse placenta acts results in changes in gene expression patterns that modulate placental growth.

Chromosome 19 microRNAs exert antiviral activity independent from type III interferon signaling.

INTRODUCTION: Cultured primary human trophoblasts (PHT), derived from term placentas, are relatively resistant to infection by diverse viruses. The resistance can be conferred to non-trophoblastic cells by pre-exposing them to medium that was conditioned by PHT cells. This antiviral effect is mediated, at least in part, by microRNAs (miRNA) expressed from the chromosome 19 microRNA cluster (C19MC). Recently we showed that PHT cells and cells pre-exposed to PHT medium are also resistant to infection by Zika virus (ZIKV), an effect mediated by the constitutive release of the type III interferons (IFN) IFN lambda-1 and IFN lambda-2 in trophoblastic medium. We hypothesized that trophoblastic C19MC miRNA are active against ZIKV, and assessed the interaction of this pathway with IFN lambda-1 - mediated resistance. METHODS: Term PHT cells were cultured using standard techniques. An osteosarcoma cell line (U2OS) was used as non-trophoblastic cells, which were infected with either ZIKV or vesicular stomatitis virus (VSV). Trophoblastic extracellular vesicles (EVs) were produced by gradient ultracentrifugation. RT-qPCR was used to determine viral infection, cellular or medium miRNA levels and the expression of interferon-stimulated genes. RESULTS: We showed that C19MC miRNA attenuate infection of U2OS cells by ZIKV, and that C19MC miRNA or exosomes that contain C19MC miRNA did not influence the type III IFN pathway. Similarly, cell exposure to recombinant IFN lambda-1 had no effect on miRNA expression, and these pathways did not exhibit synergistic interaction. DISCUSSION: PHT cells exert antiviral activity by at least two independent mechanisms, mediated by C19MC miRNA and by type III IFNs.

Determinants of effective lentivirus-driven microRNA expression in vivo.

Manipulation of microRNA (miRNA) levels, including overexpression of mature species, has become an important biological tool, even motivating miRNA-based therapeutics. To assess key determinants of miRNA overexpression in a mammalian system in vivo, we sought to bypass the laborious generation of a transgenic animal by exploiting placental trophoblast-specific gene manipulation using lentiviral vectors, which has been instrumental in elucidating trophoblast biology. We examined the impact of several key components of miRNA stem loops and their flanking sequences on the efficiency of mature miRNA expression in vivo. By combining established and novel approaches for miRNA expression, we engineered lentivirus-driven miRNA expression plasmids, which we tested in the mouse placenta. We found that reverse sense inserts minimized single-strand splicing and degradation, and that maintaining longer, poly-A-containing arms flanking the miRNA stem-loop markedly enhanced transgenic miRNA expression. Additionally, we accomplished overexpression of diverse mammalian, drosophila, or C. elegans miRNAs, either based on native context or using a "cassette" replacement of the mature miRNA sequence. Together, we have identified primary miRNA sequences that are paramount for effective expression of mature miRNAs, and validated their role in mice. Principles established by our findings may guide the design of efficient miRNA vectors for in vivo use.

C19MC microRNAs regulate the migration of human trophoblasts.

Early in pregnancy, trophoblast invasion into the decidua and inner myometrium is essential for establishment of proper implantation, maternal-fetal exchange, and immunological tolerance of the feto-placental allograft. Unlike villous trophoblasts (VTs), extravillous trophoblasts (EVTs) are unique in their capacity to invade the maternal decidua and myometrium. The largest human microRNA (miRNA) gene cluster, the chromosome 19 miRNA cluster (C19MC), is expressed almost exclusively in the placenta and, rarely, in certain tumors and undifferentiated cells. In the work reported here, we found that the expression of C19MC miRNAs is higher in VTs than in EVTs. Using a bacterial artificial chromosome (BAC)-mediated overexpression of C19MC miRNAs in an EVT-derived cell line, which does not naturally express these miRNAs, we found that C19MC miRNAs selectively attenuate cell migration without affecting cell proliferation or apoptosis. A microarray analysis revealed that C19MC miRNAs regulate target transcripts related to cellular movement. Our data also implicated a specific C19MC member, miR-519d, indirectly regulating the EVT invasive phenotype by targeting CXCL6, NR4A2 and FOXL2 transcripts through a 3'UTR miRNA-responsive element. Together, our data suggest a role for C19MC miRNAs in modulating the migration of EVTs.