The Design, Structure-Activity, and kinetic studies of 3-Benzyl-5-oxa-1,2,3,4-Tetrahydro-2H-chromeno-(3,4-c)pyridin-8-yl sulfamates as Steroid sulfatase inhibitors.

Steroid sulfatase inhibitors block the local production of estrogenic steroids and are attractive agents for the treatment of estrogen-dependent cancers. Inspiration of coumarin-based inhibitors, we synthesized thirty-two 5-oxa-1,2,3,4-tetrahydro-2H-chromeno-(3,4-c)pyridin-8-yl sulfamates, focusing on the substitution derivatives on the adjacent phenyl ring and evaluated their abilities to block STS from human placenta and MCF-7 cells. SAR analysis revealed that the incorporation of chlorine at either meta and/or para position of the adjacent phenyl ring of the tricyclic skeleton enhanced STS inhibition. Di-substitutions at the adjacent phenyl ring were superior to mono and tri-substitutions. Further kinetic analysis of these compounds revealed that chloride-bearing compounds, such as 19m, 19v, and 19w, had KI of 0.02 to 0.11 nM and kinact/KI ratios of 8.8-17.5 nM-1min-1, a parameter indicated for the efficiency of irreversible inhibition. We also used the docking model to illustrate the difference in STS inhibitory potency of compounds. Finally, the safety and anti-cancer activity of selected compounds 19m, 19v, and 19w were also studied, showing the results of low cytotoxicity on NHDF cell line and being more potent than irosustat on ZR-75-1 cell, which was a hormone-dependent cancer cell line with high STS expression.

Tryptophan degradation enzymes and Angiotensin (1-7) expression in human placenta.

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are key enzymes for tryptophan degradation, regulating immune tolerance during pregnancy. The intrauterine renin-angiotensin system is also involved in the progression of a healthy pregnancy. Angiotensin(1-7) maintains the integrity of fetal membranes via counteracting the pro-inflammatory actions of Angiotensin II. No data are available on placental Angiotensin(1-7) co-expression with TDO. We aimed to characterize TDO mRNA expression and its localization in different areas of the placenta of physiological pregnancies delivered at term; its co-expression with Angiotensin(1-7) and its correlation with the plasma kynurenine/tryptophan (Kyn/Trp) ratio was investigated. This prospective observational study included a nonconsecutive series of 20 singleton uncomplicated pregnancies delivered vaginally. TDO mRNA was expressed in both maternal and fetal sides of the placentas and TDO protein also in the villi and it was co-expressed with IDO1 in almost half of the placental cells at these sites. The percentage of TDO+ and IDO1+ cells appeared to be influenced by maternal pre-gestational smoking and newborn weight. A strong correlation was found between the percentage of TDO+ and IDO1+ cells in the villi. TDO+ cells also expressed Angiotensin(1-7), with a higher percentage on the fetal side and in the villi compared to the maternal one. Kyn/Trp plasma ratio was not correlated with IDO and TDO expression nor with the patient's characteristics. Collectively, our data indicate that TDO is detectable in placental tissue and is co-expressed with IDO and with Angiotensin(1-7)+ on the fetal side and in the villi.

MAP3K4 promotes fetal and placental growth by controlling the receptor tyrosine kinases IGF1R/IR and Akt signaling pathway.

Disruption of fetal growth results in severe consequences to human health, including increased fetal and neonatal morbidity and mortality, as well as potential lifelong health problems. Molecular mechanisms promoting fetal growth represent potential therapeutic strategies to treat and/or prevent fetal growth restriction (FGR). Here, we identify a previously unknown role for the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) in promoting fetal and placental growth. We demonstrate that inactivation of MAP3K4 kinase activity causes FGR due in part to placental insufficiency. Significantly, MAP3K4 kinase-inactive mice display highly penetrant lethality prior to weaning and persistent growth reduction of surviving adults. Additionally, we elucidate molecular mechanisms by which MAP3K4 promotes growth through control of the insulin-like growth factor 1 receptor (IGF1R), insulin receptor (IR), and Akt signaling pathway. Specifically, MAP3K4 kinase inactivation in trophoblast stem (TS) cells results in reduced IGF1R and IR expression and decreased Akt activation. We observe these changes in TS cells also occur in differentiated trophoblasts created through in vitro differentiation of cultured TS cells and in vivo in placental tissues formed by TS cells. Furthermore, we show that MAP3K4 controls this pathway by promoting Igf1r transcript expression in TS cells through activation of CREB-binding protein (CBP). In the MAP3K4 kinase-inactive TS cells, Igf1r transcripts are repressed because of reduced CBP activity and increased histone deacetylase 6 expression and activity. Together, these data demonstrate a critical role for MAP3K4 in promoting fetal and placental growth by controlling the activity of the IGF1R/IR and Akt signaling pathway.

