Villous Placental Tissue Research Articles

Acute Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Pregnancy Is Associated with Placental Angiotensin-Converting Enzyme 2 Shedding

While the human placenta may be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the rate of fetal transmission is low, suggesting a barrier at the maternal-fetal interface. Angiotensin-converting enzyme (ACE)2, the main receptor for SARS-CoV-2, is regulated by a metalloprotease cleavage enzyme, a disintegrin and metalloprotease domain 17 (ADAM17). ACE2 is expressed in the human placenta, but its regulation in relation to maternal SARS-CoV-2 infection in pregnancy is not well understood. This study evaluated ACE2 expression, ADAM17 activity, and serum ACE2 abundance in a cohort of matched villous placental and maternal serum samples from control pregnancies (SARS-CoV-2 negative, n = 8) and pregnancies affected by symptomatic maternal SARS-CoV-2 infections in the second trimester [2nd Tri coronavirus disease (COVID), n = 8] and third trimester (3rd Tri COVID, n = 8). In 3rd Tri COVID compared with control and 2nd Tri COVID villous placental tissues, ACE2 mRNA expression was remarkably elevated; however, ACE2 protein expression was significantly decreased with a parallel increase in ADAM17 activity. Soluble ACE2 was also significantly increased in the maternal serum from 3rd Tri COVID infections compared with control and 2nd Tri COVID pregnancies. These data suggest that in acute maternal SARS-CoV-2 infections, decreased placental ACE2 protein may be the result of ACE2 shedding and highlights the importance of ACE2 for studies on SARS-CoV-2 responses at the maternal-fetal interface.

Maternal Obesity and Diet Quality Modulate the Villous Placental Metabolome

Abstract Objectives Maternal diet and body composition are important determinants of offspring obesity risk, which may be mediated via changes in placental function. Here we examined the influence of maternal obesity and diet quality on the placental metabolome using untargeted metabolomics. Methods Placenta were collected from a longitudinal observational study in which maternal anthropometrics, body composition, dietary intake (3 d food records) and other variables were collected every trimester. Untargeted metabolomics of villous placental tissues from normal weight (NW, n = 72), overweight (OW, n = 49), and obese women (OB, n = 29) based on BMI at first trimester, were performed using GC-TOF MS. Differential metabolite features were identified using univariate comparisons (ANOVA/Mann Whitney U tests) and partial least squares discriminant analysis (PLS-DA). Results Principal component analysis did not reveal global shifts in the metabolome with maternal OW or OB. While no differences survived multiple testing corrections (P < 0.05), univariate analysis indicated that OW/OB subjects showed increased abundance of 2-deoxypentitol and decreases in maltotriose and 3-hydroxybutanoic acid relative to NW subjects (un-adjusted P < 0.05). Alterations in a number of non-annotated metabolites were also observed between NW, OW and OB groups. PLS-DA models predicted maternal OB status with ∼80% accuracy in held out samples. Predictive metabolites were enriched for aspartate metabolism and ammonia recycling. The global placental metabolome was significantly associated with maternal diet quality (Healthy Eating Index 2010 score averaged over pregnancy; PC1 ANOVA P < 0.01) which was characterized by differences in 138 metabolites (ANOVA, un-adjusted P < 0.05). Abundance of heptadecanoic acid and glycerol-3-galactoside increased with HEI score. Conclusions Maternal obesity appears to alter amino acid or protein metabolism, whereas diet quality seemed to affect metabolism of fatty acids and BCAA. These results suggest a potential shift in nutrient delivery to the offspring. Funding Sources Supported by USDA-ARS Project 6026–51,000-010–05S.

Maternal BMI Modulates Placental Expression of Lipid Transport, Metabolism and Innate Immune Response Genes at Term (OR35-01-19)

ObjectivesMaternal body composition during pregnancy is an important determinant of offspring obesity risk. One hypothesized mechanism linking maternal and offspring obesity is via alterations in placental gene expression and function. Here we examined the influence of early pregnancy maternal obesity on placental gene expression at term using transcriptomic approaches. MethodsWe examined placenta collected from a longitudinal observational study of mothers and infants where maternal anthropometrics, body composition, dietary intake (3 d food records) and other variables were collected every trimester in the mothers. Placentas were collected within 30 min of delivery and archived for molecular and histologic analysis. For the current analyses, transcriptomic analysis of villous placental tissues from normal weight (NW, N = 71), and overweight/obese women (OW, N = 70, based on BMI < 10 wk of pregnancy) were performed using strand-specific mRNA-seq. Data analyses were carried out using the STAR-Seqmonk-Limma pipeline. ResultsRNA-seq analysis provided quantitative information on expression of 13,831 transcripts (> 2 counts/million in 50% of samples). Assessment via principal component analysis identified significant effects of maternal BMI (in male infants), infant sex and mode of delivery on placental gene expression. Linear regression of mRNA expression with early pregnancy maternal BMI (adjusting for mode of delivery and sex) identified significant association with 230 transcripts (P < 0.05). Likewise, regression analysis of maternal body fat identified significant associations with 271 genes, with 99 genes shared in common between BMI and body fat. These transcripts were enriched for cell morphogenesis, fatty acid biosynthetic and lipid transport related pathways. Of note, expression of ABCG1 (cholesterol transporter), MMP1, CEBPα and PRKAG2 (AMPKγ2) were negatively associated with maternal BMI (P < 0.05). mRNA expression of MHC Class II antigens DRB1 and DRB5, critical in antigen presentation and immune response was increased (> 1.8-fold) in male placenta of OW women. ConclusionsIn a large cohort of well-characterized pregnant women maternal adiposity status in early pregnancy influences term placental expression of genes involved in lipid transport, metabolism and innate immune response in a sexually dimorphic manner. Funding SourcesSupported by USDA-ARS Project 6026-51000-010-05S.

