Prenatal alcohol exposure contributes to negative pregnancy outcomes by altering fetal vascular dynamics and the placental transcriptome.

BACKGROUND: Prenatal alcohol exposure (PAE) has been shown to alter fetal blood flow in utero and is also associated with placental insufficiency and intrauterine growth restriction (IUGR), suggesting an underlying connection between perturbed circulation and pregnancy outcomes. METHODS: Timed-pregnant C57/BL6NHsd mice, bred in-house, were exposed by gavage on gestational day 10 (GD10) to ethanol (3 g/kg) or purified water, as a control. Pulse-wave Doppler ultrasound measurements for umbilical arteries and ascending aorta were obtained post-gavage (GD12, GD14, GD18) on 2 fetuses/litter. RNA from the non-decidual (labyrinthine and junctional zone) portion of placentas was isolated and processed for RNA-seq and subsequent bioinformatic analyses, and the association between transcriptomic changes and fetal phenotypes assessed. RESULTS: Exposure to ethanol in pregnant mice on GD10 attenuates umbilical cord blood flow transiently during gestation, and is associated with indices of IUGR, specifically decreased fetal weight and morphometric indices of cranial growth. Moreover, RNA-seq of the fetal portion of the placenta demonstrated that this single exposure has lasting transcriptomic changes, including upregulation of Tet3, which is associated with spontaneous abortion. Weighted gene co-expression network analysis (WGCNA) identified erythrocyte differentiation and homeostasis as important pathways associated with improved umbilical cord blood flow as gestation progresses. WGCNA also identified sensory perception of chemical stimulus/odorant and receptor activity as important pathways associated with cranial growth. CONCLUSION: Our data suggest that PAE perturbs the expression of placental genes relevant for placental hematopoiesis and environmental sensing, resulting in transient impairment of umbilical cord blood flow and, subsequently, IUGR.

Ethanol Exposure Increases miR-140 in Extracellular Vesicles: Implications for Fetal Neural Stem Cell Proliferation and Maturation.

BACKGROUND: Neural stem cells (NSCs) generate most of the neurons of the adult brain in humans, during the mid-first through second-trimester period. This critical neurogenic window is particularly vulnerable to prenatal alcohol exposure, which can result in diminished brain growth. Previous studies showed that ethanol (EtOH) exposure does not kill NSCs, but, rather, results in their depletion by influencing cell cycle kinetics and promoting aberrant maturation, in part, by altering NSC expression of key neurogenic miRNAs. NSCs reside in a complex microenvironment rich in extracellular vesicles, shown to traffic miRNA cargo between cells. METHODS: We profiled the miRNA content of extracellular vesicles from control and EtOH-exposed ex vivo neurosphere cultures of fetal NSCs. We subsequently examined the effects of one EtOH-sensitive miRNA, miR-140-3p, on NSC growth, survival, and maturation. RESULTS: EtOH exposure significantly elevates levels of a subset of miRNAs in secreted extracellular vesicles. Overexpression of one of these elevated miRNAs, miR-140-3p, and its passenger strand relative, miR-140-5p, significantly increased the proportion of S-phase cells while decreasing the proportion of G0 /G1 cells compared to controls. In contrast, while miR-140-3p knockdown had minimal effects on the proportion of cells in each phase of the cell cycle, knockdown of miR-140-5p significantly decreased the proportion of cells in G2 /M phase. Furthermore, miR-140-3p overexpression, during mitogen-withdrawal-induced NSC differentiation, favors astroglial maturation at the expense of neural and oligodendrocyte differentiation. CONCLUSIONS: Collectively, the dysregulated miRNA content of extracellular vesicles following EtOH exposure may result in aberrant neural progenitor cell growth and maturation, explaining brain growth deficits associated with prenatal alcohol exposure.

Sustained descemetocele management with Prosthetic Replacement of the Ocular Surface Ecosystem (PROSE) treatment.

