Amniotic Fluid Modifies Esophageal Epithelium Differentiation and Inflammatory Responses.

The interplay between genetic and environmental factors during pregnancy can predispose to inflammatory diseases postnatally, including eosinophilic esophagitis, a chronic allergic disease triggered by food. Herein, we examined the effects of amniotic fluid (AF) on esophageal epithelial differentiation and responsiveness to pro-allergic stimuli. Multiplex analysis of AF revealed the expression of 66 cytokines, whereas 5 cytokines including IL-4 and TSLP were not detected. Several pro-inflammatory cytokines including TNFa and IL-12 were highly expressed in the AF from women who underwent preterm birth, while EGF was the highest in term birth samples. Exposure of esophageal epithelial cells to AF resulted in transient phosphorylation of ERK1/2 and the transcription of early response genes, highlighting the direct impact of AF on esophageal epithelial cells. In a 3-dimensional spheroid model, AF modified the esophageal epithelial differentiation program and enhanced the transcription of IL-13-target genes, including CCL26 and CAPN14, which encodes for a major genetic susceptibility locus for eosinophilic esophagitis. Notably, CAPN14 exhibited upregulation in spheroids exposed to preterm but not term AF following differentiation. Collectively, our findings call attention to the role of AF as a potential mediator of the intrauterine environment that influences subsequent esophageal disorders.

A genome scale transcriptional regulatory model of the human placenta.

Gene regulation is essential to placental function and fetal development. We built a genome-scale transcriptional regulatory network (TRN) of the human placenta using digital genomic footprinting and transcriptomic data. We integrated 475 transcriptomes and 12 DNase hypersensitivity datasets from placental samples to globally and quantitatively map transcription factor (TF)-target gene interactions. In an independent dataset, the TRN model predicted target gene expression with an out-of-sample R2 greater than 0.25 for 73% of target genes. We performed siRNA knockdowns of four TFs and achieved concordance between the predicted gene targets in our TRN and differences in expression of knockdowns with an accuracy of >0.7 for three of the four TFs. Our final model contained 113,158 interactions across 391 TFs and 7712 target genes and is publicly available. We identified 29 TFs which were significantly enriched as regulators for genes previously associated with preterm birth, and eight of these TFs were decreased in preterm placentas.

Lipid nanoparticle structure and delivery route during pregnancy dictate mRNA potency, immunogenicity, and maternal and fetal outcomes.

Treating pregnancy-related disorders is exceptionally challenging because the threat of maternal and/or fetal toxicity discourages the use of existing medications and hinders new drug development. One potential solution is the use of lipid nanoparticle (LNP) RNA therapies, given their proven efficacy, tolerability, and lack of fetal accumulation. Here, we describe LNPs for efficacious mRNA delivery to maternal organs in pregnant mice via several routes of administration. In the placenta, our lead LNP transfected trophoblasts, endothelial cells, and immune cells, with efficacy being structurally dependent on the ionizable lipid polyamine headgroup. Next, we show that LNP-induced maternal inflammatory responses affect mRNA expression in the maternal compartment and hinder neonatal development. Specifically, pro-inflammatory LNP structures and routes of administration curtailed efficacy in maternal lymphoid organs in an IL-1ß-dependent manner. Further, immunogenic LNPs provoked the infiltration of adaptive immune cells into the placenta and restricted pup growth after birth. Together, our results provide mechanism-based structural guidance on the design of potent LNPs for safe use during pregnancy.

High-yield and rapid isolation of extracellular vesicles by flocculation via orbital acoustic trapping: FLOAT.

Extracellular vesicles (EVs) have been identified as promising biomarkers for the noninvasive diagnosis of various diseases. However, challenges in separating EVs from soluble proteins have resulted in variable EV recovery rates and low purities. Here, we report a high-yield ( > 90%) and rapid ( < 10 min) EV isolation method called FLocculation via Orbital Acoustic Trapping (FLOAT). The FLOAT approach utilizes an acoustofluidic droplet centrifuge to rotate and controllably heat liquid droplets. By adding a thermoresponsive polymer flocculant, nanoparticles as small as 20 nm can be rapidly and selectively concentrated at the center of the droplet. We demonstrate the ability of FLOAT to separate urinary EVs from the highly abundant Tamm-Horsfall protein, addressing a significant obstacle in the development of EV-based liquid biopsies. Due to its high-yield nature, FLOAT reduces biofluid starting volume requirements by a factor of 100 (from 20 mL to 200 µL), demonstrating its promising potential in point-of-care diagnostics.

