Search

N6-methyladenosine (m6A) is the most abundant modification controlling RNA metabolism and cellular functions, but its roles in placental development are still poorly understood. Here, we characterized the synchronization of m6A modifications and placental functions by mapping the m6A methylome in human placentas (n = 3, each trimester), revealing that the dynamic patterns of m6A were associated with gene expression homeostasis and different biological pathways in placental development. Then, we generated trophoblast-specific knockout mice of Wtap, a critical component of methyltransferase complex, and demonstrated that Wtap was essential for trophoblast proliferation, placentation and perinatal growth. Further in vitro experiments which includes cell viability assays and series molecular binding assays demonstrated that WTAP-m6A-IGF2BP3 axis regulated the RNA stability and translation of Anillin (ANLN) and VEGFA, promoting trophoblast proliferation and secretion. Dysregulation of this regulatory axis was observed in placentas from pregnancies with fetal growth restriction (FGR) or preeclampsia, revealing the pathogenic effects of imbalanced m6A modifications. Therefore, our findings provide novel insights into the functions and regulatory mechanisms of m6A modifications in placental development and placental-related gestational diseases., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Chronic histiocytic intervillositis (CHI) is a recurrent placental lesion where maternal macrophages infiltrate the intervillous space. Its cause is unknown, though due to similarities to rejected allografts one hypothesis is that CHI represents maternal-fetal rejection. Here, virtual crossmatching was applied to healthy pregnancies and those with a history of CHI. Anti-HLA antibodies, measured by Luminex, were present in slightly more controls than CHI (8/17 (47.1%) vs 5/14 (35.7%)), but there was no significant difference in levels of sensitisation or fetal specific antibodies. Quantification of immunohistochemical staining for HLA-Class II was increased in syncytiotrophoblast of placentas with CHI (Grade 0.44 [IQR 0.1-0.7]) compared to healthy controls (0.06 [IQR 0-0.2]) and subsequent pregnancies (0.13 [IQR 0-0.3]) (P = 0.0004). HLA-Class II expression was positively related both to the severity of CHI (r = 0.67) and C4d deposition (r = 0.48). There was no difference in overall C4d and HLA-Class I immunostaining. Though increased anti-HLA antibodies were not evident in CHI, increased expression of HLA-Class II at the maternal-fetal interface suggests that they may be relevant in its pathogenesis. Further investigation of antibodies immediately after diagnosis is warranted in a larger cohort of CHI cases to better understand the role of HLA in its pathophysiology., (© 2024. The Author(s).)

In Brief: Placental oxidative stress contributes to both normal and abnormal placentation during pregnancy. This review discusses the potential consequence of oxidative stress-induced placental dysfunction on pregnancies complicated by fetal death and pregnancies with a high risk of fetal death., Abstract: The placenta is a source of reactive oxygen free radicals due to the oxidative metabolism required to meet the demands of the growing fetus. The placenta has an array of efficient antioxidant defense systems to deal with rising oxidative stress created by free radicals during pregnancy. Properly controlled physiological (low-level) free radical production is a necessary part of cellular signaling pathways and downstream activities during normal placental development; however, poorly controlled oxidative stress can cause aberrant placentation, immune disturbances and placental dysfunction. Abnormal placental function and immune disturbances are linked to many pregnancy-related disorders, including early and recurrent pregnancy loss, fetal death, spontaneous preterm birth, preeclampsia and fetal growth restriction. This review discusses the role of placental oxidative stress in both normal and pathological settings. Finally, based on previously published work, this review presents multiple lines of evidence for the strong association between oxidative stress and adverse pregnancy outcomes, including fetal death and pregnancies with a high risk of fetal death.

Background: Placental disorders contribute to pregnancy complications, including preeclampsia and fetal growth restriction (FGR), but debate regarding their specific pathobiology persists. Our objective was to apply transcriptomics with weighted gene correlation network analysis to further clarify the placental dysfunction in these conditions., Methods: We performed RNA sequencing with weighted gene correlation network analysis using human placental samples (n=30), separated into villous tissue and decidua basalis, and clinically grouped as follows: (1) early-onset preeclampsia (EOPE)+FGR (n=7); (2) normotensive, nonanomalous preterm FGR (n=5); (2) EOPE without FGR (n=8); (4) spontaneous idiopathic preterm birth (n=5) matched for gestational age; and (5) uncomplicated term births (n=5). Our data was compared with RNA sequencing data sets from public databases (GSE114691, GSE148241, and PRJEB30656; n=130 samples)., Results: We identified 14 correlated gene modules in our specimens, of which most were significantly correlated with birthweight and maternal blood pressure. Of the 3 network modules consistently predictive of EOPE±FGR across data sets, we prioritized a coexpression gene group enriched for hypoxia-response and metabolic pathways for further investigation. Cluster analysis based on transcripts from this module and the glycolysis/gluconeogenesis metabolic pathway consistently distinguished a subset of EOPE±FGR samples with an expression signature suggesting modified tissue bioenergetics. We demonstrated that the expression ratios of LDHA / LDHB and PDK1 / GOT1 could be used as surrogate indices for the larger panels of genes in identifying this subgroup., Conclusions: We provide novel evidence for a molecular subphenotype consistent with a glycolytic metabolic shift that occurs more frequently but not universally in placental specimens of EOPE±FGR., Competing Interests: Disclosures None.

Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) transcripts are elevated in the circulation of individuals whose pregnancies are complicated by preterm fetal growth restriction (FGR). In this paper, we show that the cases with preeclampsia (PE) have increased circulating NR4A2 transcripts compared to those with normotensive FGR. We aimed to establish whether the dysfunctional placenta mirrors the increase in NR4A2 transcripts and further, to uncover the function of placental NR4A2. NR4A2 expression was detected in preterm and term placental tissue; expressed higher at term. NR4A2 mRNA expression and protein were not altered in placentas from preterm FGR or PE pregnancies. Hypoxia (1% O 2 compared to 8% O 2 ) significantly reduced cytotrophoblast NR4A2 mRNA expression, but not placental explant NR4A2 expression. Silencing cytotrophoblast NR4A2 expression under hypoxia (via short interfering (si)RNAs) did not alter angiogenic Placental Growth Factor, nor anti-angiogenic sFlt-1 mRNA expression or protein secretion, but increased expression of cellular antioxidant, oxidative stress, inflammatory, and growth genes. NR4A2 expression was also not altered in a model of tumour necrosis factor-α-induced endothelial dysfunction, or with pravastatin treatment. Further studies are required to identify the origin of the circulating transcripts in pathological pregnancies, and investigate the function of placental NR4A2., (© 2021. The Author(s).)

A well-functioning placenta is crucial for normal gestation and regulates the nutrient, gas, and waste exchanges between the maternal and fetal circulations and is an important endocrine organ producing hormones that regulate both the maternal and fetal physiologies during pregnancy. Placental insufficiency is implicated in spontaneous preterm birth (SPTB). We proposed that deficits in the capacity of the placenta to maintain bioenergetic and metabolic stability during pregnancy may ultimately result in SPTB. To explore our hypothesis, we performed a RNA-seq study in male and female placentas from women with SPTB (<36 weeks gestation) compared to normal pregnancies (≥38 weeks gestation) to assess the alterations in the gene expression profiles. We focused exclusively on Black women (cases and controls), who are at the highest risk of SPTB. Six hundred and seventy differentially expressed genes were identified in male SPTB placentas. Among them, 313 and 357 transcripts were increased and decreased, respectively. In contrast, only 61 differentially expressed genes were identified in female SPTB placenta. The ingenuity pathway analysis showed alterations in the genes and canonical pathways critical for regulating inflammation, oxidative stress, detoxification, mitochondrial function, energy metabolism, and the extracellular matrix. Many upstream regulators and master regulators important for nutrient-sensing and metabolism were also altered in SPTB placentas, including the PI3K complex, TGFB1/SMADs, SMARCA4, TP63, CDKN2A, BRCA1, and NFAT. The transcriptome was integrated with published human placental metabolome to assess the interactions of altered genes and metabolites. Collectively, significant and biologically relevant alterations in the transcriptome were identified in SPTB placentas with fetal sex disparities. Altered energy metabolism, mitochondrial function, inflammation, and detoxification may underly the mechanisms of placental dysfunction in SPTB.