Purpose: To report a case of a 67-year-old male who was successfully managed over a 7-year period for descemetocele secondary to ocular graft versus host disease (oGVHD) using Prosthetic Replacement of the Ocular Surface Ecosystem (PROSE) treatment. Observations: We previously reported on a patient managed with a PROSE device for severe dry eyes secondary to oGVHD, who subsequently developed a central corneal descemetocele. The patient was deemed a poor surgical candidate due to limbal stem cell deficiency secondary to oGVHD. Therefore, we elected to closely monitor the descemetocele as the patient continued PROSE therapy. The patient's descemetocele has been managed successfully without perforation throughout a 7-year follow-up period with corrected distance visual acuity remaining stable at 20/50 in the affected eye. Conclusions and importance: Descemetoceles are an uncommon complication of ocular graft versus host disease. This is the longest published report of a corneal descemetocele managed with PROSE. Our report suggests that in appropriate patients who are at high-risk for post-surgical complications, PROSE in conjunction with other medical management should be considered as an alternative to corneal transplantation.

Maternal circulating miRNAs contribute to negative pregnancy outcomes by altering placental transcriptome and fetal vascular dynamics.

Circulating miRNAs the in blood are promising biomarkers for predicting pregnancy complications and adverse birth outcomes. Previous work identified 11 gestationally elevated maternal circulating miRNAs (HEamiRNAs) that predicted infant growth deficits following prenatal alcohol exposure and regulated epithelial-mesenchymal transition in the placenta. Here we show that a single intravascular administration of pooled murine-conserved HEamiRNAs to pregnant mice on gestational day 10 (GD10) attenuates umbilical cord blood flow during gestation, explaining the observed intrauterine growth restriction (IUGR), specifically decreased fetal weight, and morphometric indices of cranial growth. Moreover, RNAseq of the fetal portion of the placenta demonstrated that this single exposure has lasting transcriptomic changes, including upregulation of members of the Notch pathway (Dll4, Rfng, Hey1), which is a pathway important for trophoblast migration and differentiation. Weighted gene co-expression network analysis also identified chemokine signaling, which is responsible for regulating immune cell-mediated angiogenesis in the placenta, as an important predictor of fetal growth and head size. Our data suggest that HEamiRNAs perturb the expression of placental genes relevant for angiogenesis, resulting in impaired umbilical cord blood flow and subsequently, IUGR.

A novel Oct4/Pou5f1-like non-coding RNA controls neural maturation and mediates developmental effects of ethanol.

Prenatal ethanol exposure can result in loss of neural stem cells (NSCs) and decreased brain growth. Here, we assessed whether a noncoding RNA (ncRNA) related to the NSC self-renewal factor Oct4/Pou5f1, and transcribed from a processed pseudogene locus on mouse chromosome 9 (mOct4pg9), contributed to the loss of NSCs due to ethanol. Mouse fetal cortical-derived NSCs, cultured ex vivo to mimic the early neurogenic environment of the fetal telencephalon, expressed mOct4pg9 ncRNA at significantly higher levels than the parent Oct4/Pou5f1 mRNA. Ethanol exposure increased expression of mOct4pg9 ncRNA, but decreased expression of Oct4/Pou5f1. Gain- and loss-of-function analyses indicated that mOct4pg9 overexpression generally mimicked effects of ethanol exposure, resulting in increased proliferation and expression of transcripts associated with neural maturation. Moreover, mOct4pg9 associated with Ago2 and with miRNAs, including the anti-proliferative miR-328-3p, whose levels were reduced following mOct4pg9 overexpression. Finally, mOct4pg9 inhibited Oct4/Pou5f1-3'UTR-dependent protein translation. Consistent with these observations, data from single-cell transcriptome analysis showed that mOct4pg9-expressing progenitors lack Oct4/Pou5f1, but instead overexpress transcripts for increased mitosis, suggesting initiation of transit amplification. Collectively, these data suggest that the inhibitory effects of ethanol on brain development are explained, in part, by a novel ncRNA which promotes loss of NSC identity and maturation.

Infant circulating MicroRNAs as biomarkers of effect in fetal alcohol spectrum disorders.