Strikingly High Activity of 15-Lipoxygenase Towards Di-Polyunsaturated Arachidonoyl/Adrenoyl-Phosphatidylethanolamines Generates Peroxidation Signals of Ferroptotic Cell Death.

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non-oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA-PLs, particularly sn2-arachidonoyl(AA)- and sn2-adrenoyl(AdA)-containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1-2 mol %) of doubly PUFA-acylated phospholipids (di-PUFA-PLs) whose role in ferroptosis remains enigmatic. Here we report that 15-lipoxygenase (15LOX) exhibits unexpectedly high pro-ferroptotic peroxidation activity towards di-PUFA-PEs. We revealed that peroxidation of several molecular species of di-PUFA-PEs occurred early in ferroptosis. Ferrostatin-1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di-PUFA-PEs. Furthermore, co-incubation of cells with di-AA-PE and 15LOX produced PUFA-PE peroxidation and induced ferroptotic death. The decreased contents of di-PUFA-PEs in ACSL4 KO A375 cells was associated with lower levels of di-PUFA-PE peroxidation and enhanced resistance to ferroptosis. Thus, di-PUFA-PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death.

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 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.

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.

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.

A solution to the biophysical fractionation of extracellular vesicles: Acoustic Nanoscale Separation via Wave-pillar Excitation Resonance (ANSWER).

High-precision isolation of small extracellular vesicles (sEVs) from biofluids is essential toward developing next-generation liquid biopsies and regenerative therapies. However, current methods of sEV separation require specialized equipment and time-consuming protocols and have difficulties producing highly pure subpopulations of sEVs. Here, we present Acoustic Nanoscale Separation via Wave-pillar Excitation Resonance (ANSWER), which allows single-step, rapid (<10 min), high-purity (>96% small exosomes, >80% exomeres) fractionation of sEV subpopulations from biofluids without the need for any sample preprocessing. Particles are iteratively deflected in a size-selective manner via an excitation resonance. This previously unidentified phenomenon generates patterns of virtual, tunable, pillar-like acoustic field in a fluid using surface acoustic waves. Highly precise sEV fractionation without the need for sample preprocessing or complex nanofabrication methods has been demonstrated using ANSWER, showing potential as a powerful tool that will enable more in-depth studies into the complexity, heterogeneity, and functionality of sEV subpopulations.

Nitric Oxide Production and Effects in Group B Streptococcus Chorioamnionitis.

Intrauterine infection, or chorioamnionitis, due to group B Streptococcus (GBS) is a common cause of miscarriage and preterm birth. To cause chorioamnionitis, GBS must bypass maternal-fetal innate immune defenses including nitric oxide (NO), a microbicidal gas produced by nitric oxide synthases (NOS). This study examined placental NO production and its role in host-pathogen interactions in GBS chorioamnionitis. In a murine model of ascending GBS chorioamnionitis, placental NOS isoform expression quantified by RT-qPCR revealed a four-fold expression increase in inducible NOS, no significant change in expression of endothelial NOS, and decreased expression of neuronal NOS. These NOS expression results were recapitulated ex vivo in freshly collected human placental samples that were co-incubated with GBS. Immunohistochemistry of wild type C57BL/6 murine placentas with GBS chorioamnionitis demonstrated diffuse inducible NOS expression with high-expression foci in the junctional zone and areas of abscess. Pregnancy outcomes between wild type and inducible NOS-deficient mice did not differ significantly although wild type dams had a trend toward more frequent preterm delivery. We also identified possible molecular mechanisms that GBS uses to survive in a NO-rich environment. In vitro exposure of GBS to NO resulted in dose-dependent growth inhibition that varied by serovar. RNA-seq on two GBS strains with distinct NO resistance phenotypes revealed that both GBS strains shared several detoxification pathways that were differentially expressed during NO exposure. These results demonstrate that the placental immune response to GBS chorioamnionitis includes induced NO production and indicate that GBS activates conserved stress pathways in response to NO exposure.

Small extracellular vesicles from plasma of women with preeclampsia increase myogenic tone and decrease endothelium-dependent relaxation of mouse mesenteric arteries.