Biomarkers for placental dysfunction are currently lacking. We recently identified SPINT1 as a novel biomarker; SPINT2 is a functionally related placental protease inhibitor. This study aimed to characterise SPINT2 expression in placental insufficiency. Circulating SPINT2 was assessed in three prospective cohorts, collected at the following: (1) term delivery ( n = 227), (2) 36 weeks ( n = 364), and (3) 24-34 weeks' ( n = 294) gestation. SPINT2 was also measured in the plasma and placentas of women with established placental disease at preterm (<34 weeks) delivery. Using first-trimester human trophoblast stem cells, SPINT2 expression was assessed in hypoxia/normoxia (1% vs. 8% O2), and following inflammatory cytokine treatment (TNFα, IL-6). Placental SPINT2 mRNA was measured in a rat model of late-gestational foetal growth restriction. At 36 weeks, circulating SPINT2 was elevated in patients who later developed preeclampsia ( p = 0.028; median = 2233 pg/mL vs. controls, median = 1644 pg/mL), or delivered a small-for-gestational-age infant ( p = 0.002; median = 2109 pg/mL vs. controls, median = 1614 pg/mL). SPINT2 was elevated in the placentas of patients who required delivery for preterm preeclampsia ( p = 0.025). Though inflammatory cytokines had no effect, hypoxia increased SPINT2 in cytotrophoblast stem cells, and its expression was elevated in the placental labyrinth of growth-restricted rats. These findings suggest elevated SPINT2 is associated with placental insufficiency.

Cervical remodeling is a critical component in both term and preterm labor in eutherian mammals. However, the molecular mechanisms underlying cervical remodeling remain poorly understood in the mare. The current study compared the transcriptome of the equine cervix (cervical mucosa (CM) and stroma (CS)) during placentitis (placentitis group, n = 5) and normal prepartum mares (prepartum group, n = 3) to normal pregnant mares (control group, n = 4). Transcriptome analysis identified differentially expressed genes (DEGs) during placentitis (5310 in CM and 907 in CS) and during the normal prepartum period (189 in CM and 78 in CS). Our study revealed that cervical remodeling during placentitis was dominated by inflammatory signaling as reflected by the overrepresented toll-like receptor signaling, interleukin signaling, T cell activation, and B cell activation pathways. These pathways were accompanied by upregulation of several proteases, including matrix metalloproteinases (MMP1, MMP2, and MMP9), cathepsins (CTSB, CTSC, and CTSD) and a disintegrin and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1, ADAMTS4, and ADAMTS5), which are crucial for degradation of cervical collagens during remodeling. Cervical remodeling during placentitis was also associated with upregulation of water channel-related transcripts (AQP9 and RLN), angiogenesis-related transcripts (NOS3, ENG1, THBS1, and RAC2), and aggrecan (ACAN), a hydrophilic glucosaminoglycan, with subsequent cervical hydration. The normal prepartum cervix was associated with upregulation of ADAMTS1, ADAMTS4, NOS3 and THBS1, which might reflect an early stage of cervical remodeling taking place in preparation for labor. In conclusion, our findings revealed the possible key regulators and mechanisms underlying equine cervical remodeling during placentitis and the normal prepartum period.

Improved understanding of the molecular mechanisms underlying ascending equine placentitis holds the potential for the development of new diagnostic tools and therapies to forestall placentitis-induced preterm labor. The current study characterized the equine placental transcriptome (chorioallantois [CA] and endometrium [EN]) during placentitis (placentitis group, n = 6) in comparison to gestationally-matched controls (control group, n = 6). Transcriptome analysis identified 2953 and 805 differentially expressed genes in CA and EN during placentitis, respectively. Upstream regulator analysis revealed the central role of toll-like receptors (TLRs) in triggering the inflammatory signaling, and consequent immune-cell chemotaxis. Placentitis was associated with the upregulation of matrix metalloproteinase (MMP1, MMP2, and MMP9) and apoptosis-related genes such as caspases (CASP3, CASP4, and CASP7) in CA. Also, placentitis was associated with downregulation of transcripts coding for proteins essential for placental steroidogenesis (SRD5A1 and AKR1C1), progestin signaling (PGRMC1 and PXR) angiogenesis (VEGFA, VEGFR2, and VEGFR3), and nutrient transport (GLUT12 and SLC1A4), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could explain placental insufficiency during placentitis. Placentitis was also associated with aberrant expression of several placenta-regulatory genes, such as PLAC8, PAPPA, LGALS1, ABCG2, GCM1, and TEPP, which could negatively affect placental functions. In conclusion, our findings revealed for the first time the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during equine placentitis, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pregnancy complications are a major cause of fetal and maternal morbidity and mortality in humans. The majority of pregnancy complications initiate due to abnormal placental development and function. During the last decade, the role of microRNAs (miRNAs) in regulating placental and fetal development has become evident. Dysregulation of miRNAs in the placenta not only affects placental development and function, but these miRNAs can also be exported to both maternal and fetal compartments and affect maternal physiology and fetal growth and development. Due to their differential expression in the placenta and maternal circulation during pregnancy complications, miRNAs can be used as diagnostic biomarkers. However, the differential expression of a miRNA in the placenta may not always be reflected in maternal circulation, which makes it difficult to find a reliable biomarker for placental dysfunction. In this review, we provide an overview of differentially expressed miRNAs in the placenta and/or maternal circulation during preeclampsia (PE) and intrauterine growth restriction (IUGR), which can potentially serve as biomarkers for prediction or diagnosis of pregnancy complications. Using different bioinformatics tools, we also identified potential target genes of miRNAs associated with PE and IUGR, and the role of miRNA-mRNA networks in the regulation of important signaling pathways and biological processes.

Growing evidence indicates that reduced uterine perfusion pressure (RUPP) triggers the cascade of events leading to preeclampsia. Edaravone is a powerful free radical scavenger used for the treatment of ischemia/reperfusion diseases due to its anti-oxidative stress and anti-inflammatory properties. Here we investigate the effect of edaravone (3 mg/kg) on different maternal and fetal outcomes of RUPP-induced placental ischemia mice model. RUPP surgery was performed on gestation day (GD) 13 followed by edaravone injection from GD14 to GD18, sacrifice day. The results showed that edaravone injection significantly decreased the maternal blood pressure (113.2 ± 2.3 mmHg) compared with RUPP group (131.5 ± 1.9 mmHg). Edaravone increased fetal survival rate (75.4%) compared with RUPP group (54.4%), increased fetal length, weights, and feto-placental ratio (7.2 and 5.7 for RUPP and RUPP-Edaravone groups, respectively) compared with RUPP group. In addition, RUPP resulted in many fetal morphological abnormalities as well as severe delayed ossification, however edaravone decreased the morphological abnormalities and increased the ossification of the fetal endoskeleton. Edaravone improved the histopathological structure of the maternal kidney and heart as well as decreased the elevated blood urea and creatinine levels (31.5 ± 0.15 mg/dl (RUPP), 25.6 ± 0.1 mg/dl (RUPP+edaravone) for urea and 5.4 ± 0.1 mg/dl (RUPP), 3.5 ± 0.1 mg/dl (RUPP+edaravone) for creatinine) and decreased cleaved caspase-3 expression in the maternal kidney. In conclusion, this study demonstrated that our RUPP mice model recapitulated preeclampsia symptoms and edaravone injection ameliorated most of these abnormalities suggesting its effectiveness and potential application in preeclampsia treatment regimes.

Monitoring the health of a pregnancy is of utmost importance to both the fetus and the mother. The diagnosis of pregnancy complications typically occurs after the manifestation of symptoms, and limited preventative measures or effective treatments are available. Traditionally, pregnancy health is evaluated by analyzing maternal serum hormone levels, genetic testing, ultrasonographic imaging, and monitoring maternal symptoms. However, researchers have reported a difference in extracellular vesicle (EV) quantity and cargo between healthy and at-risk pregnancies. Thus, placental EVs (PEVs) may help to understand normal and aberrant placental development, monitor pregnancy health in terms of developing placental pathologies, and assess the impact of environmental influences, such as infection, on pregnancy. The diagnostic potential of PEVs could allow for earlier detection of pregnancy complications via noninvasive sampling and frequent monitoring. Understanding how PEVs serve as a means of communication with maternal cells and recognizing their potential utility as a readout of placental health have sparked a growing interest in basic and translational research. However, to date, PEV research with animal models lags behind human studies. The strength of animal pregnancy models is that they can be used to assess placental pathologies in conjunction with isolation of PEVs from fluid samples at different time points throughout gestation. Assessing PEV cargo in animals within normal and complicated pregnancies will accelerate the translation of PEV analysis into the clinic for potential use in prognostics. We propose that appropriate animal models of human pregnancy complications must be established in the PEV field., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Preeclampsia is a pregnancy-specific disease of high prevalence characterized by the onset of hypertension, among other maternal or fetal signs. Its etiopathogenesis remains elusive, but it is widely accepted that abnormal placentation results in the release of soluble factors that cause the clinical manifestations of the disease. An increased level of soluble endoglin (sEng) in plasma has been proposed to be an early diagnostic and prognostic biomarker of this disease. A pathogenic function of sEng involving hypertension has also been reported in several animal models with high levels of plasma sEng not directly dependent on pregnancy. The aim of this work was to study the functional effect of high plasma levels of sEng in the pathophysiology of preeclampsia in a model of pregnant mice, in which the levels of sEng in the maternal blood during pregnancy replicate the conditions of human preeclampsia. Our results show that wild type pregnant mice carrying human sEng-expressing transgenic fetuses (f WT ( hsEng + )) present high plasma levels of sEng with a timing profile similar to that of human preeclampsia. High plasma levels of human sEng (hsEng) are associated with hypertension, proteinuria, fetal growth restriction, and the release of soluble factors to maternal plasma. In addition, f WT ( hsEng + ) mice also present placental alterations comparable to those caused by the poor remodeling of the spiral arteries characteristic of preeclampsia. In vitro and ex vivo experiments, performed in a human trophoblast cell line and human placental explants, show that sEng interferes with trophoblast invasion and the associated pseudovasculogenesis, a process by which cytotrophoblasts switch from an epithelial to an endothelial phenotype, both events being related to remodeling of the spiral arteries. Our findings provide a novel and useful animal model for future research in preeclampsia and reveal a much more relevant role of sEng in preeclampsia than initially proposed.