Prenatal alcohol exposure (PAE) can result in cognitive and behavioral disabilities and growth deficits. Because alcohol-related neurobehavioral deficits may occur in the absence of overt dysmorphic features or growth deficits, there is a need to identify biomarkers of PAE that can predict neurobehavioral impairment. In this study, we assessed infant plasma extracellular, circulating miRNAs (exmiRNAs) obtained from a heavily exposed Cape Town cohort to determine whether these can be used to predict PAE-related growth restriction and cognitive impairment. PAE, controlling for smoking as a covariate, altered 27% of expressed exmiRNAs with clinically-relevant effect sizes (Cohen's d ≥ 0.4). Moreover, at 2 weeks, PAE increased correlated expression of exmiRNAs across chromosomes, suggesting potential co-regulation. In confirmatory factor analysis, the variance in expression for PAE-altered exmiRNAs at 2 weeks and 6.5 months was best described by three-factor models. Pathway analysis found that factors at 2 weeks were associated with (F1) cell maturation, cell cycle inhibition, and somatic growth, (F2) cell survival, apoptosis, cardiac development, and metabolism, and (F3) cell proliferation, skeletal development, hematopoiesis, and inflammation, and at 6.5 months with (F1) neurodevelopment, neural crest/mesoderm-derivative development and growth, (F2) immune system and inflammation, and (F3) somatic growth and cardiovascular development. Factors F3 at 2 weeks and F2 at 6.5 months partially mediated PAE-induced growth deficits, and factor F3 at 2 weeks partially mediated effects of PAE on infant recognition memory at 6.5 months. These findings indicate that infant exmiRNAs can help identify infants who will exhibit PAE-related deficits in growth and cognition.

Association between fetal sex and maternal plasma microRNA responses to prenatal alcohol exposure: evidence from a birth outcome-stratified cohort.

Most persons with fetal alcohol spectrum disorders (FASDs) remain undiagnosed or are diagnosed in later life. To address the need for earlier diagnosis, we previously assessed miRNAs in the blood plasma of pregnant women who were classified as unexposed to alcohol (UE), heavily exposed with affected infants (HEa), or heavily exposed with apparently unaffected infants (HEua). We reported that maternal miRNAs predicted FASD-related growth and psychomotor deficits in infants. Here, we assessed whether fetal sex influenced alterations in maternal circulating miRNAs following prenatal alcohol exposure (PAE). To overcome the loss of statistical power due to disaggregating maternal samples by fetal sex, we adapted a strategy of iterative bootstrap resampling with replacement to assess the stability of statistical parameter estimates. Bootstrap estimates of parametric and effect size tests identified male and female fetal sex-associated maternal miRNA responses to PAE that were not observed in the aggregated sample. Additionally, we observed, in HEa mothers of female, but not male fetuses, a network of co-secreted miRNAs whose expression was linked to miRNAs encoded on the X-chromosome. Interestingly, the number of significant miRNA correlations for the HEua group mothers with female fetuses was intermediate between HEa and UE mothers at mid-pregnancy, but more similar to UE mothers by the end of pregnancy. Collectively, these data show that fetal sex predicts maternal circulating miRNA adaptations, a critical consideration when adopting maternal miRNAs as diagnostic biomarkers. Moreover, a maternal co-secretion network, predominantly in pregnancies with female fetuses, emerged as an index of risk for adverse birth outcomes due to PAE.

Maternal circulating miRNAs that predict infant FASD outcomes influence placental maturation.

Prenatal alcohol exposure (PAE), like other pregnancy complications, can result in placental insufficiency and fetal growth restriction, although the linking causal mechanisms are unclear. We previously identified 11 gestationally elevated maternal circulating miRNAs (HEamiRNAs) that predicted infant growth deficits following PAE. Here, we investigated whether these HEamiRNAs contribute to the pathology of PAE, by inhibiting trophoblast epithelial-mesenchymal transition (EMT), a pathway critical for placental development. We now report for the first time that PAE inhibits expression of placental pro-EMT pathway members in both rodents and primates, and that HEamiRNAs collectively, but not individually, mediate placental EMT inhibition. HEamiRNAs collectively, but not individually, also inhibited cell proliferation and the EMT pathway in cultured trophoblasts, while inducing cell stress, and following trophoblast syncytialization, aberrant endocrine maturation. Moreover, a single intravascular administration of the pooled murine-expressed HEamiRNAs, to pregnant mice, decreased placental and fetal growth and inhibited the expression of pro-EMT transcripts in the placenta. Our data suggest that HEamiRNAs collectively interfere with placental development, contributing to the pathology of PAE, and perhaps also, to other causes of fetal growth restriction.