S-Nitrosoglutathione Reductase Deficiency Causes Aberrant Placental S-Nitrosylation and Preeclampsia.

Background Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S-nitrosylated proteins and reactive oxygen species, suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress. Methods and Results Here, we show that mice lacking S-nitrosoglutathione reductase (GSNOR-/-), a denitrosylase regulating protein S-nitrosylation, exhibit a preeclampsia phenotype, including hypertension, proteinuria, renal pathology, cardiac concentric hypertrophy, decreased placental vascularization, and fetal growth retardation. Reactive oxygen species, NO, and peroxynitrite levels are elevated. Importantly, mass spectrometry reveals elevated placental S-nitrosylated amino acid residues in GSNOR-/- mice. Ascorbate reverses the phenotype except for fetal weight, reduces the difference in the S-nitrosoproteome, and identifies a unique set of S-nitrosylated proteins in GSNOR-/- mice. Importantly, human preeclamptic placentas exhibit decreased GSNOR activity and increased nitrosative stress. Conclusions Therefore, deficiency of GSNOR creates dysregulation of placental S-nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for preeclampsia.

Maternal 11-Ketoandrostenedione Rises Through Normal Pregnancy and Is the Dominant 11-Oxygenated Androgen in Cord Blood.

CONTEXT: Adrenal-derived 11-oxygenated androgens (11oAs) are known important contributors to human physiology and disease but have not been studied in pregnancy. OBJECTIVE: We characterize 11oAs in normal human pregnancy and neonatal period and assess the ratios between 11oAs and compare with ratios of other steroids that undergo placental metabolism. DESIGN: Prospective cohort study, 2010-2018. SETTING: Academic institution. PATIENTS: Pairs of pregnant women and newborns (n = 120) were studied. Inclusion criteria were maternal age between 18 and 42 years old, spontaneous singleton pregnancies, and intention to deliver at University of Michigan. INTERVENTION: Maternal venous blood was collected during first trimester and at term. Neonatal cord blood was collected following delivery. Steroids were measured via liquid chromatography-tandem mass spectrometry. MAIN OUTCOME MEASURES: Levels of 11ß-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), 11ß-hydroxytestosterone, and 11-ketotestoterone (11KT) in maternal first trimester, maternal term, and neonatal cord blood were compared. 11OHA4-to-11KA4 ratios were correlated with cortisol-to-cortisone ratios. RESULTS: Dominant 11oAs in pregnancy and the cord blood are 11OHA4 and 11KA4, compared to 11OHA4 and 11KT in adult men and nonpregnant women. We found a rise in 11oA concentrations, particularly 11KA4, from first to third trimester. In cord blood, the concentration of 11KA4 exceeded those of both 11OHA4 and 11KT, reflecting placental 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) and 17ß-hydroxysteroid dehydrogenase (17ßHSD2) activities, respectively. 11OHA4-to-11KA4 ratios are concordant with cortisol-to-cortisone ratios across all maternal and fetal compartments, reflecting placental 11ßHSD2 activity. CONCLUSIONS: Placental 17ßHSD2 activity defends the fetus against the androgen 11KT. Our normative values may be used in future studies of 11oAs in complicated pregnancies.

Characterization of the temporal, cell-specific and interferon-inducible patterns of indoleamine 2,3 dioxygenase 1 (IDO1) expression in the human placenta across gestation.

