Potential Association of Gut Microbial Metabolism and Circulating mRNA Based on Multiomics Sequencing Analysis in Fetal Growth Restriction.

Objective: Fetal growth restriction (FGR) is a significant contributor to negative pregnancy and postnatal developmental outcomes. Currently, the exact pathological mechanism of FGR remains unknown. This study aims to utilize multiomics sequencing technology to investigate potential relationships among mRNA, gut microbiota, and metabolism in order to establish a theoretical foundation for diagnosing and understanding the molecular mechanisms underlying FGR. Methods: In this study, 11 healthy pregnant women and nine pregnant women with FGR were divided into Control group and FGR group based on the health status. Umbilical cord blood, maternal serum, feces, and placental tissue samples were collected during delivery. RNA sequencing, 16S rRNA sequencing, and metabolomics methods were applied to analyze changes in umbilical cord blood circulating mRNA, fecal microbiota, and metabolites. RT-qPCR, ELISA, or western blot were used to detect the expression of top 5 differential circulating mRNA in neonatal cord blood, maternal serum, or placental tissue samples. Correlation between differential circulating mRNA, microbiota, and metabolites was analyzed by the Spearman coefficient. Results: The top 5 mRNA genes in FGR were altered with the downregulation of TRIM34, DEFA3, DEFA1B, DEFA1, and QPC, and the upregulation of CHPT1, SMOX, FAM83A, GDF15, and NAPG in newborn umbilical cord blood, maternal serum, and placental tissue. The abundance of Bacteroides, Akkermansia, Eubacterium_coprostanoligenes_group, Phascolarctobacterium, Parasutterella, Odoribacter, Lachnospiraceae_UCG_010, and Dielma were significantly enriched in the FGR group. Metabolites such as aspartic acid, methionine, alanine, L-tryptophan, 3-methyl-2-oxovalerate, and ketoleucine showed notable functional alterations. Spearman correlation analysis indicated that metabolites like methionine and alanine, microbiota (Tyzzerella), and circulating mRNA (TRIM34, SMOX, FAM83A, NAPG) might play a role as mediators in the communication between the gut and circulatory system interaction in FGR. Conclusion: Metabolites (METHIONINE, alanine) as well as microbiota (Tyzzerella) and circulating mRNA (TRIM34, SMOX, FAM83A, NAPG) were possible mediators that communicated the interaction between the gut and circulatory systems in FGR.

Effects of foetal size, sex and developmental stage on adaptive transcriptional responses of skeletal muscle to intrauterine growth restriction in pigs.

Intrauterine growth restriction (IUGR) occurs both in humans and domestic species. It has a particularly high incidence in pigs, and is a leading cause of neonatal morbidity and mortality as well as impaired postnatal growth. A key feature of IUGR is impaired muscle development, resulting in decreased meat quality. Understanding the developmental origins of IUGR, particularly at the molecular level, is important for developing effective strategies to mitigate its economic impact on the pig industry and animal welfare. The aim of this study was to characterise transcriptional profiles in the muscle of growth restricted pig foetuses at different gestational days (GD; gestational length ~ 115 days), focusing on selected genes (related to development, tissue injury and metabolism) that were previously identified as dysregulated in muscle of GD90 fetuses. Muscle samples were collected from the lightest foetus (L) and the sex-matched foetus with weight closest to the litter average (AW) from each of 22 Landrace x Large White litters corresponding to GD45 (n = 6), GD60 (n = 8) or GD90 (n = 8), followed by analyses, using RT-PCR and protein immunohistochemistry, of selected gene targets. Expression of the developmental genes, MYOD, RET and ACTN3 were markedly lower, whereas MSTN expression was higher, in the muscle of L relative to AW littermates beginning on GD45. Levels of all tissue injury-associated transcripts analysed (F5, PLG, KNG1, SELL, CCL16) were increased in L muscle on GD60 and, most prominently, on GD90. Among genes involved in metabolic regulation, KLB was expressed at higher levels in L than AW littermates beginning on GD60, whereas both IGFBP1 and AHSG were higher in L littermates on GD90 but only in males. Furthermore, the expression of genes specifically involved in lipid, hexose sugar or iron metabolism increased or, in the case of UCP3, decreased in L littermates on GD60 (UCP3, APOB, ALDOB) or GD90 (PNPLA3, TF), albeit in the case of ALDOB this only involved females. In conclusion, marked dysregulation of genes with critical roles in development in L foetuses can be observed from GD45, whereas for a majority of transcripts associated with tissue injury and metabolism differences between L and AW foetuses were apparent by GD60 or only at GD90, thus identifying different developmental windows for different types of adaptive responses to IUGR in the muscle of porcine foetuses.

CircTHBS1 promotes trophoblast cell migration and invasion and inhibits trophoblast apoptosis by regulating miR-136-3p/IGF2R axis.