Gestational diabetes mellitus (GDM) is a disease of pregnancy that is associated with d-glucose intolerance and foeto-placental vascular dysfunction. GMD causes mitochondrial dysfunction in the placental endothelium and trophoblast. Additionally, GDM is associated with reduced placental oxidative phosphorylation due to diminished activity of the mitochondrial F 0 F 1 -ATP synthase (complex V). This phenomenon may result from a higher generation of reactive superoxide anion and nitric oxide. Placental mitochondrial biogenesis and mitophagy work in concert to maintain cell homeostasis and are vital mechanisms securing the efficient generation of ATP, whose demand is higher in pregnancy, ensuring foetal growth and development. Additional factors disturbing placental ATP synthase activity in GDM include pre-gestational maternal obesity or overweight, intracellular pH, miRNAs, fatty acid oxidation, and foetal (and 'placental') sex. GDM is also associated with maternal and foetal hyperinsulinaemia, altered circulating levels of adiponectin and leptin, and the accumulation of extracellular adenosine. Here, we reviewed the potential interplay between these molecules or metabolic conditions on the mechanisms of mitochondrial dysfunction in the foeto-placental unit in GDM pregnancies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: An adequate development of the placenta includes trophoblast differentiation with the processes of trophoblast migration, invasion, cellular senescence and apoptosis which are all crucial to establishing a successful pregnancy. Altered placental development and function lead to placental diseases such as preeclampsia (PE) which is mainly characterized by insufficient trophoblast invasion and abnormally invasive placenta (AIP) disorders ( Placenta accreta , increta , or percreta) which are characterized by excessive trophoblast invasion. Both of them will cause maternal and fetal morbidity/mortality. However, the etiology of these diseases is still unclear. Our previous study has shown that the matricellular protein nephroblastoma overexpressed (NOV, CCN3) induces G0/G1 cell cycle arrest, drives trophoblast cells into senescence and activates FAK and Akt kinases resulting in reduced cell proliferation and enhanced migration capability of the human trophoblast cell line SGHPL-5. The present study focuses on whether CCN3 can alter cell cycle-regulated pathways associated with trophoblast senescence and invasion activity in pathological versus gestational age-matched control placentas., Methods: Cell cycle regulator proteins were investigated by immunoblotting and qPCR. For localization of CCN3, p16, p21, and Cyclin D1 proteins, co-immunohistochemistry was performed., Results: In early-onset PE placentas, CCN3 was expressed at a significantly lower level compared to gestational age-matched controls. The decrease of CCN3 level is associated with an increase in p53, Cyclin E1 and pRb protein expression, whereas the level of cleaved Notch-1, p21, Cyclin D1, pFAK, pAKT, and pmTOR protein decreased. In term AIP placentas, the expression of CCN3 was significantly increased compared to matched term controls. This increase was correlated to an increase in p53, p16, p21, Cyclin D1, cleaved Notch-1, pFAK, pAkt, and pmTOR whereas pRb was significantly decreased. However, in late PE and early AIP placentas, no significant differences in CCN3, p16, p21, Cyclin D1, p53, and cleaved Notch-1 expression were found when matched to appropriate controls., Conclusions: CCN3 expression levels are correlated to markers of cell cycle arrest oppositely in PE and AIP by activating the FAK/AKT pathway in AIP or down-regulating in PE. This may be one mechanism to explain the different pathological features of placental diseases, PE and AIP., (Copyright © 2020 Duan, Schimmelmann, Wu, Reisch, Faas, Kimmig, Winterhager, Köninger and Gellhaus.)

Peroxisome proliferator-activated receptors (PPARs) belong to the family of ligand-activated nuclear receptors. The PPAR family consists of three subtypes encoded by three separate genes: PPARα (NR1C1), PPARβ/δ (NR1C2), and PPARγ (NR1C3). PPARs are critical regulators of metabolism and exhibit tissue and cell type-specific expression patterns and functions. Specific PPAR ligands have been proposed as potential therapies for a variety of diseases such as metabolic syndrome, cancer, neurogenerative disorders, diabetes, cardiovascular diseases, endometriosis, and retinopathies. In this review, we focus on the knowledge of PPAR function in angiogenesis, a complex process that plays important roles in numerous pathological conditions for which therapeutic use of PPAR modulation has been suggested.

The placenta performs a range of crucial functions that support fetal growth during pregnancy, including facilitating the supply of oxygen and nutrients to the fetus, removal of waste products from the fetus and the endocrine modulation of maternal physiology. The placenta also stores glucose in the form of glycogen, the function of which remains unknown. Aberrant placental glycogen storage in humans is associated with maternal diabetes during pregnancy and pre-eclampsia, thus linking placental glycogen storage and metabolism to pathological pregnancies. To understand the role of placental glycogen in normal and complicated pregnancies, we must turn to animal models. Over 40 targeted mutations in mice demonstrate the defects in placental cells that store glycogen and suggest that placental glycogen represents a source of readily mobilized glucose required during periods of high fetal demand. However, direct functional evidence is currently lacking. Here, we evaluate these genetic mouse models with placental phenotypes that implicate glycogen trophoblast cell differentiation and function to illuminate the common molecular pathways that emerge and to better understand the relationship between placental glycogen and fetal growth. We highlight the current limitations in exploring the key questions regarding placental glycogen storage and metabolism and define how to experimentally overcome these constraints.

The key event in placentitis-induced preterm labor is myometrial activation with the subsequent initiation of labor. However, the molecular mechanisms underlying myometrial activation are not fully understood in the mares. Therefore, the equine myometrial transcriptome was characterized during placentitis (290.0 ± 1.52 days of GA, n = 5) and the prepartum period (330 days of GA, n = 3) in comparison to normal pregnant mares (289.8 ± 2.18 days of GA, n = 4). Transcriptome analysis identified 596 and 290 DEGs in the myometrium during placentitis and the prepartum period, respectively, with 138 DEGs in common. The placentitis DEGs included eight genes (MMP1, MMP8, S100A9, S100A8, PI3, APOBEC3Z1B, RETN, and CXCL2) that are exclusively expressed in the inflamed myometrium. Pathway analysis elucidated that inflammatory signaling, Toll-like receptor signaling, and apoptosis pathways dominate myometrial activation during placentitis. The prepartum myometrium was associated with overexpression of inflammatory signaling, oxidative stress, and 5-hydroxytryptamine degradation. Gene ontology enrichment analysis identified several chemoattractant factors in the myometrium during placentitis and prepartum period, including CCL2, CXCL1, CXCL3, and CXCL6 in common. Upstream regulator analysis revealed 19 potential upstream regulators in placentitis dataset including transcription regulators (E2F1, FOXM1, HIF1A, JUNB, NFKB1A, and STAT1), transmembrane receptors (FAS, ICAM1, SELP, TLR2, and TYROBP), growth factors (HGF and TGFB3), enzymes (PTGS2 and PRKCP), and others (S100A8, S100A9, CD44, and C5AR1). Additionally, three upstream regulators (STAT3, EGR1, and F2R) were identified in the prepartum dataset. These findings revealed the key regulators and pathways underlying myometrial activation during placentitis, which aid in understanding the disease and facilitate the development of efficacious therapies., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy of reproductive-aged women. In pregnancy, women with PCOS experience increased risk of miscarriage, gestational diabetes, preeclampsia, and extremes of fetal birth weight, and their offspring are predisposed to reproductive and cardiometabolic dysfunction in adulthood. Pregnancy complications, adverse fetal outcomes, and developmental programming of long-term health risks are known to have placental origins. These findings highlight the plausibility of placental compromise in pregnancies of women with PCOS., Evidence Synthesis: A comprehensive PubMed search was performed using terms "polycystic ovary syndrome," "placenta," "developmental programming," "hyperandrogenism," "androgen excess," "insulin resistance," "hyperinsulinemia," "pregnancy," and "pregnancy complications" in both human and animal experimental models., Conclusions: There is limited human placental research specific to pregnancy of women with PCOS. Gestational androgen excess and insulin resistance are two clinical hallmarks of PCOS that may contribute to placental dysfunction and underlie the higher rates of maternal-fetal complications observed in pregnancies of women with PCOS. Additional research is needed to prevent adverse maternal and developmental outcomes in women with PCOS and their offspring., (Copyright © 2019 Endocrine Society.)