INTRODUCTION: The human placenta performs multiple functions necessary for successful pregnancy, but the metabolic pathways and molecular mechanisms responsible for regulating placental development and functions remain incompletely understood. Catabolism of the essential amino acid tryptophan has numerous critical roles in normal physiology, including inflammation. The kynurenine pathway, which accounts for ∼90% of tryptophan breakdown, is mediated by indoleamine 2,3 dioxygenase 1 (IDO1) in the placenta. In pregnant mice, alterations of IDO1 activity or expression result in fetal resorption and a preeclampsia-like phenotype. Decreased IDO1 expression at the maternal-fetal interface has also been linked to preeclampsia, in utero growth restriction and recurrent miscarriage in humans. These collective observations suggest essential role(s) for IDO1 in maintaining healthy pregnancy. Despite these important roles, the precise temporal, cell-specific and inflammatory cytokine-mediated patterns of IDO1 expression in the human placenta have not been thoroughly characterized across gestation. METHODS: Western blot and whole mount immunofluorescence (WMIF) were utilized to characterize and quantify basal and interferon (IFN)-inducible IDO1 expression in 1st trimester (7-13 weeks), 2nd trimester (14-22 weeks) and term (39-41 weeks) placental villi. RESULTS: IDO1 expression is activated in the human placenta between the 13th and 14th weeks of pregnancy, increases through the 2nd trimester and remains elevated at term. Constitutive IDO1 expression is restricted to placental endothelial cells. Interestingly, different types of IFNs have distinct effects on IDO1 expression in the human placenta. DISCUSSION: Our collective results are consistent with potential role(s) for IDO1 in the regulation of vascular functions in placental villi.

TNFα blockade reverses vascular and uteroplacental matrix metalloproteinases imbalance and collagen accumulation in hypertensive pregnant rats.

Preeclampsia is a pregnancy-related disorder of maternal hypertension-in-pregnancy (HTN-Preg) and often fetal growth restriction (FGR). Placental ischemia could be an initiating event leading to inadequate vascular and uteroplacental remodeling and HTN-Preg; however, the molecular targets are unclear. To test the hypothesis that placental ischemia-induced release of proinflammatory cytokines target vascular and uteroplacental matrix metalloproteinases (MMPs), we tested if infusing TNFα (200 ng/kg/day) in day-14 pregnant (Preg) rats causes MMP imbalance and collagen accumulation, and if infusing TNFα decoy receptor Etanercept (0.4 mg/kg/day) in HTN-Preg rats with reduced uteroplacental perfusion pressure (RUPP) reverses MMP imbalance and collagen accumulation. On gestational day-19, blood pressure (BP) was higher in Preg + TNFα and RUPP vs Preg rats, and restored in RUPP + Etanercept rats. Gelatin zymography and Western blots revealed decreases in MMP-2 and MMP-9 and increases in MMP-1 and MMP-7 in aorta, uterus and placenta of Preg + TNFα and RUPP, that were reversed in RUPP + Etanercept rats. Collagen-I and IV were abundant in Preg + TNFα and RUPP, and were decreased in RUPP + Etanercept rats. The litter size, uterine, placenta, and pup weight were markedly reduced in RUPP, insignificantly reduced in Preg + TNFα, and slightly improved in RUPP + Etanercept rats. Thus TNFα blockade reverses the decreases in vascular and uteroplacental MMP-2 and MMP-9, and the increases in MMP-1, MMP-7 and accumulation of collagen-I and IV induced by placental ischemia and TNFα in HTN-Preg rats. Targeting TNFα using cytokine antagonists, or MMPs using MMP modulators could rectify MMP imbalance and collagen accumulation, restore vascular and uteroplacental remodeling, and improve BP in HTN-Preg and preeclampsia.

ACE2: a key modulator of the renin-angiotensin system and pregnancy.