The precise molecular mechanism behind fetal growth restriction (FGR) is still unclear, although there is a strong connection between placental dysfunction, inadequate trophoblast invasion, and its etiology and pathogenesis. As a new type of non-coding RNA, circRNA has been shown to play a crucial role in the development of FGR. This investigation identified the downregulation of hsa_circ_0034533 (circTHBS1) in FGR placentas through high-sequencing analysis and confirmed this finding in 25 clinical placenta samples using qRT-PCR. Subsequent in vitro functional assays demonstrated that silencing circTHBS1 inhibited trophoblast proliferation, migration, invasion, and epithelial mesenchymal transition (EMT) progression and promoted apoptosis. Furthermore, when circTHBS1 was overexpressed, cell function experiments showed the opposite result. Analysis using fluorescence in situ hybridization revealed that circTHBS1 was primarily found in the cytoplasmic region. Through bioinformatics analysis, we anticipated the involvement of miR-136-3p and IGF2R in downstream processes, which was subsequently validated through qRT-PCR and dual-luciferase assays. Moreover, the inhibition of miR-136-3p or the overexpression of IGF2R partially reinstated proliferation, migration, and invasion abilities following the silencing of circTHBS1. In summary, the circTHBS1/miR-136-3p/IGF2R axis plays a crucial role in the progression and development of FGR, offering potential avenues for the exploration of biological indicators and treatment targets.

Placental MFSD2A expression in fetal growth restriction and maternal and fetal DHA status.

INTRODUCTION: Fetal growth restriction (FGR) may affect placental transfer of key nutrients to the fetus, such as the fatty acid docosahexaenoic acid (DHA). Major facilitator superfamily domain containing 2A (MFSD2A) has been described as a specific DHA carrier in placenta, but its expression has not been studied in FGR. The aim of this study was to evaluate for the first time the placental MFSD2A levels in late-FGR pregnancies and the maternal and cord plasma DHA. METHODS: 87 pregnant women from a tertial reference center were classified into late-FGR (N = 18) or control (N = 69). Fatty acid profile was determined in maternal and cord venous plasma, as well as placental levels of MFSD2A and of insulin mediators like phospho-protein kinase B (phospho-AKT) and phospho-extracellular regulated kinase (phospho-ERK). RESULTS: Maternal fatty acid profile did not differ between groups. Nevertheless, late-FGR cord vein presented higher content of saturated fatty acids than control, producing a concomitant decrease in the percentage of some unsaturated fatty acids. In the late-FGR group, a lower DHA fetal/maternal ratio was observed when using percentages, but not with concentrations. No alterations were found in the expression of MFSD2A in late-FGR placentas, nor in phospho-AKT or phospho-ERK. DISCUSSION: MFSD2A protein expression was not altered in late-FGR placentas, in line with no differences in cord DHA concentration between groups. The increase in the saturated fatty acid content of late-FGR cord might be a compensatory mechanism to ensure fetal energy supply, decreasing other fatty acids percentage. Future studies are warranted to elucidate if altered saturated fatty acid profile in late-FGR fetuses might predispose them to postnatal catch-up and to long-term health consequences.

First trimester maternal tryptophan metabolism and embryonic and fetal growth: the Rotterdam Periconceptional Cohort (Predict Study).

STUDY QUESTION: What is the association between first trimester maternal tryptophan (TRP) metabolites and embryonic and fetal growth? SUMMARY ANSWER: Higher 5-hydroxytryptophan (5-HTP) concentrations are associated with reduced embryonic growth and fetal growth and with an increased risk of small-for-gestational age (SGA), while higher kynurenine (KYN) concentrations are associated with a reduced risk of SGA. WHAT IS KNOWN ALREADY: The maternal TRP metabolism is involved in many critical processes for embryonic and fetal growth, including immune modulation and regulation of vascular tone. Disturbances in TRP metabolism are associated with adverse maternal and fetal outcomes. STUDY DESIGN, SIZE, DURATION: This study was embedded within the Rotterdam Periconceptional Cohort (Predict Study), an ongoing prospective observational cohort conducted at a tertiary hospital from November 2010 onwards. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1115 women were included before 11 weeks of gestation between November 2010 and December 2020. Maternal serum samples were collected between 7 and 11 weeks of gestation, and TRP metabolites (TRP, KYN, 5-HTP, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid) were determined using a validated liquid chromatography (tandem) mass spectrometry method. Serial 3D ultrasound scans were performed at 7, 9, and 11 weeks of gestation to accurately assess features of embryonic growth, including crown-rump length (CRL) and embryonic volume (EV) offline using virtual reality systems. Fetal growth parameters were retrieved from medical records and standardized according to Dutch reference curves. Mixed models were used to assess associations between maternal TRP metabolites and CRL and EV trajectories. Linear and logistic regression models were utilized to investigate associations with estimated fetal weight (EFW) and birthweight, and with SGA, respectively. All analyses were adjusted for potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: Maternal 5-HTP concentrations and the maternal 5-HTP/TRP ratio were inversely associated with embryonic growth (5-HTP, √CRL: ß = -0.015, 95% CI = -0.028 to -0.001; 5-HTP 3√EV: ß = -0.009, 95% CI = -0.016 to -0.003). An increased maternal 5-HTP/TRP ratio was also associated with lower EFW and birthweight, and with an increased risk of SGA (odds ratio (OR) = 1.006, 95% CI = 1.00-1.013). In contrast, higher maternal KYN concentrations were associated with a reduced risk of SGA in the unadjusted models (OR = 0.548, 95% CI = 0.320-0.921). LIMITATIONS, REASONS FOR CAUTION: Residual confounding cannot be ruled out because of the observational design of this study. Moreover, this study was conducted in a single tertiary hospital, which assures high internal validity but may limit external validity. WIDER IMPLICATIONS OF THE FINDINGS: The novel finding that maternal 5-HTP concentrations are associated with a smaller embryo and fetus implies that disturbances of the maternal serotonin pathway in the first trimester of pregnancy are potentially involved in the pathophysiology of fetal growth restriction. The association between higher maternal KYN concentrations and a reduced risk of SGA substantiate the evidence that the KYN pathway has an important role in fetal growth. More research is needed to delve deeper into the potential role of the maternal TRP metabolism during the periconception period and pregnancy outcome for mother and offspring. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Department of Obstetrics and Gynecology and the Department of Clinical Chemistry of the Erasmus MC, University Medical Center, Rotterdam, the Netherlands. The authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER: N/A.