Preeclampsia (PE) is a common syndrome of pregnancy, characterized by new-onset hypertension and proteinuria after gestational week 20, or new onset of hypertension and significant end-organ dysfunction. In the worst cases, it can threaten the survival of both mother and baby. Extracellular vesicles (EVs) are lipid-bilayer nanoparticles released from cells. They are involved in cell-cell communication and transport of diverse cargo molecules. Small extracellular vesicles (sEVs, exosomes) are defined by their size and biogenesis within the endocytic compartment of the cell or reverse budding of the plasma membrane. The function of circulating gestational EVs, released from maternal organs or the placenta, remains to be explored. Here, we focused on sEVs that circulate in the maternal blood in the third trimester of human pregnancy and hypothesized that sEVs from pregnant women with PE play a role in regulation of vessel tone. When compared to sEVs from women with uncomplicated pregnancies, ex vivo exposure of isolated mouse mesenteric arteries to sEVs purified from the plasma of pregnant women with PE led to constriction in response to intraluminal pressure. This effect was not observed using microvesicles from the plasma of women with PE or using PE plasma that was depleted of EVs. Blood vessels exposed to sEVs from women with PE were also more resistant to methacholine-stimulated relaxation. Immunofluorescence microscopy confirmed the presence of sEVs within the vessel wall. Together, these data support the notion that circulating sEVs from pregnant women play a role in the regulation of arterial tone.

Assessing hypoxic damage to placental trophoblasts by measuring membrane viscosity of extracellular vesicles.

INTRODUCTION: As highly sophisticated intercellular communication vehicles in biological systems, extracellular vesicles (EVs) have been investigated as both promising liquid biopsy-based disease biomarkers and drug delivery carriers. Despite tremendous progress in understanding their biological and physiological functions, mechanical characterization of these nanoscale entities remains challenging due to the limited availability of proper techniques. Especially, whether damage to parental cells can be reflected by the mechanical properties of their EVs remains unknown. METHODS: In this study, we characterized membrane viscosities of different types of EVs collected from primary human trophoblasts (PHTs), including apoptotic bodies, microvesicles and small extracellular vesicles, using fluorescence lifetime imaging microscopy (FLIM). The biochemical origin of EV membrane viscosity was examined by analyzing their phospholipid composition, using mass spectrometry. RESULTS: We found that different EV types derived from the same cell type exhibit different membrane viscosities. The measured membrane viscosity values are well supported by the lipidomic analysis of the phospholipid compositions. We further demonstrate that the membrane viscosity of microvesicles can faithfully reveal hypoxic injury of the human trophoblasts. More specifically, the membrane of PHT microvesicles released under hypoxic condition is less viscous than its counterpart under standard culture condition, which is supported by the reduction in the phosphatidylethanolamine-to-phosphatidylcholine ratio in PHT microvesicles. DISCUSSION: Our study suggests that biophysical properties of released trophoblastic microvesicles can reflect cell health. Characterizing EV's membrane viscosity may pave the way for the development of new EV-based clinical applications.

Placental miRNAs Targeting Cellular Stress Response Pathways Are Highly Expressed in Non-Hispanic Black People.

Non-Hispanic Black (NHB) people have a 2.5-fold higher risk of maternal mortality when compared to non-Hispanic White (NHW) people. Neonates of NHB people are more likely to be born preterm and small for gestational age, which may be driven by structural racism. The placenta is very sensitive to the maternal environment and may play a critical role in the translation of environmental stressors to pregnancy outcomes. Our aim was to assess the placental miRNA expression profile in both NHB and NHW people and the association between differentially expressed miRNAs and pregnancy outcomes. Placentas were collected from 50 NHB and 74 NHW people with a normal singleton pregnancy undergoing elective cesarean section at term prior to the onset of labor. Placental miRNA expression was measured via whole-genome small RNA-sequencing in a subset of 77 placentas. Fifteen miRNAs were more highly expressed in the placentas of NHB people. Several of these miRNAs were associated with cellular stress response pathways, suggesting that they may be responding to environmental stressors. Placental miR-192-5p expression was lower among NHB people and was positively associated with neonatal adiposity, suggesting it may be sensitive to structural racism with potential impacts on fetal growth.

Extracellular vesicles and immune response during pregnancy: A balancing act.