Clinical end-stage parameters define the pregnancy disorders preeclampsia and fetal growth restriction while classification of the underlying placental dysfunction is missing and urgently needed. Flt-1 (FMS-like tyrosine kinase receptor 1) is the most promising placenta-derived predictive biomarker for preeclampsia. We aimed to classify placental dysfunction in preeclampsia and fetal growth restriction at delivery by metabolic profiling and authenticate the biomarker Flt-1 for placental dysfunction. We studied 143 pregnancies with or without preeclampsia and/or fetal growth restriction delivered by cesarean section. Metabolic placenta profiles were created by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy and the resulting placental phenotypes obtained by hierarchical clustering. Placental Flt-1 expression (membrane-bound and soluble isoforms combined) and maternal serum Flt-1 expression (soluble isoforms) were analyzed by immunohistochemistry and ELISA, respectively. We identified 3 distinct placenta groups by 21 metabolites and diagnostic outcome parameters; normal placentas, moderate placental dysfunction, and severe placental dysfunction. Increased placental Flt-1 was associated with severe placental dysfunction, and increased serum Flt-1 was associated with moderate and severe placental dysfunction. The preeclamptic pregnancies with and without placental dysfunction could be distinguished by 5 metabolites and placental Flt-1. Placental Flt-1 alone could separate normal pregnancies with and without placental dysfunction. In conclusion, metabolomics could classify placental dysfunction and provide information not identified by traditional diagnostics and metabolites with biomarker potential were identified. Flt-1 was confirmed as precision biomarker for placental dysfunction, substantiating its usefulness for identification of high-risk pregnancies for preeclampsia and fetal growth restriction with placental involvement.

Podoplanin, a reliable marker of lymphatic endothelium, is a mucin-type transmembrane protein. Although the human placenta is devoid of a lymphatic system, chorionic villous stromal (CVS) cells express podoplanin. In this study, the pattern of podoplanin expression in normal and pathological placental tissues and the biological role of podoplanin were investigated. In total, 198 placental tissues belonging to 184 patients, seen at the Department of Pathology of Bulent Ecevit University Education and Research Hospital, Zonguldak, Turkey, were evaluated histopathologically and determined to meet the study criteria. The tissues were assigned to control, cisternal placental disorders, inflammation and hypoxic-ischemic pathology groups. Podoplanin expression in CVS cells was graded from 0 to 3 depending on the staining intensity, as determined by an immunohistochemical evaluation of chorionic villi in the most intensively stained tissue region. Podoplanin levels in control CVS cells increased in parallel with placental maturation, whereas in molar pregnancies podoplanin expression was lower than in control tissues. In the acute placental inflammation group, podoplanin immunoreactivity was similar to that in the control group, whereas in the preeclampsia group, podoplanin expression was higher than in all other groups. Our study showed an increase in podoplanin expression in CVS cells during pregnancy. In preeclamptic patients, the increase in podoplanin expression may be a response to hypoxic-ischemic conditions, whereas in molar pregnancies the decrease in podoplanin levels may cause villous swelling by disrupting intercellular fluid homeostasis.

The current study aimed to elucidate the mechanisms underlying myometrial activation during equine placentitis related to progestogens and the progesterone receptor signaling pathways. Placentitis was induced via intracervical inoculation with Streptococcus equi ssp zooepidemicus in mares at approximately 290 days of gestation (placentitis group; n = 6) with uninoculated gestationally matched mares as controls (n = 4). Mares in the placentitis and control groups were euthanized, and myometrial samples were collected from two regions: region 1-parallel to active placentitis lesion with placental separation in placentitis group (P1) or caudal pole of the placenta in control group (C1); and region 2-parallel to apparently normal placenta without separation in placentitis group (P2) or uterine body in control group (C2). In the current study, SRD5A1 and AKR1C23, which encode for the key P4 metabolizing enzymes, were downregulated in P1 in comparison to C1, C2, and P2, and this was associated with a decline (P < 0.05) in 5αDHP, allopregnanolone (3αDHP), and 20αDHP in P1 in comparison to C1. Further, myometrial expression of PR was downregulated (P < 0.05) in P1 in comparison to C1 and P2, and this was associated with activation of the inflammatory cascade as reflected by significant upregulation of IL-1β and IL-8 in P1 in comparison to C1, C2, and P2, and supported by increased tissue leukocytes in P1 in comparison to C1. In conclusion, equine placentitis is associated with a localized withdrawal of progestins and a downregulation of the PR in the myometrium concomitant with upregulation of inflammatory cytokines and subsequent myometrial activation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Exposure to intrauterine inflammation (IUI) is associated with short- and long-term adverse perinatal outcomes. However, little data exist on utilizing placenta to prognosticate fetal injury in this scenario. Our study aimed to utilize imaging modalities to evaluate mechanisms contributing to placental injury following IUI exposure and correlated it to concomitant fetal brain injury. CD1 pregnant dams underwent laparotomies and received intrauterine injections of either lipopolysaccharide (LPS; a model of IUI) or phosphate-buffered saline (PBS). In utero ultrasound Doppler velocimetry of uterine and umbilical arteries and magnetic resonance imaging (MRI) of placental volumes with confirmatory immunohistochemical (vimentin) and histochemistry (fibrin) analyses were performed. ELISA for thrombosis markers, fibrinogen and fibrin was performed to analyze thrombi in placenta. Fetal brain immunohistochemistry was performed to detect microglial activation (ionized calcium-binding adaptor molecule 1, Iba1). On ultrasound, LPS group demonstrated elevated resistance indices, pulsatility indices and a greater occurrence of absent end-diastolic flow in the umbilical and uterine arteries. In the fetus, there was an increased cardiac Tei indices in the LPS group. MRI revealed decreased volume of placenta in the LPS group associated with placental thinning and placental endothelial damage on immunohistochemistry. Decreased fibrinogen content and more thrombi staining in placenta exposed to maternal LPS indicated the hypercoagulability. Furthermore, the expression of Iba1was significantly associated with placental thickness (r = -0.7890, Pearson correlation coefficient). Our data indicate that IUI can trigger events leading to maternal placental malperfusion and fetal vessel resistance, as well as predispose the developing fetus to cardiac dysfunction and brain damage. Furthermore, our data suggest that prenatal ultrasound can be a real-time clinical tool for assessing fetal risk for adverse neurologic outcomes following the potential IUI exposure., Competing Interests: The authors have declared that no competing interests exist.

Preeclampsia is a pregnancy-specific disorder of new-onset hypertension linked to placental ischemia. While obesity is a major risk factor for preeclampsia, not all obese pregnant women develop pregnancy-induced hypertension or preeclampsia. Previously, we reported that placental ischemia-induced hypertension is dependent upon intact signaling of the sympathetic nervous system. Moreover, in various models of obesity, blockade of MC4R (melanocortin-4 receptor) signaling protects against the development of hypertension via suppression of the sympathetic nervous system. Less is known about this pathway during obese pregnancy. Although blockade of MC4R may lead to increased body weight during pregnancy, we tested the hypothesis that placental ischemia-induced hypertension is attenuated in obese MC4R-deficient pregnant rats. On gestational day 14, MC4R wild-type or heterozygous-deficient (MC4R-def) rats were subjected to chronic placental ischemia via the reduced uterine perfusion pressure procedure or Sham surgery then examined on gestational day 19. In Sham MC4R-def versus Sham wild-type pregnant rats, there was increased body weight, fat mass, and circulating leptin levels but they had similar fetus weights. Reduced uterine perfusion pressure reduced fetus weights in both strains. Reduced uterine perfusion pressure increased blood pressure in wild-type rats but this response was significantly attenuated in MC4R-def rats, although blood pressure was elevated in Sham MC4R-def over Sham wild-type. These data indicate that while obese MC4R-def pregnant rats have higher blood pressure during pregnancy, placental ischemia-induced hypertension is attenuated in obese MC4R-def pregnant rats. Thus, obese women with abnormal MC4R signaling may be less susceptible to the development of placental ischemia-induced hypertension.