Placentae of small appropriately-grown-for-gestational-age neonates exhibit sexually dimorphic transcriptomic changes representative of placental insufficiency.

INTRODUCTION: Previous studies have reported that neonates less than the 25th BWC especially if they were male, were more likely to be associated with birth complications suggesting small neonates often identified as appropriately grown are at risk of adverse outcomes. We have questioned whether smaller neonates not typically categorized as "small for gestational age" may not reach their genetically determined growth due to placental insufficiency. METHODS: RNA-Seq was performed on the Illumina NovaSeq 600 using term placentae from neonates that were less than the 10th birthweight centile (BWC) (n = 39), between the 10th and the 30th BWC (n = 15) or greater than the 30th BWC (n = 23). Bioinformatic analyses were conducted and statistical significance was assessed at a level of P < 0.05 for single comparisons or FDR <0.05 unless otherwise noted. RESULTS: Gene set enrichment analysis revealed differences between BWC groups and in relation to the sex of the placenta. Genes associated with hypoxia, inflammatory responses, estrogen responsive genes, and androgen responsive genes were enriched (FDR <0.1) for in placentae of neonates <10th BWC regardless of sex and also in male placentae of neonates between the 10th-30th BWC. Female placenta of neonates between the 10th-30th BWC were comparable to placentae of neonates >30th BWC. DISCUSSION: These findings provide evidence that small male neonates may be at a greater risk of an adverse outcome than females due to changes in gene expression that are associated with placental dysfunction. The current data raises questions of whether placental pathology for smaller appropriately grown neonates should be scientifically and clinically examined in more depth.

Exosomal small RNA profiling in first-trimester maternal blood explores early molecular pathways of preterm preeclampsia.

Introduction: Preeclampsia (PE) is a severe obstetrical syndrome characterized by new-onset hypertension and proteinuria and it is often associated with fetal intrauterine growth restriction (IUGR). PE leads to long-term health complications, so early diagnosis would be crucial for timely prevention. There are multiple etiologies and subtypes of PE, and this heterogeneity has hindered accurate identification in the presymptomatic phase. Recent investigations have pointed to the potential role of small regulatory RNAs in PE, and these species, which travel in extracellular vesicles (EVs) in the circulation, have raised the possibility of non-invasive diagnostics. The aim of this study was to investigate the behavior of exosomal regulatory small RNAs in the most severe subtype of PE with IUGR. Methods: We isolated exosomal EVs from first-trimester peripheral blood plasma samples of women who later developed preterm PE with IUGR (n=6) and gestational age-matched healthy controls (n=14). The small RNA content of EVs and their differential expression were determined by next-generation sequencing and further validated by quantitative real-time PCR. We also applied the rigorous exceRpt bioinformatics pipeline for small RNA identification, followed by target verification and Gene Ontology analysis. Results: Overall, >2700 small RNAs were identified in all samples and, of interest, the majority belonged to the RNA interference (RNAi) pathways. Among the RNAi species, 16 differentially expressed microRNAs were up-regulated in PE, whereas up-regulated and down-regulated members were equally found among the six identified Piwi-associated RNAs. Gene ontology analysis of the predicted small RNA targets showed enrichment of genes in pathways related to immune processes involved in decidualization, placentation and embryonic development, indicating that dysregulation of the induced small RNAs is connected to the impairment of immune pathways in preeclampsia development. Finally, the subsequent validation experiments revealed that the hsa_piR_016658 piRNA is a promising biomarker candidate for preterm PE associated with IUGR. Discussion: Our rigorously designed study in a homogeneous group of patients unraveled small RNAs in circulating maternal exosomes that act on physiological pathways dysregulated in preterm PE with IUGR. Therefore, our small RNA hits are not only suitable biomarker candidates, but the revealed biological pathways may further inform us about the complex pathology of this severe PE subtype.