Angiotensin-converting enzyme 2 (ACE2) is a membrane-bound protein containing 805 amino acids. ACE2 shows approximately 42% sequence similarity to somatic ACE but has different biochemical activities. The key role of ACE2 is to catalyze the vasoconstrictor peptide angiotensin (ANG) II to Ang-(1-7), thus regulating the two major counterbalancing pathways of the renin-angiotensin system (RAS). In this way, ACE2 plays a protective role in end-organ damage by protecting tissues from the proinflammatory actions of ANG II. The circulating RAS is activated in normal pregnancy and is essential for maintaining fluid and electrolyte homeostasis and blood pressure. Renin-angiotensin systems are also found in the conceptus. In this review, we summarize the current knowledge on the regulation and function of circulating and uteroplacental ACE2 in uncomplicated and complicated pregnancies, including those affected by preeclampsia and fetal growth restriction. Since ACE2 is the receptor for SARS-CoV-2, and COVID-19 in pregnancy is associated with more severe disease and increased risk of abnormal pregnancy outcomes, we also discuss the role of ACE2 in mediating some of these adverse consequences. We propose that dysregulation of ACE2 plays a critical role in the development of preeclampsia, fetal growth restriction, and COVID-19-associated pregnancy pathologies and suggest that human recombinant soluble ACE2 could be a novel therapeutic to treat and/or prevent these pregnancy complications.

Elevated placental histone H3K4 methylation via upregulated histone methyltransferases SETD1A and SMYD3 in preeclampsia and its possible involvement in hypoxia-induced pathophysiological process.

INTRODUCTION: Disturbance in placental epigenetic regulation contributes to the pathogenesis of preeclampsia (PE). Although aberrant placental DNA methylation status in PE has been thoroughly studied, the role of histone modifications, including histone methylation, in PE remains unclear. Moreover, no study has ever reported the association between PE and placental histone methylation status by focusing on histone methyltransferases. The present study aimed to investigate the possible involvement of placental epigenetic regulation by histone methylation via histone methyltransferases in the pathophysiology of PE. METHODS: Placental mRNA expression of histone methyltransferases was examined using quantitative RT-PCR. Protein expression of histone methyltransferases and histone methylation status in placentas and trophoblast cell lines were assessed by immunoblotting and immunohistochemistry. RESULTS: Expression profile of histone methyltransferases in the placentas using quantitative RT-PCR revealed that the mRNA expression levels of histone 3 lysine 4 (H3K4) methyltransferases, SETD1A and SMYD3, were significantly increased in placentas from PE patients. Immunoblotting and immunohistochemistry revealed that not only protein expression levels of SETD1A and SMYD3, but also H3K4 methylation status was increased in the trophoblasts from PE placentas. In vitro studies using HTR-8/SV-neo and BeWo cells showed that hypoxia induced the expression levels of SETD1A and SMYD3, and subsequently enhanced H3K4 methylation. Furthermore, the overexpression of SETD1A and SMYD3 in HTR-8/SV-neo cells enhanced H3K4 methylation in response to hypoxia. DISCUSSION: Our study results suggest that placental epigenetic alteration by enhanced histone H3K4 methylation through upregulated SETD1A and SMYD3 might play a role in the pathophysiological process of PE associated with hypoxia.

Viral model of maternal immune activation alters placental AMPK and mTORC1 signaling in rats.

INTRODUCTION: Maternal immune activation (MIA) is associated with neurodevelopmental disorders in offspring. We previously demonstrated that poly(I:C)-mediated MIA alters placental and fetal brain amino acid transporter expression in rats, which could potentially play a role in altered neurodevelopment; however, the mechanism(s) underlying these changes in amino acid transporter expression remain unknown. The objective of the current study was to investigate the mechanism(s) underlying poly(I:C)-mediated changes in the expression of the amino acid transporters in the placenta. METHODS: Pregnant rats received poly(I:C) on gestational day 14 and placentas were collected 6 h later. Mass spectrometry-based proteomics of placentas was performed followed by pathway enrichment analysis. Activation of mTORC1 and its upstream regulator, AMPK, was investigated using immunoblotting. Finally, the role of mTORC1 and AMPK in regulating the expression and localization of the amino acid transporters EAAT2 and ASCT1 was investigated in the human choriocarcinoma cell line JAR. RESULTS: The impact of poly(I:C) on the placental proteome was highly sexually dimorphic. While proteomics-based pathway enrichment analysis indicated enrichment of mTOR signaling in male placentas only, further investigation revealed inhibition of mTORC1 in both male and female placentas in addition to activation of AMPK. In vitro, activation of AMPK and inhibition of mTORC1 decreased membrane localization of EAAT2 and ASCT1. DISCUSSION: Poly(I:C)-mediated MIA activates AMPK and inhibits mTORC1 in rat placenta, both of which decrease expression and membrane localization of EAAT2 and ASCT1 in vitro. Thus, AMPK/mTORC1 signaling could be a novel treatment target for alleviating MIA-mediated changes in placental amino acid transport.