Differentiation of first trimester human placental cytotrophoblast (CTB) from an anchorage-dependent epithelial phenotype into the mesenchymal-like invasive extravillous trophoblast (EVT) is crucial in the development of the maternal-fetal interface. We showed previously that differentiation of first trimester CTB to EVT involves an epithelial-mesenchymal transition (EMT). Here we compare the epithelial-mesenchymal characteristics of CTB and EVT derived from normal third trimester placenta or placenta previa versus abnormally invasive placenta (AIP). CTB and EVT were isolated from normal term placenta or placenta previa following Caesarean section and EVT from AIP following Caesarean hysterectomy. Cell identity was validated by measurement of cytokeratin-7 and HLA-G. Comparing normal term CTB with EVT from normal term placenta or placenta previa for differential expression analysis of genes associated with the EMT showed changes in >70% of the genes probed. While demonstrating a mesenchymal phenotype relative to CTB, many of the gene expression changes in third trimester EVT were reduced relative to the first trimester EVT. We suggest that third trimester EVT are in a more constrained, metastable state compared to first trimester equivalents. By contrast, EVT from AIP demonstrate characteristics that are more mesenchymal than normal third trimester EVT, placing them closer to first trimester EVT on the EMT spectrum, consistent with a more invasive phenotype.

Preeclampsia is a persistent hypertensive gestational disease characterized by high blood pressure and proteinuria, which presents from the second trimester of pregnancy. At the cellular level, preeclampsia has largely been associated with the release of free radicals by the placenta. Placenta-borne oxidative and nitrosative stresses are even sometimes considered as the major molecular determinants of the maternal disease. In this review, we present the recent literature evaluating free radical production in both normal and pathological placentas (including preeclampsia and other major pregnancy diseases), in humans and animal models. We then assess the putative effects of these free radicals on the placenta and maternal endothelium. This analysis was conducted with regard to recent papers and possible therapeutic avenues.

Background: Placental dysfunction is related to many pregnancy complications, but collecting placental specimens for investigation in large scale epidemiologic studies is often infeasible. Standard procedures involving immediate collection after birth and snap freezing are often cost prohibitive. We aimed to collect pilot data regarding the feasibility and precision of a simpler approach, the collection of tissue samples following 24 hours of refrigeration of whole placentae at 4°C, as compared to the "gold standard" of snap freezing excised tissue within 40 minutes of delivery for the assessment of inflammatory cytokines., Methods: Placentae were collected from 12 women after delivering live-born singleton babies via uncomplicated vaginal delivery. Two placentae were utilized to establish laboratory tissue processing and assay protocols. The other 10 placentae were utilized in a comparison of three tissue collection conditions. Specifically, key inflammatory cytokines were measured in 3 sections, representing three collection conditions. Sections 1 (full thickness) and 2 (excised prior to freezing) were obtained within 40 minutes of delivery and snap frozen in liquid nitrogen, and section 3 (full thickness) was obtained after refrigerating the placenta at 4°C for 24 hours., Results: IL-6, IL-10, and IL-8 all had comparable concentrations and variability overall in all three section types. Levels of tumor necrosis factor alpha (TNF-α) were too low among samples to reliably measure using immunoassay., Conclusions: Refrigeration of placentae prior to processing does not appear to compromise detection of these cytokines for purposes of large scale studies. These findings provide a framework and preliminary data for the study of inflammatory cytokines within the placenta in large scale and/or resource-limited settings.

The objective of our study was to investigate quantitative parameters of vimentin in fibroblasts and endotheliocytes of the chorionic villi by means of immunohistochemical examination of placenta with preterm maturation of the chorionic tree with iron-deficiency anemia of pregnancy in two different terms of gestation - 29-32 weeks and 33-36 weeks. 182 placentas were examined. The study design included two main groups of investigation of the above terms of gestation and three groups of comparison. Quantitative parameters of vimentin in the cytoplasm of fibroblasts and endotheliocytes of the placenta intermediate and terminal villi were considered on the basis of staining optic density measured by means of computer microdensitometry method. Immunohistochemical staining on vimentin was determined in the cytoplasm of fibroblasts and endotheliocytes of the placenta intermediate and terminal villi in all the groups of the study. The main results are presented in the conclusions. Optic density of immunohistochemical staining on vimentin in the cytoplasm of fibroblasts and endotheliocytes of the placenta intermediate and terminal villi was found to be a criterion to determine maturation of the placenta chorionic tree. Iron-deficiency anemia paradoxically causes immaturity of fibroblasts and endotheliocytes of the placenta intermediate and terminal villi even in those placentas where preterm maturation of the chorionic tree is determined.

Preeclampsia is a major pregnancy complication associated with poor placental perfusion and placental hypoxia. Systemic and placental inflammation and elevated placental secretion of the antiangiogenic factors sFlt-1 (soluble fms-like tyrosine kinase 1) and sEng (soluble endoglin) are hallmarks of preeclampsia, causing endothelial dysfunction and multiorgan injury. A molecule that links placental hypoxia, inflammation, and antiangiogenic factor release has not been described. ATF3 (activating transcription factor 3) is highly expressed in placenta. We assessed whether placental ATF3 is dysregulated in preterm preeclampsia, is altered by hypoxia, and regulates proinflammatory cytokine and antiangiogenic factor production. ATF3 mRNA and protein expression was significantly reduced in preterm preeclamptic placentas compared with gestation-matched controls. Hypoxia reduced ATF3 expression in primary cytotrophoblast and placental explants. Silencing ATF3 in primary cytotrophoblast increased proinflammatory cytokine (IL-6 [interleukin 6], TNF-α [tumor necrosis factor α]) and NF-κB (nuclear factor κB) expression. In silico analysis identified an ATF3-binding site in the promoter of Flt-1 (the transcript from which sFlt-1 is produced). Silencing ATF3 increased sFlt-1 and sEng secretion from primary cytotrophoblast possibly by increasing Rab11a and Arf1, cargo proteins that facilitate exosomal release of sFlt-1. ATF3 knockout mice did not have a preeclampsia phenotype, suggesting that these pathways may be specific to humans (preeclampsia is a uniquely human condition). To conclude, we have shown that ATF3 is decreased in preeclamptic placentas and that this decrease is likely to occur after prolonged hypoxia. We show that ATF3 is a regulator of placental proinflammatory cytokines and antiangiogenic factors sFlt-1 and sEng. Therefore, reduced ATF3 may be centrally involved in the pathology of preeclampsia., (© 2017 American Heart Association, Inc.)

Fetal growth restriction (FGR) threatens perinatal health and is correlated with increased incidence of fetal original adult diseases. Most cases of FGR were idiopathic, which were supposed to be associated with placental abnormality. Decreased circulating placental growth factor (PGF) was recognized as an indication of placental deficiency in FGR. In this study, the epigenetic regulation of PGF in FGR placentas and the involvement of PGF in modulation of trophoblast activity were investigated. The expression level of PGF in placental tissues was determined by RT-qPCR, immunohistochemistry and ELISA. DNA methylation profile of PGF gene was analyzed by bisulfite sequencing. Trophoblastic cell lines were treated with ZM-306416, an inhibitor of PGF receptor FLT1, to observe the effect of PGF/FLT1 signaling on cell proliferation and migration. We demonstrated that PGF was downregulated in placentas from FGR pregnancies compared with normal controls. The villous expression of PGF was positively correlated with placental and fetal weight. The CpG island inside PGF promoter was hypomethylated without obvious difference in both normal and FGR placentas. However, the higher DNA methylation at another CpG island downstream exon 7 of PGF was demonstrated in FGR placentas. Additionally, we found FLT1 was expressed in trophoblast cells. Inhibition of PGF/FLT1 signaling by a selective inhibitor impaired trophoblast proliferation and migration. In conclusion, our data suggested that the PGF expression was dysregulated, and disrupted PGF/FLT1 signaling in trophoblast might contribute to placenta dysfunction in FGR. Thus, our results support the significant role of PGF in the pathogenesis of FGR., (© 2017 Society for Reproduction and Fertility.)