Placenta-anchored tadalafil liposomes rescues intrauterine growth restriction through continuous placental blood perfusion improvement.

Physiological or pathological hypoperfusion of the placenta is one of the main causes of intrauterine growth restriction (IUGR) which poses a significant risk to the health of the fetus and newborn. Tadalafil, a 5-type phosphodiesterase inhibitor, has previously been found to improve the symptoms of IUGR in various clinical studies. Unfortunately, its clinical utility is hindered by its limited water solubility, rapid metabolism, and lack of specific distribution in target tissues rendering tadalafil unable to maintain long-term placental perfusion. In this study, iRGD-modified tadalafil-loaded liposomes (iRGD-lipo@Tad) featuring a size of approximately 480 nm were designed to rectify the shortcomings of tadalafil. The prepared iRGD-lipo@Tad exhibited superior stability, sustained drug release capacity, and low cytotoxicity. The fluorescence study, tissue slice study, and drug biodistribution study together demonstrated the placenta-anchored ability of iRGD-modified liposomes. This was achieved by a dual approach consisting of the iRGD-mediated placenta-targeting effect and special particle size-mediated placenta resident effect. The pharmacokinetic study revealed a significant improvement in the in vivo process of tadalafil encapsulated by the iRGD-modified liposomes. In comparison to the tadalafil solution, the peak plasma concentration of iRGD-lipo@Tad was significantly increased, and the area under the curve was increased by about 7.88 times. In the pharmacodynamic study, iRGD-lipo@Tad achieved a continuous and efficient improvement of placental blood perfusion. This was achieved by decreasing the ratio of plasma soluble fms-like tyrosine kinase to placental growth factor and increasing the levels of cyclic guanosine monophosphate and nitric oxide. Consequently, iRGD-lipo@Tad resulted in a significant increase in embryo weight and a reduction in the miscarriage rate of N-Nitro-L-arginine methyl ester-induced IUGR pregnant mice without detectable toxicity. In summary, the nanotechnology-assisted therapy strategy presented here not only overcomes the limitations of tadalafil in the clinical treatment of IUGR but also offers new avenues to address the treatment of other placenta-originated diseases.

Overexpression of Human sFLT1 in the Spongiotrophoblast Is Sufficient to Induce Placental Dysfunction and Fetal Growth Restriction in Transgenic Mice.

Preeclampsia (PE) is characterized by maternal hypertension and placental dysfunction, often leading to fetal growth restriction (FGR). It is associated with an overexpression of the anti-angiogenic sFLT1 protein, which originates from the placenta and serves as a clinical biomarker to predict PE. To analyze the impact of sFLT1 on placental function and fetal growth, we generated transgenic mice with placenta-specific human sFLT1 (hsFLT1) overexpression. Immunohistochemical, morphometrical, and molecular analyses of the placentas on 14.5 dpc and 18.5 dpc were performed with a focus on angiogenesis, nutrient transport, and inflammation. Additionally, fetal development upon placental hsFLT1 overexpression was investigated. Dams exhibited a mild increase in serum hsFLT1 levels upon placental hsFLT1 expression and revealed growth restriction of the fetuses in a sex-specific manner. Male FGR fetuses expressed higher amounts of placental hsFLT1 mRNA compared to females. FGR placentas displayed an altered morphology, hallmarked by an increase in the spongiotrophoblast layer and changes in labyrinthine vascularization. Further, FGR placentas showed a significant reduction in placental glycogen storage and nutrient transporter expression. Moreover, signs of hypoxia and inflammation were observed in FGR placentas. The transgenic spongiotrophoblast-specific hsFLT1 mouse line demonstrates that low hsFLT1 serum levels are sufficient to induce significant alterations in fetal and placental development in a sex-specific manner.

The differential regulation of placenta trophoblast bisphosphoglycerate mutase in fetal growth restriction: preclinical study in mice and observational histological study of human placenta.