O-GlcNAcylation in Hyperglycemic Pregnancies: Impact on Placental Function.

The dynamic cycling of N-acetylglucosamine, termed as O-GlcNAcylation, is a post-translational modification of proteins and is involved in the regulation of fundamental cellular processes. It is controlled by two essential enzymes, O-GlcNAc transferase and O-GlcNAcase. O-GlcNAcylation serves as a modulator in placental tissue; furthermore, increased levels of protein O-GlcNAcylation have been observed in women with hyperglycemia during pregnancy, which may affect the short-and long-term development of offspring. In this review, we focus on the impact of O-GlcNAcylation on placental functions in hyperglycemia-associated pregnancies. We discuss the following topics: effect of O-GlcNAcylation on placental development and its association with hyperglycemia; maternal-fetal nutrition transport, particularly glucose transport, via the mammalian target of rapamycin and AMP-activated protein kinase pathways; and the two-sided regulatory effect of O-GlcNAcylation on inflammation. As O-GlcNAcylation in the placental tissues of pregnant women with hyperglycemia influences near- and long-term development of offspring, research in this field has significant therapeutic relevance.

Connecting the dots on vertical transmission of SARS-CoV-2 using protein-protein interaction network analysis - Potential roles of placental ACE2 and ENDOU.

We conducted a protein-protein interaction (PPI) network study searching for proteins relevant to pregnancy-associated COVID-19 in pregnancy complicated with severe preeclampsia (sPE) and intra-amniotic infection and/or inflammation (Triple-I). PPI networks from sPE and Triple-I were intersected with the PPI network from coronavirus infection. Common proteins included the SARS-CoV-2 entry receptor ACE2 and ENDOU, a placental endoribonuclease homologous to Nsp15, a protein produced by the virus to escape host immunity. Remarkably, placental ENDOU mRNA expression far exceeded that of ACE2. Immunohistochemistry confirmed ENDOU localization at the hemochorial maternal-fetal interface. Investigation of ENDOU's relevance to vertical transmission of SARS-CoV-2 is further warranted.

A heretical view: rather than a solely placental protective function, placental 11ß hydroxysteroid dehydrogenase 2 also provides substrate for fetal peripheral cortisol synthesis in obese pregnant ewes.

Exposure to glucocorticoid levels higher than appropriate for current developmental stages induces offspring metabolic dysfunction. Overfed/obese (OB) ewes and their fetuses display elevated blood cortisol, while fetal Adrenocorticotropic hormone (ACTH) remains unchanged. We hypothesized that OB pregnancies would show increased placental 11ß hydroxysteroid dehydrogenase 2 (11ß-HSD2) that converts maternal cortisol to fetal cortisone as it crosses the placenta and increased 11ß-HSD system components responsible for peripheral tissue cortisol production, providing a mechanism for ACTH-independent increase in circulating fetal cortisol. Control ewes ate 100% National Research Council recommendations (CON) and OB ewes ate 150% CON diet from 60 days before conception until necropsy at day 135 gestation. At necropsy, maternal jugular and umbilical venous blood, fetal liver, perirenal fat, and cotyledonary tissues were harvested. Maternal plasma cortisol and fetal cortisol and cortisone were measured. Fetal liver, perirenal fat, cotyledonary 11ß-HSD1, hexose-6-phosphate dehydrogenase (H6PD), and 11ß-HSD2 protein abundance were determined by Western blot. Maternal plasma cortisol, fetal plasma cortisol, and cortisone were higher in OB vs. CON (p < 0.01). 11ß-HSD2 protein was greater (p < 0.05) in OB cotyledonary tissue than CON. 11ß-HSD1 abundance increased (p < 0.05) in OB vs. CON fetal liver and perirenal fat. Fetal H6PD, an 11ß-HSD1 cofactor, also increased (p < 0.05) in OB vs. CON perirenal fat and tended to be elevated in OB liver (p < 0.10). Our data provide evidence for increased 11ß-HSD system components responsible for peripheral tissue cortisol production in fetal liver and adipose tissue, thereby providing a mechanism for an ACTH-independent increase in circulating fetal cortisol in OB fetuses.