Receptor activity-modifying protein 2 (Ramp2) is a single-pass transmembrane protein that heterodimerizes with several family B G-protein coupled receptors to alter their function. Ramp2 has been primarily characterized in association with calcitonin receptor-like receptor (Calcrl, CLR), forming the canonical receptor complex for the endocrine peptide adrenomedullin (Adm, AM). However, we previously demonstrated that Ramp2+/- female mice display a constellation of endocrine-related phenotypes that are distinct from those of Adm+/- and Calcrl+/- mice, implying that RAMP2 has physiological functions beyond its canonical complex. Here, we localize Ramp2 expression in the mouse placenta, finding that Ramp2 is robustly expressed in the fetal labyrinth layer, and then characterize the effects of loss of Ramp2 on placental development. Consistent with the expression pattern of Ramp2 in the placenta, Ramp2-/- placentas have a thinner labyrinth layer with significantly fewer trophoblast cells secondary to a reduction in trophoblast proliferation. We also find that absence of Ramp2 leads to failed spiral artery remodeling unaccompanied by changes in the uterine natural killer cell population. Furthermore, we assess changes in gene expression of other RAMP2-associated G-protein coupled receptors (GPCRs), concluding that Ramp2 loss decreases parathyroid hormone 1 receptor (Pthr1) expression and causes a blunted response to systemic parathyroid hormone (PTH) administration in mice. Ultimately, these studies provide in vivo evidence of a role for RAMP2 in placental development distinct from the RAMP2-CLR/AM signaling paradigm and identify additional pathways underlying the endocrine and fertility defects of the previously characterized Ramp2 heterozygous adult females.

Preeclampsia (PE) results in placental dysfunction and is one of the primary causes of maternal and fetal mortality and morbidity. During pregnancy, estrogen is produced primarily in the placenta by conversion of androgen precursors originating from maternal and fetal adrenal glands. These processes lead to increased plasma estrogen concentrations compared with levels in nonpregnant women. Aberrant production of estrogens could play a key role in PE symptoms because they are exclusively produced by the placenta and they promote angiogenesis and vasodilation. Previous assessments of estrogen synthesis during PE yielded conflicting results, possibly because of the lack of specificity of the assays. However, with the introduction of reliable analytical protocols using liquid chromatography/mass spectrometry or gas chromatography/mass spectrometry, more recent studies suggest a marked decrease in estradiol levels in PE. The aim of this review is to summarize current knowledge of estrogen synthesis, regulation in the placenta, and biological effects during pregnancy and PE. Moreover, this review highlights the links among the occurrence of PE, estrogen biosynthesis, angiogenic factors, and cardiovascular risk factors. A close link between estrogen dysregulation and PE occurrence might validate estrogen levels as a biomarker but could also reveal a potential approach for prevention or cure of PE., (Copyright © 2017 Endocrine Society.)

Waddlia chondrophila is a Gram-negative intracellular bacterial organism that is related to classical chlamydial species and has been implicated as a cause of abortion in cattle. Despite an increasing number of observational studies linking W. chondrophila infection to cattle abortion, little direct experimental evidence exists. Given this paucity of direct evidence the current study was carried out to investigate whether experimental challenge of pregnant cattle with W. chondrophila would result in infection and abortion. Nine pregnant Friesian-Holstein heifers received 2 × 10 8 inclusion forming units (IFU) W. chondrophila intravenously on day 105-110 of pregnancy, while four negative-control animals underwent mock challenge. Only one of the challenged animals showed pathogen-associated lesions, with the organism being detected in the diseased placenta. Importantly, the organism was re-isolated and its identity confirmed by whole genome sequencing, confirming Koch's third and fourth postulates. However, while infection of the placenta was observed, the experimental challenge in this study did not confirm the abortifacient potential of the organism., Competing Interests: ES is the CEO and VF the lead in bacteriology at BioSellal which manufactures and supplies the commercial Waddlia chondrophila qPCR kit used during this study. The remaining authors declare no competing financial interests.

Luteolin is a natural compound known for its anticancer effects on various human cancers by regulating signal transduction cascades. However, the effects of luteolin on human placental choriocarcinoma are not known. Results of the present study revealed that luteolin decreased viability of JAR and JEG-3 cells, which are valuable placental models, in a dose-dependent manner, and it induced apoptosis and loss of mitochondrial membrane potential in JAR and JEG-3 cells. The results also suggested that the PI3K/AKT pathway was inhibited by luteolin treatment of JAR and JEG-3 cells in a dose- and time-dependent manner. Next, we established effects of luteolin in the presence of pharmacological inhibitors of PI3K/AKT, ERK1/2 MAPK, and mTOR on proliferation of JAR and JEG-3 cells. In addition, these inhibitors were used to verify phosphorylation of AKT, GSK3beta, and ERK1/2 and to confirm mechanisms regulated by luteolin in JAR and JEG-3 cells. We also determined levels of SREBP1 and SREBP2 expression to investigate regulatory functions of luteolin in lipid metabolism in JAR and JEG-3 cells. Expression levels of both SREBP1 and SREBP2 mRNAs were significantly reduced, but only SREBP1 protein was influenced by luteolin. We compared viability of JAR and JEG-3 cells in response to luteolin alone or in combination with other chemotherapeutic drugs (etoposide, cisplatin, and paclitaxel) and found that luteolin has synergistic effects with the conventional chemotherapeutic drugs as an anticancer agent. Collectively, these results showed that luteolin plays an important role in the treatment of human choriocarcinoma cells by inhibiting the PI3K/AKT/mTOR/SREBP cascade and expression of lipogenic genes., (© 2016 by the Society for the Study of Reproduction, Inc.)

Fetoplacental glucocorticoid overexposure is a significant mechanism underlying fetal growth restriction and the programming of adverse health outcomes in the adult. Placental glucocorticoid inactivation by 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) plays a key role. We previously discovered that Hsd11b2(-/-) mice, lacking 11β-HSD2, show marked underdevelopment of the placental vasculature. We now explore the consequences for fetal cardiovascular development and whether this is reversible. We studied Hsd11b2(+/+), Hsd11b2(+/-), and Hsd11b2(-/-) littermates from heterozygous (Hsd11b(+/-)) matings at embryonic day (E)14.5 and E17.5, where all three genotypes were present to control for maternal effects. Using high-resolution ultrasound, we found that umbilical vein blood velocity in Hsd11b2(-/-) fetuses did not undergo the normal gestational increase seen in Hsd11b2(+/+) littermates. Similarly, the resistance index in the umbilical artery did not show the normal gestational decline. Surprisingly, given that 11β-HSD2 absence is predicted to initiate early maturation, the E/A wave ratio was reduced at E17.5 in Hsd11b2(-/-) fetuses, suggesting impaired cardiac function. Pravastatin administration from E6.5, which increases placental vascular endothelial growth factor A and, thus, vascularization, increased placental fetal capillary volume, ameliorated the aberrant umbilical cord velocity, normalized fetal weight, and improved the cardiac function of Hsd11b2(-/-) fetuses. This improved cardiac function occurred despite persisting indications of increased glucocorticoid exposure in the Hsd11b2(-/-) fetal heart. Thus, the pravastatin-induced enhancement of fetal capillaries within the placenta and the resultant hemodynamic changes correspond with restored fetal cardiac function. Statins may represent a useful therapeutic approach to intrauterine growth retardation due to placental vascular hypofunction.

Placental malaria caused by Plasmodium falciparum contributes to ~200,000 child deaths annually, mainly due to low birth weight (LBW). Parasitized erythrocyte sequestration and consequent inflammation in the placenta are common attributes of placental malaria. The precise molecular details of placental changes leading to LBW are still poorly understood. We hypothesized that placental malaria may disturb maternofetal exchange of vitamins, lipids, and hormones mediated by the multi-ligand (n ~ 50) scavenging/signaling receptor megalin, which is abundantly expressed in placenta but was not previously analyzed in pregnancy outcomes. We studied abundance of megalin and its intracellular adaptor protein Dab2 by immunofluorescence microscopy in placental biopsies from Ugandan women with (n = 8) and without (n = 20) active placental malaria. We found that: (a) abundances of both megalin (p = 0.01) and Dab2 (p = 0.006) were significantly reduced in brush border of syncytiotrophoblast of infected placentas; (b) amounts of megalin and Dab2 were strongly correlated (Spearman's r = 0.53, p = 0.003); (c) abundances of megalin and Dab2 (p = 0.046) were reduced in infected placentas from women with LBW deliveries. This study provides first evidence that placental malaria infection is associated with reduced abundance of megalin transport/signaling system and indicate that these changes may contribute to the pathology of LBW.