Background: Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown. Methods: Pregnant mice were subjected to acute or chronic hypoxia (12.5% O2) resulting in reduced fetal weight. Placenta oxygen transport was assessed by blood oxygenation level dependent (BOLD) contrast magnetic resonance imaging (MRI). The placentae were analyzed via immunohistochemistry and in situ hybridization. Human placentae were selected from FGR and matched controls and analyzed by immunohistochemistry (IHC). Maternal and cord sera were analyzed by mass spectrometry. Results: We show that murine acute and chronic gestational hypoxia recapitulates FGR phenotype and affects placental structure and morphology. Gestational hypoxia decreased labyrinth area, increased the incidence of red blood cells (RBCs) in the labyrinth while expanding the placental spiral arteries (SpA) diameter. Hypoxic placentae exhibited higher hemoglobin-oxygen affinity compared to the control. Placental abundance of Bisphosphoglycerate mutase (BPGM) was upregulated in the syncytiotrophoblast and spiral artery trophoblast cells (SpA TGCs) in the murine gestational hypoxia groups compared to the control. Hif1α levels were higher in the acute hypoxia group compared to the control. In contrast, human FGR placentae exhibited reduced BPGM levels in the syncytiotrophoblast layer compared to placentae from healthy uncomplicated pregnancies. Levels of 2,3 BPG, the product of BPGM, were lower in cord serum of human FGR placentae compared to control. Polar expression of BPGM was found in both human and mouse placentae syncytiotrophoblast, with higher expression facing the maternal circulation. Moreover, in the murine SpA TGCs expression of BPGM was concentrated exclusively in the apical cell side, in direct proximity to the maternal circulation. Conclusions: This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR. Funding: This work was supported by the Weizmann Krenter Foundation and the Weizmann - Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.

Ddx5 Targeted Epigenetic Modification of Pericytes in Pulmonary Hypertension After Intrauterine Growth Restriction.

Newborns with intrauterine growth restriction (IUGR) have a higher likelihood of developing pulmonary arterial hypertension (PAH) in adulthood. Although there is increasing evidence suggesting that pericytes play a role in regulating myofibroblast transdifferentiation and angiogenesis in malignant and cardiovascular diseases, their involvement in the pathogenesis of IUGR-related pulmonary hypertension and the underlying mechanisms remain incompletely understood. To address this issue, a study was conducted using a Sprague-Dawley rat model of IUGR-related pulmonary hypertension. Our investigation revealed increased proliferation and migration of pulmonary microvascular pericytes in IUGR-related pulmonary hypertension, accompanied by weakened endothelial-pericyte interactions. Through whole-transcriptome sequencing, Ddx5 (DEAD-box protein 5) was identified as one of the hub genes in pericytes. DDX5, a member of the RNA helicase family, plays a role in the regulation of ATP-dependent RNA helicase activities and cellular function. MicroRNAs have been implicated in the pathogenesis of PAH, and microRNA-205 (miR-205) regulates cell proliferation, migration, and angiogenesis. The results of dual-luciferase reporter assays confirmed the specific binding of miR-205 to Ddx5. Mechanistically, miR-205 negatively regulates Ddx5, leading to the degradation of ß-catenin by inhibiting the phosphorylation of Gsk3ß at serine 9. In vitro experiments showed the addition of miR-205 effectively ameliorated pericyte dysfunction. Furthermore, in vivo experiments demonstrated that miR-205 agomir could ameliorate pulmonary hypertension. Our findings indicated that the downregulation of miR-205 expression mediates pericyte dysfunction through the activation of Ddx5. Therefore, targeting the miR-205/Ddx5/p-Gsk3ß/ß-catenin axis could be a promising therapeutic approach for IUGR-related pulmonary hypertension.

Gut microbiota contributes to bisphenol A-induced maternal intestinal and placental apoptosis, oxidative stress, and fetal growth restriction in pregnant ewe model by regulating gut-placental axis.

BACKGROUND: Bisphenol A (BPA) is an environmental contaminant with endocrine-disrupting properties that induce fetal growth restriction (FGR). Previous studies on pregnant ewes revealed that BPA exposure causes placental apoptosis and oxidative stress (OS) and decreases placental efficiency, consequently leading to FGR. Nonetheless, the response of gut microbiota to BPA exposure and its role in aggravating BPA-mediated apoptosis, autophagy, mitochondrial dysfunction, endoplasmic reticulum stress (ERS), and OS of the maternal placenta and intestine are unclear in an ovine model of gestation. RESULTS: Two pregnant ewe groups (n = 8/group) were given either a subcutaneous (sc) injection of corn oil (CON group) or BPA (5 mg/kg/day) dissolved in corn oil (BPA group) once daily, from day 40 to day 110 of gestation. The maternal colonic digesta and the ileum and placental tissue samples were collected to measure the biomarkers of autophagy, apoptosis, mitochondrial dysfunction, ERS, and OS. To investigate the link between gut microbiota and the BPA-induced FGR in pregnant ewes, gut microbiota transplantation (GMT) was conducted in two pregnant mice groups (n = 10/group) from day 0 to day 18 of gestation after removing their intestinal microbiota by antibiotics. The results indicated that BPA aggravates apoptosis, ERS and autophagy, mitochondrial function injury of the placenta and ileum, and gut microbiota dysbiosis in pregnant ewes. GMT indicated that BPA-induced ERS, autophagy, and apoptosis in the ileum and placenta are attributed to gut microbiota dysbiosis resulting from BPA exposure. CONCLUSIONS: Our findings indicate the underlying role of gut microbiota dysbiosis and gut-placental axis behind the BPA-mediated maternal intestinal and placental apoptosis, OS, and FGR. The findings further provide novel insights into modulating the balance of gut microbiota through medication or probiotics, functioning via the gut-placental axis, to alleviate gut-derived placental impairment or FGR. Video Abstract.