Catechol-O-methyltransferase and Pregnancy Outcome: an Appraisal in Rat.

Catechol-O-methyltransferase (COMT) has been shown to be a key regulator of pregnancy outcomes in mouse, and its deficiency is causative in the development of a preeclampsia-like disease process. Preeclampsia is a human pregnancy disorder associated with failure of intrauterine trophoblast cell invasion and trophoblast-guided uterine spiral artery remodeling, which are not well-developed in mouse. The purpose of this study was to investigate COMT in rat, a species with deep intrauterine trophoblast invasion. To accomplish this task, we used clustered regularly interspaced short palindromic repeats/Cas9-mediated genome editing of the rat Comt gene. A Comt null rat model was established and its fertility characterized. Comt null male and female rats were viable and fertile. COMT deficiency did not significantly impact pregnancy outcomes, including litter size, placental and fetal weights, Mendelian and sex ratios, or pregnancy-dependent adaptations to hypoxia. Collectively, our findings indicate that pregnancy-associated phenotypic outcomes of COMT deficiency are not equivalent in mouse and rat. In rat, COMT is not required for a successful pregnancy outcome.

Effect of the IDO Gene on Pregnancy in Mice with Recurrent Pregnancy Loss.

The aim of this study is to investigate the effect of the IDO (indoleamine 2,3-dioxygenase) gene on pregnancy outcome in mice with recurrent pregnancy loss (RPL) and its mechanism of action in the maternal-fetal interface. An RPL model was established via natural mating of female CBA/J mice with male DBA/2 mice; thereafter, the female mice were randomly divided into groups treated with LV-EGFP (enhanced green fluorescent protein)-IDO (lentivirus vector carrying IDO-EGFP gene), LV-EGFP (negative control lentivirus vector), or phosphate-buffered saline (control). The mice were sacrificed at 13.5 days of pregnancy, and the embryo absorption rate was determined. Peripheral blood regulatory T cells (Tregs) from the pregnant mice were detected using flow cytometry. Placental and decidual tissue IDO expression was detected using immunofluorescence and Western blotting. Inflammatory cell infiltration of the placental and decidual tissue was observed using hematoxylin-eosin (HE) staining. The LV-EGFP-IDO group had a significantly lower embryo absorption rate than the LV-EGFP and control groups (P = 0.0006 and P = 0.0049, respectively) and significantly more Tregs than the LV-EGFP and control groups (P = 0.0151 and P = 0.0392, respectively). Placental and decidual IDO protein levels correlated positively with peripheral blood Treg expression levels. The LV-EGFP-IDO group had significantly higher placental and decidual IDO protein levels than the LV-EGFP and control groups (P < 0.005), and it had significantly less inflammatory cell infiltration than the LV-EGFP and control groups. The IDO gene may reduce the embryo absorption rate in an RPL mouse model, possibly improving pregnancy outcome by upregulating Tregs and reducing the inflammatory response.

Influence of labor on direct and indirect determinants of placental 11beta-hydroxysteroid dehydrogenase activity.