The aim of the current study was to examine the expression level and possibilities of apoptotic markers in realization of placental insufficiency in pregnant women with urogenital infections. The study was conducted on 250 pregnant women with urogenital infections (1-st group - 50 pregnant women with bacterial infections (Chlamydia, ureaplasma, mycoplasma), 2-nd group - 50 pregnant women with viral infections (CMV and herpes simplex virus), 3-rd group - 150 patients with mixed viral and bacterial infections) and 50 pregnant women with normal pregnancy. The content of apoptosis inducers: sFasL and TNF-α in blood serum of pregnant women was determined; the level of caspase-3 in placental sample was analyzed; sonographic examination of the placenta was performed. Maximal indices of apoptosis inducers were observed in the 3-rd group (with mixed viral and bacterial infections). Changes in the placenta according to ultrasound data were determined in all pregnant women with urogenital infections. It was suggested that increased placental cell death in apoptosis might be one of the key points, triggering the development of placental dysfunction.

Placental villi play pivotal roles in feto-maternal transportation and phospholipids constitute a major part of the villous membrane. We have been developing and optimizing an imaging system based on a matrix-assisted laser desorption/ionization (MALDI)-based mass spectrometer, which provides clear two-dimensional molecular distribution patterns using highly sensitive mass spectrometry from mixtures of ions generated on tissue surfaces. We recently applied this technology to normal human uncomplicated term placentas and detected the specific distribution of sphingomyelin (SM) (d18:1/16:0) in stem villi and phosphatidylcholine (PC) (16:0/20:4) in terminal villi. In the present study, we applied this technology to nine placentas with maternal or fetal complications, and determined whether a relationship existed between these specific distribution patterns of phospholipid molecules and the six representative pathological findings of placentas, i.e., villitis of unknown etiology (VUE), thrombus, atherosis, chorioamnionitis (CAM), immature terminal villi, and multiple branched terminal villi. In two placentas with the first and second largest total number of positive pathological findings, i.e., five and three positive findings, the specific distribution of SM (d18:1/16:0) in stem villi and PC (16:0/20:4) in terminal villi disappeared. The common pathological findings in these two placentas were atherosis, immature terminal villi, and multiple branched terminal villi, suggesting the possible involvement of the underperfusion of maternal blood into the intervillous space. On the other hand, the number of pathological findings were two or less in the seven other placentas, in which no specific relationships were observed between the differential expression patterns of these two phospholipids in stem and terminal villi and the pathological findings of the placentas; however, the specific distribution pattern of SM (d18:1/16:0) in stem villi disappeared in four placentas, while that of PC (16:0/20:4) in terminal villi was preserved. These results suggested that the absence of the specific distribution of PC (16:0/20:4) in terminal villi, possibly in combination with the absence of SM (d18:1/16:0) in stem villi, was linked to placental morphological changes in response to maternal underperfusion of the placenta.

Compelling evidence indicates that stress in utero, as manifested by low birth weight (LBW), increases the risk of metabolic syndrome in adulthood. Singletons conceived by assisted reproductive technology (ART) display a significant increase in LBW risk and ART offspring have a different metabolic profile starting at birth. Here, used mouse as a model, we found that ART resulted in reduced fetal weight and placental overgrowth at embryonic day 18.5 (E18.5). The ART placentae exhibited histomorphological alterations with defects in placental layer segregation and glycogen cells migration at E18.5. Further, ART treatments resulted in downregulation of a majority of placental nutrient transporters and reduction in placental efficiency. Moreover, the ART placentae were associated with increased methylation levels at imprinting control regions of H19, KvDMR1 and disrupted expression of a majority of imprinted genes important for placental development and function at E18.5. Our results from the mouse model show the first piece of evidence that ART treatment could affect fetal growth by disrupting placental development and function, suggests that perturbation of genomic imprinting resulted from embryo manipulation may contribute to these problems.

It is increasingly recognized that vitamin D3 (VitD3) has an anti-inflammatory activity. The present study investigated the effects of maternal VitD3 supplementation during pregnancy on LPS-induced placental inflammation and fetal intrauterine growth restriction (IUGR). All pregnant mice except controls were intraperitoneally injected with LPS (100 μg/kg) daily from gestational day (GD)15-17. In VitD3 + LPS group, pregnant mice were orally administered with VitD3 (25 μg/kg) before LPS injection. As expected, maternal LPS exposure caused placental inflammation and fetal IUGR. Interestingly, pretreatment with VitD3 repressed placental inflammation and protected against LPS-induced fetal IUGR. Further analysis showed that pretreatment with VitD3, which activated placental vitamin D receptor (VDR) signaling, specifically suppressed LPS-induced activation of nuclear factor kappa B (NF-κB) and significantly blocked nuclear translocation of NF-κB p65 subunit in trophoblast gaint cells of the labyrinth layer. Conversely, LPS, which activated placental NF-κB signaling, suppressed placental VDR activation and its target gene expression. Moreover, VitD3 reinforced physical interaction between placental VDR and NF-κB p65 subunit. The further study demonstrates that VitD3 inhibits placental NF-κB signaling in VDR-dependent manner. These results provide a mechanistic explanation for VitD3-mediated anti-inflammatory activity. Overall, the present study provides evidence for roles of VDR as a key regulator of placental inflammation.

Maternal intrauterine inflammation or infection is an important risk factor for neonatal cerebral white matter injury (WMI) and future neurological deficits. Activated protein C (APC), a natural anticoagulant, has been shown to exhibit anti-inflammatory, anti-apoptotic, profibrinolytic and cytoprotective activities. Recent studies have demonstrated that the novel prothrombinase, fibrinogen-like protein 2 (fgl2), contributes to the pathogenesis of a number of inflammatory diseases through the generation of fibrin. Thus, we hypothesized that APC may regulate coagulant and inflammatory processes and improve brain injury in an experimental rat model of intrauterine inflammation-induced WMI. The animal model was established by the administration of an intraperitoneal injection of lipopolysaccharide (LPS) to pregnant Sprague-Dawley rats on embryonic day (E)17 and E18. APC was administered intraperitoneally 30 min after the second LPS injection. The expression of fgl2 and the pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β expression in the placentas and fetal brains was determined on E19. Nerve cell death, the brain water content and protease-activated receptor 1 (PAR1) and nuclear factor κB (NF-κB) p65 expression was detected in the fetal brains. WMI in the neonatal rat brains was evaluated by hematoxylin and eosin (H&E) staining and immunohistochemistry for myelin basic protein (MBP). The results revealed that APC markedly reduced the LPS-induced increase in fgl2 expression and fibrin deposition, as well as the production of the pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β, in the placentas and fetal brains. In addition, APC attenuated cerebral apoptosis and brain edema, downregulated PAR1 and NF-κB p65 expression in the fetal brains, and improved hypomyelination and structural disturbances in the periventricular area of the neonatal rat brains. Our observations provide evidence that APC attenuates fetal neuroinflammation and the associated secondary WMI in the developing brain by inhibiting the expression of fgl2 and pro-inflammatory mediators, suggesting that APC may be a potential therapeutic approach for intrauterine inflammation-induced neonatal brain injury.

The thyroid hormone, 3,3,5-triiodo-L-thyronine (T3), modulates several physiological processes, including cellular growth, differentiation, metabolism, inflammation and proliferation, via interactions with thyroid hormone response elements (TREs) in the regulatory regions of target genes. Infection and inflammation are critical processes in placental development and pregnancy-related diseases. In particular, infection is the leading cause of neonatal mortality and morbidity worldwide. However, to date, no successful approach has been developed for the effective diagnosis of infection in preterm infants. Pre-eclampsia (PE) is a serious disorder that adversely affects ~5% of human pregnancies. Recent studies identified a multiprotein complex, the inflammasome, including the Nod-like receptor (NLR) family of cytosolic pattern recognition receptors, the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1, which plays a vital role in the placenta. The thyroid hormone modulates inflammation processes and is additionally implicated in placental development and disease. Therefore, elucidation of thyroid hormone receptor-regulated inflammation-related molecules, and their underlying mechanisms in placenta, should facilitate the identification of novel predictive and therapeutic targets for placental disorders. This review provides a detailed summary of current knowledge with respect to identification of useful biomarkers and their physiological significance in placenta.