Nonprotein-coding RNAs in Fetal Alcohol Spectrum Disorders.

Early developmental exposure to ethanol, a known teratogen, can result in a range of neurodevelopmental disorders, collectively referred to as Fetal Alcohol Spectrum Disorders (FASDs). Changes in the environment, including exposure to teratogens, can result in long term alterations to the epigenetic landscape of a cell, thereby altering gene expression. Noncoding RNAs (ncRNAs) can affect transcription and translation of networks of genes. ncRNAs are dynamically expressed during development and have been identified as a target of alcohol. ncRNAs therefore make for attractive targets for novel therapeutics to address the developmental deficits associated with FASDs.

The BAF (BRG1/BRM-Associated Factor) chromatin-remodeling complex exhibits ethanol sensitivity in fetal neural progenitor cells and regulates transcription at the miR-9-2 encoding gene locus.

Fetal alcohol spectrum disorders are a leading cause of intellectual disability worldwide. Previous studies have shown that developmental ethanol exposure results in loss of microRNAs (miRNAs), including miR-9, and loss of these miRNAs, in turn, mediates some of ethanol's teratogenic effects in the developing brain. We previously found that ethanol increased methylation at the miR-9-2 encoding gene locus in mouse fetal neural stem cells (NSC), advancing a mechanism for epigenetic silencing of this locus and consequently, miR-9 loss in NSCs. Therefore, we assessed the role of the BAF (BRG1/BRM-Associated Factor) complex, which disassembles nucleosomes to facilitate access to chromatin, as an epigenetic mediator of ethanol's effects on miR-9. Chromatin immunoprecipitation and DNAse I-hypersensitivity analyses showed that the BAF complex was associated with both transcriptionally accessible and heterochromatic regions of the miR-9-2 locus, and that disintegration of the BAF complex by combined knockdown of BAF170 and BAF155 resulted in a significant decrease in miR-9. We hypothesized that ethanol exposure would result in loss of BAF-complex function at the miR-9-2 locus. However, ethanol exposure significantly increased mRNA transcripts for maturation-associated BAF-complex members BAF170, SS18, ARID2, BAF60a, BRM/BAF190b, and BAF53b. Ethanol also significantly increased BAF-complex binding within an intron containing a CpG island and in the terminal exon encoding precursor (pre)-miR-9-2. These data suggest that the BAF complex may adaptively respond to ethanol exposure to protect against a complete loss of miR-9-2 in fetal NSCs. Chromatin remodeling factors may adapt to the presence of a teratogen, to maintain transcription of critical miRNA regulatory pathways.

Plasma miRNA Profiles in Pregnant Women Predict Infant Outcomes following Prenatal Alcohol Exposure.

Fetal alcohol spectrum disorders (FASD) are difficult to diagnose since many heavily exposed infants, at risk for intellectual disability, do not exhibit craniofacial dysmorphology or growth deficits. Consequently, there is a need for biomarkers that predict disability. In both animal models and human studies, alcohol exposure during pregnancy resulted in significant alterations in circulating microRNAs (miRNAs) in maternal blood. In the current study, we asked if changes in plasma miRNAs in alcohol-exposed pregnant mothers, either alone or in conjunction with other clinical variables, could predict infant outcomes. Sixty-eight pregnant women at two perinatal care clinics in western Ukraine were recruited into the study. Detailed health and alcohol consumption histories, and 2nd and 3rd trimester blood samples were obtained. Birth cohort infants were assessed by a geneticist and classified as unexposed (UE), heavily prenatally exposed and affected (HEa) or heavily exposed but apparently unaffected (HEua). MiRNAs were assessed in plasma samples using qRT-PCR arrays. ANOVA models identified 11 miRNAs that were all significantly elevated in maternal plasma from the HEa group relative to HEua and UE groups. In a random forest analysis classification model, a combination of high variance miRNAs, smoking history and socioeconomic status classified membership in HEa and UE groups, with a misclassification rate of 13%. The RFA model also classified 17% of the HEua group as UE-like, whereas 83% were HEa-like, at least at one stage of pregnancy. Collectively our data indicate that maternal plasma miRNAs predict infant outcomes, and may be useful to classify difficult-to-diagnose FASD subpopulations.