PURPOSE: Labor is a complex process involving multiple para-, auto- and endocrine cascades. The interaction of cortisol, corticotropin-releasing hormone (CRH) and progesterone is essential. The action of cortisol on the human feto-placental unit is regulated by 11beta-hydroxysteroid dehydrogenase type 2 (11ß-HSD2/HSD11B2) that converts cortisol into inactive cortisone. The majority of studies on the assessment of placental 11ß-HSD2 function determined indirect activity parameters. It remains elusive if indirect measurements correlate with enzymatic function and if these parameters are affected by potential confounders (e.g., mode of delivery). Thus, we compared determinants of indirect 11ß-HSD2 tissue activity with its direct enzymatic turnover rate in placental samples from spontaneous births and cesarean (C)-sections. METHODS: Using LC-MS/MS, we determined CRH, cortisol, cortisone, progesterone and 17-hydroxy(OH)-progesterone in human term placentas (spontaneous birth vs. C-section, n = 5 each) and measured the enzymatic glucocorticoid conversion rates in placental microsomes. Expression of HSD11B1, 2 and CRH was determined via qRT-PCR in the same samples. RESULTS: Cortisol-cortisone ratio correlated with direct microsomal enzymatic turnover. While this observation seemed independent of sampling site, a strong influence of mode of delivery on tissue steroids was observed. The mRNA expression of HSD11B2 correlated with indirect and direct cortisol turnover rates in C-section placentas only. In contrast to C-sections, CRH, cortisol and cortisone levels were significantly increased in placental samples following spontaneous birth. CONCLUSION: Labor involves a series of complex hormonal processes including activation of placental CRH and glucocorticoid metabolism. This has to be taken into account when selecting human cohorts for comparative analysis of placental steroids.

Single-cell RNA expression profiling of SARS-CoV-2-related ACE2 and TMPRSS2 in human trophectoderm and placenta.

OBJECTIVES: To examine the characteristics and distribution of possible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target cells in the human trophectoderm (TE) and placenta. METHODS: Bioinformatics analysis was performed based on published single-cell transcriptomic datasets of early TE and first- and second-trimester human placentae. We conducted the transcriptomic analysis of 4198 early TE cells, 1260 first-trimester placental cells and 189 extravillous trophoblast cells (EVTs) from 24-week placentae (EVT_24W) using the SMART-Seq2 method. In addition, to confirm the bioinformatic results, we performed immunohistochemical staining of three first-trimester, three second-trimester and three third-trimester placentae from nine women recruited prospectively to this study. We evaluated the expression of the SARS-CoV-2-related molecules angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). RESULTS: Via bioinformatic analysis, we identified the existence of ACE2 and TMPRSS2 expression in human TE as well as in first- and second-trimester placentae. In the human TE, 54.4% of TE1 cells, 9.0% of cytotrophoblasts (CTBs), 3.2% of EVTs and 29.5% of syncytiotrophoblasts (STBs) were ACE2-positive. In addition, 90.7% of TE1 cells, 31.5% of CTBs, 22.1% of EVTs and 70.8% of STBs were TMPRSS2-positive. In placental cells, 20.4% of CTBs, 44.1% of STBs, 3.4% of EVTs from 8-week placentae (EVT_8W) and 63% of EVT_24W were ACE2-positive, while 1.6% of CTBs, 26.5% of STBs, 1.9% of EVT_8W and 20.1% of EVT_24W were TMPRSS2-positive. Pathway analysis revealed that EVT_24W cells that were positive for both ACE2 and TMPRSS2 (ACE2 + TMPRSS2-positive) were associated with morphogenesis of branching structure, extracellular matrix interaction, oxygen binding and antioxidant activity. The ACE2 + TMPRSS2-positive TE1 cells were correlated with an increased capacity for viral invasion, epithelial-cell proliferation and cell adhesion. Expression of ACE2 and TMPRSS2 was observed on immunohistochemical staining in first-, second- and third-trimester placentae. CONCLUSIONS: ACE2- and TMPRSS2-positive cells are present in the human TE and placenta in all three trimesters of pregnancy, which indicates the possibility that SARS-CoV-2 could spread via the placenta and cause intrauterine fetal infection. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Inflammasome components and ADAMTS4 in premature rupture of membranes.