Study Question: Are the fetal membranes of women affected with endometriosis similar to those from disease-free women?, Summary Answer: Decidua of women with endometriosis is able to generate endometriotic-like lesions in contact with the fetal membranes., What Is Known Already: Eutopic endometrium of women affected with endometriosis presents compromised properties. Endometrium undergoes decidualisation to accept and to further control the conceptus development during pregnancy. Decidualized endometrium is in close contact with the chorionic membrane and forms the choriodecidual layer, a major maternal-fetal interface., Study Design, Size, Duration: This is a laboratory case-control study involving diseased versus control samples. Eleven case samples and 11 control samples were collected from women in a tertiary care/research center between November 2011 and December 2013., Participants/materials, Setting, Methods: Participants were consecutive pregnant women affected with confirmed endometriosis and disease free women, who underwent Cesarean section before labor for obstetrical indication. The choriodecidual tissues were characterized using histology, immunohistochemistry, transcriptomic and whole genome CpG methylation analyses., Main Results and the Role of Chance: We demonstrate for the first time the presence of endometriotic-like lesions within the decidual side of the choriodecidua of the fetal membranes from women affected with severe endometriosis. Fetal membranes from women affected with endometriosis exhibited glandular components in the choriodecidual layer surrounded by enlarged decidualized cells disseminated along the entire membrane surface. Significant deregulation (variation of expression ≥2, P-value ≤0.05) was observed for 2773 genes known to be enriched in processes involved in glandular function, endocrine and nervous system, neoangiogenesis, and autoimmune disease. CpG methylation analysis revealed 5999 differentially methylated regions with a P-value ≤0.05., Limitations, Reasons for Caution: We studied women who delivered at term by Cesarean section before labor, following an uneventful pregnancy. Notwithstanding this, one cannot exclude that the presence of disseminated endometriotic lesions within the choriodecidual layer of the fetal membranes may disturb the anatomical integrity and/or the function of the membranes in some women with endometriosis., Wider Implications of the Findings: Our results shed new light on the capability of the diseased decidua to develop lesions not only at ectopic autologous locations, but also on the semi-allogenous fetal membranes, a particularly immunotolerant environment., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Placental inflammatory response (PIR) is associated with adverse neonatal outcomes such as sepsis, cerebral palsy, low birth weight, preterm birth, and neonatal mortality. However, there is an urgent need for noninvasive and sensitive biomarkers for prediction of PIR. In this study, we evaluated the clinical usefulness of maternal serum inflammatory markers for prediction of PIR in women with impending preterm birth. We conducted a retrospective cohort study of 483 patients who delivered preterm neonates. Serum levels of leukocyte differential counts, C-reactive protein (CRP), and neutrophil to lymphocyte ratio (NLR) were compared between women with no placental inflammation and women with PIR. The mean neutrophil counts, CRP levels, and NLR in both the patients with histologic chorioamnionitis (HCA) alone and those with HCA with funisitis were significantly higher than those in women with no placental inflammation. Compared to leukocyte subset or CRP, NLR in women with funisitis was significantly higher than in women with HCA alone and showed higher predictive accuracy, along with 71.4% sensitivity, 77.9% specificity, 80.7% positive predictive value, and 67.8% negative predictive value for prediction of PIR. On Kaplan-Meier survival analysis, women with both an elevated level of CRP and a high NLR had a shorter admission-to-delivery interval compared to women with either an elevated level of CRP or a high NLR alone. NLR may be a predictive marker of PIR and could be used as a cost-effective parameter for identifying women at risk of PIR.

Preeclampsia (PE) is the major pregnancy-induced hypertensive disorder responsible for maternal and fetal morbidity and mortality that can be associated with intrauterine growth restriction (IUGR). PE and IUGR are thought to be due to a placental defect, occurring early during pregnancy. Several placental microRNAs (miRNAs) have been shown to be deregulated in the context of placental diseases and could thus play a role in the pathophysiology of PE. Here, we show that pri-miR-34a is overexpressed in preeclamptic placentas and that its placental expression is much higher during the first trimester of pregnancy than at term, suggesting a possible developmental role. We explored pri-miR-34a regulation and showed that P53, a known activator of miR-34a, is reduced in all pathological placentas and that hypoxia can induce pri-miR-34a expression in JEG-3 cells. We also studied the methylation status of the miR-34a promoter and revealed hypomethylation in all preeclamptic placentas (associated or not with IUGR), whereas hypoxia induced a hypermethylation in JEG-3 cells at 72 h. Despite the overexpression of pri-miR-34a in preeclampsia, there was a striking decrease of the mature miR-34a in this condition, suggesting preeclampsia-driven alteration of pri-miR-34a maturation. SERPINA3, a protease inhibitor involved in placental diseases, is elevated in IUGR and PE. We show here that miR-34a overexpression in JEG-3 downregulates SERPINA3. The low level of mature miR-34a could thus be an important mechanism contributing to SERPINA3 upregulation in placental diseases. Overall, our results support a role for miR-34a in the pathophysiology of preeclampsia, through deregulation of the pri-miRNA expression and its altered maturation.

Background: The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine γ-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension., Methods and Results: Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8-12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production., Conclusions: These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia.

Placental malaria (PM) can lead to poor neonatal outcomes, including low birthweight due to fetal growth restriction (FGR), especially when associated with local inflammation (intervillositis or IV). The pathogenesis of PM-associated FGR is largely unknown, but in idiopathic FGR, impaired transplacental amino acid transport, especially through the system A group of amino acid transporters, has been implicated. We hypothesized that PM-associated FGR could result from impairment of transplacental amino acid transport triggered by IV. In a cohort of Malawian women and their infants, the expression and activity of system A (measured by Na⁺-dependent ¹⁴C-MeAIB uptake) were reduced in PM, especially when associated with IV, compared to uninfected placentas. In an in vitro model of PM with IV, placental cells exposed to monocyte/infected erythrocytes conditioned medium showed decreased system A activity. Amino acid concentrations analyzed by reversed phase ultra performance liquid chromatography in paired maternal and cord plasmas revealed specific alterations of amino acid transport by PM, especially with IV. Overall, our data suggest that the fetoplacental unit responds to PM by altering its placental amino acid transport to maintain adequate fetal growth. However, IV more profoundly compromises placental amino acid transport function, leading to FGR. Our study offers the first pathogenetic explanation for FGR in PM.

Background: Ischemia in the placenta is considered the base of the pathogenesis of preeclampsia, a pregnancy-specific syndrome in which soluble endoglin (sEng) is a prognostic marker and plays a pathogenic role. Here, we investigated the effects of hypoxia and the downstream pathways in the release of sEng., Methods and Results: Under hypoxic conditions, the trophoblast-like cell line JAR showed an increase in sEng parallel to an elevated formation of reactive oxygen species. Because reactive oxygen species are related to the formation of oxysterols, we assessed the effect of 22-(R)-hydroxycholesterol, a natural ligand of the liver X receptor (LXR), and the LXR synthetic agonist T0901317. Treatment of JAR cells or human placental explants with 22-(R)-hydroxycholesterol or T0901317 resulted in a clear increase in sEng that was dependent on LXR. These LXR agonists induced an increased matrix metalloproteinase-14 expression and activity and a significant reduction of its endogenous inhibitor, tissue inhibitor of metalloproteinase-3. In addition, mice treated with LXR agonists underwent an increase in the plasma sEng levels, concomitant with an increase in arterial pressure. Moreover, transgenic mice overexpressing sEng displayed high blood pressure. Finally, administration of an endoglin peptide containing the consensus matrix metalloproteinase-14 cleavage site G-L prevented the oxysterol-dependent increase in arterial pressure and sEng levels in mice., Conclusions: These studies provide a clue to the involvement of the LXR pathway in sEng release and its pathogenic role in vascular disorders such as preeclampsia.

SERPINA3 (Serpin peptidase inhibitor clade A member 3), also known as a1-antichymotrypsin, is a serine protease inhibitor involved in a wide range of biological processes. Recently, it has been shown to be up-regulated in human placental diseases in association with a hypomethylation of the 5' region of the gene. In the present study, we show that the promoter of SERPINA3 is transcriptionally activated by three transcription factors (TFs) (SP1, MZF1 and ZBTB7B), the level of induction being dependent on the rs1884082 single nucleotide polymorphism (SNP) located inside the promoter, the T allele being consistently induced to a higher level than the G, with or without added TFs. When the promoter was methylated, the response to ZBTB7B was allele specific (the G allele was strongly induced, while the T allele was strongly down-regulated). We propose an adaptive model to explain the interest of such a regulation for placental function and homeostasis. Overexpression of SERPINA3 in JEG-3 cells, a trophoblast cell model, decreased cell adhesion to the extracellular matrix and to neighboring cells, but protects them from apoptosis, suggesting a way by which this factor could be deleterious at high doses. In addition, we show in different human populations that the T allele appears to predispose to Intra Uterine Growth Restriction (IUGR), while a G allele at a second SNP located in the second exon (rs4634) increases the risk of preeclampsia. Our results provide mechanistic views inside the involvement of SERPINA3 in placental diseases, through its regulation by a combination of epigenetic, genetic and TF-mediated regulations.