Dysfunctional Postnatal Mitochondrial Energy Metabolism in a Patient with Neurodevelopmental Defects Caused by Intrauterine Growth Restriction Due to Idiopathic Placental Insufficiency.

We report the case of a four-year-old male patient with a complex medical history born prematurely as the result of intrauterine growth restriction due to placental insufficiency. His clinical manifestations included severe neurodevelopmental deficits, global developmental delay, Pierre-Robin sequence, and intractable epilepsy with both generalized and focal features. The proband's low levels of citrulline and lactic acidosis provoked by administration of Depakoke were evocative of a mitochondrial etiology. The proband's genotype-phenotype correlation remained undefined in the absence of nuclear and mitochondrial pathogenic variants detected by deep sequencing of both genomes. However, live-cell mitochondrial metabolic investigations provided evidence of a deficient oxidative-phosphorylation pathway responsible for adenosine triphosphate (ATP) synthesis, leading to chronic energy crisis in the proband. In addition, our metabolic analysis revealed metabolic plasticity in favor of glycolysis for ATP synthesis. Our mitochondrial morphometric analysis by transmission electron microscopy confirmed the suspected mitochondrial etiology, as the proband's mitochondria exhibited an immature morphology with poorly developed and rare cristae. Thus, our results support the concept that suboptimal levels of intrauterine oxygen and nutrients alter fetal mitochondrial metabolic reprogramming toward oxidative phosphorylation (OXPHOS) leading to a deficient postnatal mitochondrial energy metabolism. In conclusion, our collective studies shed light on the long-term postnatal mitochondrial pathophysiology caused by intrauterine growth restriction due to idiopathic placental insufficiency and its negative impact on the energy-demanding development of the fetal and postnatal brain.

Implications for miR-339-5p regulation of trophoblast proliferation and migration in placentas associated with porcine intrauterine growth retardation using integrated transcriptome sequencing analysis.

Placental dysfunction is considered as one of the main etiologies of fetal intrauterine growth retardation (IUGR). MicroRNAs (miRNAs) have been demonstrated to be a vital epigenetic modification involved in regulating the placental function and pregnancy outcomes in mammals. However, the mechanisms underlying placenta-specific miRNAs involved in the occurrence and development of pig IUGR remain unclear. In this work, we compared the placental morphologies of piglets with IUGR and normal birth weight (NBW) by using histomorphological analysis and performed a miRNA-mRNA integrative analysis of the gene expression profiles of IUGR and NBW placentas through RNA sequencing. We also investigated the role of differentially expressed ssc-miR-339-5p/GRIK3 through an in vitro experiment on porcine trophoblast cells (PTr2). IUGR piglets had significantly lower birth weight, placental weight, placental efficiency, and placental villus and capillary densities compared with the NBW piglets (P < 0.05). A total of 81 differentially expressed miRNAs and 726 differentially expressed genes in the placentas were screened out between the IUGR and NBW groups. The miRNA-mRNA interaction networks revealed the key core miRNA (ssc-miR-339-5p) and its corresponding target genes. Subsequently, we found that upregulation of ssc-miR-339-5p significantly inhibited the migration and proliferation of PTr2 cells (P < 0.05). The dual-luciferase reporter system showed that GRIK3 was the target gene of ssc-miR-339-5p, and the transcription level of GRIK3 may be negatively regulated by ssc-miR-339-5p. Additionally, overexpression of ssc-miR-339-5p significantly increased (P < 0.05) the mRNA expression levels of genes involved in the cytokine-cytokine receptor interaction pathway. These results indicate that ssc-miR-339-5p may affect the migration and proliferation of trophoblast cells by regulating the expression of GRIK3 and altering the placental inflammatory response, resulting in a suboptimal morphology and function of the placenta and the development of pig IUGR.

Zika virus co-opts microRNA networks to persist in placental niches detected by spatial transcriptomics.