The mechanisms underlying maternal tolerance of the semi- or fully-allogeneic fetus are intensely investigated. Across gestation, feto-placental antigens interact with the maternal immune system locally within the trophoblast-decidual interface and distantly through shed cells and soluble molecules that interact with maternal secondary lymphoid tissues. The discovery of extracellular vesicles (EVs) as local or systemic carriers of antigens and immune-regulatory molecules has added a new dimension to our understanding of immune modulation prior to implantation, during trophoblast invasion, and throughout the course of pregnancy. New data on immune-regulatory molecules, located on EVs or within their cargo, suggest a role for EVs in negotiating immune tolerance during gestation. Lessons from the field of transplant immunology also shed light on possible interactions between feto-placentally derived EVs and maternal lymphoid tissues. These insights illuminate a potential role for EVs in major obstetrical disorders. This review provides updated information on intensely studied, pregnancy-related EVs, their cargo molecules, and patterns of fetal-placental-maternal trafficking, highlighting potential immune pathways that might underlie immune suppression or activation in gestational health and disease. Our summary also underscores the likely need to broaden the definition of the maternal-fetal interface to systemic maternal immune tissues that might interact with circulating EVs.

Ferroptosis induces membrane blebbing in placental trophoblasts.

Ferroptosis is a regulated, non-apoptotic form of cell death, characterized by hydroxy-peroxidation of discrete phospholipid hydroperoxides, particularly hydroperoxyl (Hp) forms of arachidonoyl- and adrenoyl-phosphatidylethanolamine, with a downstream cascade of oxidative damage to membrane lipids, proteins and DNA, culminating in cell death. We recently showed that human trophoblasts are particularly sensitive to ferroptosis caused by depletion or inhibition of glutathione peroxidase 4 (GPX4) or the lipase PLA2G6. Here, we show that trophoblastic ferroptosis is accompanied by a dramatic change in the trophoblast plasma membrane, with macro-blebbing and vesiculation. Immunofluorescence revealed that ferroptotic cell-derived blebs stained positive for F-actin, but negative for cytoplasmic organelle markers. Transfer of conditioned medium that contained detached macrovesicles or co-culture of wild-type target cells with blebbing cells did not stimulate ferroptosis in target cells. Molecular modeling showed that the presence of Hp-phosphatidylethanolamine in the cell membrane promoted its cell ability to be stretched. Together, our data establish that membrane macro-blebbing is characteristic of trophoblast ferroptosis and can serve as a useful marker of this process. Whether or not these blebs are physiologically functional remains to be established.

Placental miR-3940-3p Is Associated With Maternal Insulin Resistance in Late Pregnancy.

CONTEXT: An increase in maternal insulin resistance (IR) during pregnancy is essential for normal fetal growth. The mechanisms underlying this adaptation are poorly understood. Placental factors are believed to instigate and maintain these changes, as IR decreases shortly after delivery. Methylation of placental gene loci that are common targets for miRNAs are associated with maternal IR. OBJECTIVE: We hypothesized that placental miRNAs targeting methylated loci are associated with maternal IR during late pregnancy. METHODS: We collected placentas from 132 elective cesarean sections and fasting blood samples at delivery to estimate maternal homeostasis model assessment of insulin resistance (HOMA-IR). Placental miRNA expression was measured via whole genome small-RNA sequencing in a subset of 40 placentas selected by maternal pre-gravid body mass index (BMI) and neonatal adiposity. Five miRNAs correlated with maternal HOMA-IR and previously identified as targeting methylated genes were selected for validation in all 132 placenta samples via RT-qPCR. Multiple regression adjusted for relevant clinical variables. RESULTS: Median maternal age was 27.5 years, with median pre-pregnancy BMI of 24.7 kg/m2, and median HOMA-IR of 2.9. Among the 5 selected miRNA, maternal HOMA-IR correlated with the placental expression of miRNA-371b-3p (r = 0.25; P = 0.008) and miRNA-3940-3p (r = 0.32; P = 0.0004) across the 132 individuals. After adjustment for confounding variables, placental miRNA-3940-3p expression remained significantly associated with HOMA-IR (ß = 0.16; P = 0.03). CONCLUSION: Placental miRNA-3940-3p was associated with maternal IR at delivery. This placental miRNA may have an autocrine or paracrine effect-regulating placental genes involved in modulating maternal IR.

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.

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.