Inflammation may be responsible for the development of premature rupture of membranes (PROM) including preterm PROM (PPROM) and mature PROM (MPROM). A total of four classic receptor proteins have been confirmed to assemble inflammasomes: NLR family pyrin domain containing (NLRP)1, NLRP3 and NLR family CARD­domain containing 4 (NLRC4) and absent in melanoma 2 (AIM2). The activation and expression of these receptor­modulated inflammasomes in placenta and fetal membrane of PROM pregnancies requires investigation. In addition, a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a risk factor for PROM, but whether its expression is associated with inflammasome activation remains to be elucidated. In the present study, the placenta and fetal membrane tissues of patients who had suffered PPROM and MPROM and healthy pregnancies were investigated. Reverse transcription­quantitative PCR was used to determine the mRNA expression of inflammasomes and ADAMTS4. Western blotting, immunohistochemistry and ELISA were used to investigate the protein expression levels of inflammasomes and ADAMTS4. The results demonstrated that all four inflammasomes were elevated in placenta and fetal membrane of PPROMs as were mRNA and protein expression levels of IL­18 and IL­1ß (compared with controls). A further increase of inflammasomes and interleukins was observed in MPROMs compared with controls. Similar results were also observed in ADAMTS4 expression in PPROM and MPROM groups. However, immunohistochemistry results revealed no significant difference of inflammasome receptor expression in PPROMs compared with controls. Finally, a general positive correlation between ADAMTS4 and all four inflammasome receptors in placenta and fetal membrane of PPROMs and MPROMs was observed. The present study revealed that NLRP1, NLRP3, AIM2 and NLRC4 inflammasome activation in PROM was increased. Promoted ADAMTS4 level was further observed in PROM group and was significantly correlated with inflammasome expression. Inhibition of inflammasome activation may provide a therapeutic target for clinical PROM treatment.

Protein expression of angiotensin-converting enzyme 2, a SARS-CoV-2-specific receptor, in fetal and placental tissues throughout gestation: new insight for perinatal counseling.

OBJECTIVE: Pregnant women can be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), yet the incidence of perinatal infection is low. We hypothesized that this could be related to low expression of the membrane receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), in the fetoplacental unit. We evaluated protein expression of ACE2 at various gestational ages in both placentae and fetal organs from pregnancies not infected with SARS-CoV-2. METHODS: In May 2020, using samples from a registered biobank, we performed immunohistochemical analysis for ACE2 in tissue samples from fetal organs and placentae from five cases of second- or third-trimester medical termination of pregnancy in healthy women (performed between 15 and 38 weeks' gestation), as well as a further two placentae, one from a 7-week spontaneous miscarriage in a non-infected woman and one from a symptomatic pregnant woman positive for SARS-CoV-2 delivered by Cesarean section at 34 weeks. Samples were paraffin-embedded and organ tissues included kidney, brain, lung, intestinal tract, heart and testis. Matching tissues (kidney, intestinal tract, lung and testis) from autopsies of four 8-year-old children were tested as controls. Tissue sections were incubated with rabbit monoclonal anti-ACE2, and protein expression of ACE2 was detected by immunohistochemistry. RESULTS: ACE2 expression was detected in fetal kidney, rectum and ileum samples from 15 weeks onwards and in the pediatric controls. It was barely detectable in fetal lung samples at 15 + 5 weeks' gestation and not detectable thereafter, and, in the pediatric controls, ACE2 was detectable only in type-2 pneumocytes. No ACE2 expression was found in the cerebral ependymal or parenchymal tissues or in cardiac tissues. ACE2 was expressed in placental syncytiotrophoblast and cytotrophoblast samples, but not in the amnion, from 7 weeks onwards. The intensity and distribution of ACE2 staining in the placenta from the symptomatic SARS-CoV-2 woman was similar to that in the non-infected placentae. CONCLUSIONS: Marked placental expression of ACE2 provides a rationale for vertical transmission at the cellular level. Absence of ACE2 expression in the fetal brain and heart is reassuring regarding the risk of congenital malformation. Clinical follow-up of infected pregnant women and their children is needed to validate these observations. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

TAM and TIM receptors mRNA expression in Zika virus infected placentas.

To investigate the role of TYRO3, AXL and TIM1 receptors in the Zika virus (ZIKV) cycle, we determined their mRNA expression in different placental sites of ZIKV infected tissue during pregnancy. Unexpectedly, the ZIKV infection was not related with mRNA upregulation of these receptors or changes in expression of type I and III interferons in different placental sites. Instead, a decrease of TYRO3 mRNA expression was observed in positive sites of ZIKV positive placentas in comparison to negative sites. The basis of this downregulation can help to understand how ZIKV persists in placental tissue during pregnancy.