BACKGROUND: Zika virus congenital infection evades double-stranded RNA detection and may persist in the placenta for the duration of pregnancy without accompanying overt histopathologic inflammation. Understanding how viruses can persist and replicate in the placenta without causing overt cellular or tissue damage is fundamental to deciphering mechanisms of maternal-fetal vertical transmission. OBJECTIVE: Placenta-specific microRNAs are believed to be a tenet of viral resistance at the maternal-fetal interface. We aimed to test the hypothesis that the Zika virus functionally disrupts placental microRNAs, enabling viral persistence and fetal pathogenesis. STUDY DESIGN: To test this hypothesis, we used orthogonal approaches in human and murine experimental models. In primary human trophoblast cultures (n=5 donor placentae), we performed Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation to identify any significant alterations in the functional loading of microRNAs and their targets onto the RNA-induced silencing complex. Trophoblasts from same-donors were split and infected with a contemporary first-passage Zika virus strain HN16 (multiplicity of infection=1 plaque forming unit per cell) or mock infected. To functionally cross-validate microRNA-messenger RNA interactions, we compared our Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation results with an independent analysis of published bulk RNA-sequencing data from human placental disk specimens (n=3 subjects; Zika virus positive in first, second, or third trimester, CD45- cells sorted by flow cytometry) and compared it with uninfected controls (n=2 subjects). To investigate the importance of these microRNA and RNA interference networks in Zika virus pathogenesis, we used a gnotobiotic mouse model uniquely susceptible to the Zika virus. We evaluated if small-molecule enhancement of microRNA and RNA interference pathways with enoxacin influenced Zika virus pathogenesis (n=20 dams total yielding 187 fetal specimens). Lastly, placentae (n=14 total) from this mouse model were analyzed with Visium spatial transcriptomics (9743 spatial transcriptomes) to identify potential Zika virus-associated alterations in immune microenvironments. RESULTS: We found that Zika virus infection of primary human trophoblast cells led to an unexpected disruption of placental microRNA regulation networks. When compared with uninfected controls, Zika virus-infected placentae had significantly altered SLC12A8, SDK1, and VLDLR RNA-induced silencing complex loading and transcript levels (-22; adjusted P value <.05; Wald-test with false discovery rate correction q<0.05). In silico microRNA target analyses revealed that 26 of 119 transcripts (22%) in the transforming growth factor-ß signaling pathway were targeted by microRNAs that were found to be dysregulated following Zika virus infection in trophoblasts. In gnotobiotic mice, relative to mock controls, Zika virus-associated fetal pathogenesis included fetal growth restriction (P=.036) and viral persistence in placental tissue (P=.011). Moreover, spatial transcriptomics of murine placentae revealed that Zika virus-specific placental niches were defined by significant up-regulation of complement cascade components and coordinated changes in transforming growth factor-ß gene expression. Finally, treatment of Zika virus-infected mice with enoxacin abolished placental Zika virus persistence, rescued the associated fetal growth restriction, and the Zika virus-associated transcriptional changes in placental immune microenvironments were no longer observed. CONCLUSION: These results collectively suggest that (1) Zika virus infection and persistence is associated with functionally perturbed microRNA and RNA interference pathways specifically related to immune regulation in placental microenvironments and (2) enhancement of placental microRNA and RNA interference pathways in mice rescued Zika virus-associated pathogenesis, specifically persistence of viral transcripts in placental microenvironments and fetal growth restriction.

Gestational exposure to 1-NP induces ferroptosis in placental trophoblasts via CYP1B1/ERK signaling pathway leading to fetal growth restriction.

Fetal growth restriction (FGR) is a prevalent complication in obstetrics, yet its exact aetiology remains unknown. Numerous studies suggest that the degradation of the living environment is a significant risk factor for FGR. 1-Nitropyrene (1-NP) is a widespread environmental pollutant as a representative substance of nitro-polycyclic aromatic hydrocarbons. In this study, we revealed that 1-NP induced FGR in fetal mice by constructing 1-NP exposed pregnant mice models. Intriguingly, we found that placental trophoblasts of 1-NP exposed mice exhibited significant ferroptosis, which was similarly detected in placental trophoblasts from human FGR patients. In this regard, we established a 1-NP exposed cell model in vitro using two human trophoblast cell lines, HTR8/SVneo and JEG-3. We found that 1-NP not only impaired the proliferation, migration, invasion and angiogenesis of trophoblasts, but also induced severe cellular ferroptosis. Meanwhile, the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively rescued 1-NP-induced trophoblast biological function impairment. Mechanistically, we revealed that 1-NP regulated ferroptosis by activating the ERK signaling pathway. Moreover, we innovatively revealed that CYP1B1 was essential for the activation of ERK signaling pathway induced by 1-NP. Overall, our study innovatively identified ferroptosis as a significant contributor to 1-NP induced trophoblastic functional impairment leading to FGR and clarified the specific mechanism by which 1-NP induced ferroptosis via the CYP1B1/ERK signaling pathway. Our study provided novel insights into the aetiology of FGR and revealed new mechanisms of reproductive toxicity of environmental pollutants.

Lactobacillus amylovorus Promotes Lactose Utilization in Small Intestine and Enhances Intestinal Barrier Function in Intrauterine Growth Restricted Piglets.

BACKGROUND: Intrauterine growth restriction (IUGR) resulted in high mortality and many physiological defects of piglets, causing huge economic loss in the swine industry. Lactobacillus amylovorus (L. amylovorus) was identified as one of the main differential bacteria between IUGR and normal piglets. However, the effects of L. amylovorus on the growth performance and intestinal health in IUGR piglets remained unclear. OBJECTIVES: This study aimed to investigate the promoting effects of L. amylovorus Mafic1501, a new strain isolated from normal piglets, on the growth performance and intestinal barrier functions in IUGR piglets. METHODS: Newborn mice or piglets were assigned into 3 groups: CON (normal birth weight, control), IUGR (low birth weight), and IUGR+L. amy (low birth weight), administered with sterile saline or L. amylovorus Mafic1501, respectively. Growth performance, lactose content in the digesta, intestinal lactose transporter, and barrier function parameters were profiled. IPEC-J2 cells were cultured to verify the effects of L. amylovorus Mafic1501 on lactose utilization and intestinal barrier functions. RESULTS: L. amylovorus Mafic1501 elevated body weight and average daily gain of IUGR mice and piglets (P < 0.05). The lactose content in the ileum was decreased, whereas gene expression of glucose transporter 2 (GLUT2) was increased by L. amylovorus Mafic1501 in IUGR piglets during suckling period (P < 0.05). Besides, L. amylovorus Mafic1501 promoted intestinal barrier functions by increasing the villus height and relative gene expressions of tight junctions (P < 0.05). L. amylovorus Mafic1501 and its culture supernatant decreased the lactose level in the medium and upregulated gene expressions of transporter GLUT2 and tight junction protein Claudin-1 of IPEC-J2 cells (P < 0.05). CONCLUSION: L. amylovorus Mafic1501 improved the growth performance of IUGR piglets by promoting the lactose utilization in small intestine and enhancing intestinal barrier functions. Our results provided the new evidence of L. amylovorus Mafic1501 for its application in the swine industry.

Human cord plasma proteomic analysis reveals sexually dimorphic proteins associated with intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is associated with increased risk of cardiometabolic disease later in life and has been shown to affect female and male offspring differently, but the mechanisms remain unclear. The purpose of this study was to identify proteomic differences and metabolic risk markers in IUGR male and female neonates when compared to appropriate for gestational age (AGA) babies that will provide a better understanding of IUGR pathogenesis and its associated risks. Our results revealed alterations in IUGR cord plasma proteomes with most of the differentially abundant proteins implicated in peroxisome pathways. This effect was evident in females but not in males. Furthermore, we observed that catalase activity, a peroxisomal enzyme, was significantly increased in females (p < 0.05) but unchanged in males. Finally, we identified risk proteins associated with obesity, type-2 diabetes, and glucose intolerance such as EGF containing fibulin extracellular matrix protein 1 (EFEMP1), proprotein convertase subtilisin/kexin type 9 (PCSK9) and transforming growth factor beta receptor 3 (TGFBR3) proteins unique to females while coagulation factor IX (C9) and retinol binding protein 4 (RBP4) are unique in males. In conclusion, IUGR may display sexual dimorphism which may be associated with differences in lifelong risk for cardiometabolic disease between males and females.

N-acetylcysteine and Hydroxychloroquine Ameliorate ADMA-Induced Fetal Growth Restriction in Mice via Regulating Oxidative Stress and Autophagy.

Fetal growth restriction (FGR) seriously threatens perinatal health. The main cause of FGR is placental malperfusion, but the specific mechanism is still unclear, and there is no effective treatment for FGR. We constructed a FGR mouse model by adding exogenous asymmetric dimethylarginine (ADMA) through in vivo experiments and found that ADMA could cause placental dysplasia and induce the occurrence of FGR. Compared with the control group, reactive oxygen species (ROS) production in the placenta was increased in mice with FGR, and the expression of autophagy-related proteins p-AKT/AKT, p-mTOR/mTOR, and P62 was significantly decreased, while the expression of Beclin-1 and LC3-II was significantly increased in the FGR group. Furthermore, ADMA had a favorable effect in promoting the formation of autophagosomes. Hydroxychloroquine (HCQ) and N-acetylcysteine (NAC) improved ADMA-induced disorders of placental development and alleviated ADMA-induced FGR. This study found that ADMA could cause excessive autophagy of trophoblasts by increasing the level of oxidative stress, ultimately leading to the occurrence of FGR, and HCQ and NAC had therapeutic effects on ADMA-induced FGR.

Association between placental oxygen transport and fetal brain cortical development: a study in monochorionic diamniotic twins.

Normal cortical growth and the resulting folding patterns are crucial for normal brain function. Although cortical development is largely influenced by genetic factors, environmental factors in fetal life can modify the gene expression associated with brain development. As the placenta plays a vital role in shaping the fetal environment, affecting fetal growth through the exchange of oxygen and nutrients, placental oxygen transport might be one of the environmental factors that also affect early human cortical growth. In this study, we aimed to assess the placental oxygen transport during maternal hyperoxia and its impact on fetal brain development using MRI in identical twins to control for genetic and maternal factors. We enrolled 9 pregnant subjects with monochorionic diamniotic twins (30.03 ± 2.39 gestational weeks [mean ± SD]). We observed that the fetuses with slower placental oxygen delivery had reduced volumetric and surface growth of the cerebral cortex. Moreover, when the difference between placenta oxygen delivery increased between the twin pairs, sulcal folding patterns were more divergent. Thus, there is a significant relationship between placental oxygen transport and fetal brain cortical growth and folding in monochorionic twins.