Cytogenetic Analysis of Spontaneous Miscarriages Using Long-Term Culturing of Chorionic Villi

Spontaneous miscarriages are strongly associated with fetal chromosomal abnormalities. We sought to validate the occurrence of chromosomal abnormalities in Japanese patients with early spontaneous miscarriages using a conventional long-term culture method. One hundred and thirty-two patients were studied after excluding 7 cases with culture failure. Chromosomal analysis of 132 cases was performed using a long-term culture method of the villous placental tissue. Patients’ age, number of previous spontaneous abortions, fetal data from ultrasonography, and the method of conception (ART or spontaneous pregnancy) were obtained from the electronic medical records. The association between the patient’s background and the cytogenetic data was analyzed using a Chi squared test. The average maternal age of the 132 cases of early spontaneous abortion was 34.2 ± 4.7 year (range: 24–43). Chromosomal abnormalities were detected in 89 cases (67.4%), among which 52.8% represented trisomy, 1.1% represented double trisomy, 10.1% represented polyploidy, 7.9% represented monosomy, 12.4% represented structural chromosomal abnormality, and 15.7% represented mosaic chromosome abnormalities. Women older than 35 year had a higher rate of chromosomal abnormalities than younger women (76.6% vs. 58.8%; P = 0.015). Twenty-five of the 132 pregnancies were achieved with ART and were associated with a higher rate of trisomy (64.0% vs. 37.4%; P = 0.008). G-banding identified a rare form of polyploidy 94,XXYYYY. Our results confirmed previous findings that older Japanese women with spontaneous miscarriages have a higher rate of chromosomal abnormalities.

Preeclampsia Up-Regulates Angiogenesis-Associated MicroRNA (i.e., miR-17, -20a, and -20b) That Target Ephrin-B2 and EPHB4 in Human Placenta

Placental angiogenesis contributes to the pathogenesis of preeclampsia (PE) that affects 5-8% of all human pregnancies. MicroRNA (miRNA) are a class of noncoding 21- to 25-nucleotide RNA that negatively regulate gene expression posttranscriptionly. The aim of this study was to test the hypothesis that miRNA are differentially expressed in healthy term and PE placentas and a subclass of angiogenesis-associated miRNA are increased by PE. Total miRNA were extracted from villous placental tissues from healthy term and severe preeclamptic pregnancies. Differential miRNA expression was analyzed by microarray and real-time quantitative PCR. Angiogenesis-associated miRNA were analyzed by target prediction databases. In situ hybridization was used to localize miRNA. Target verification was performed by transfection of miRNA precursors or antagomirs into endothelial and BeWo cells and luciferase reporter assays. Three highly expressed miRNA (miR-17, -20a, and -20b) were found significantly increased in PE compared with healthy term placentas (n = 10 per group). They target on the same group of genes important for angiogenesis. miR-20b was expressed primarily in villous syncytiotrophoblasts in term placenta. Overexpression or inhibition of miR-20b differentially regulated mRNA expression of those genes in endothelial vs. trophoblast cells. Luciferase reporter assay showed that miR-20b targets EPHB4 and ephrin-B2 that have been shown to be critical for early human placental development. Placental ephrin-B2 mRNA was significantly down-regulated in PE compared with normotensive pregnancies. miR-17, miR-20a, and miR-20b are differentially regulated in human placentas by PE. They regulate EPHB4 and ephrin-B2 expression in trophoblast and endothelial cells via the same "seed" sequence, suggesting their roles in early placental development.

Global analysis of differentially expressed genes in early gestational decidua and chorionic villi using a 9600 human cDNA microarray.

The global gene expression profiles of the decidua and chorionic villi of early human pregnancies were analysed by using cDNA microarray technology. Decidual and villous placental tissues were obtained from first trimester abortus and mRNA was extracted for cDNA microarray analysis. The human cDNA microarray [9600 clones, including known regulatory genes and expressed sequence tags (EST)] with colorimetric detection was used to identify differentially expressed genes between early gestational decidua and villi. According to cDNA microarray analysis, we have identified 641 genes with highly expressed mRNA in both decidua and villi, 49 genes with higher expressions in decidua, and 75 genes with higher expression in chorionic villi. These differentially expressed genes were further grouped into categories by their putative functions, including: cell growth-related factors, hormones/cytokines, cell adhesion molecules, signal transduction molecules, apoptosis-related factors, cytoskeleton/extracellular matrix proteins, and EST. Immunohistochemical stainings of cathepsin L, leukaemia inhibitory factor-receptor, and proliferative cell nuclear antigen showed results consistent with the microarray data. Identification of the differentially expressed genes between decidua and villi by microarray provide a global profiling of the gene expression pattern. This work adds to our understanding of placentation by reporting the gene expression profiles during first trimester human pregnancies using cDNA microarray.

The 15-Deoxy-Δ12,14-prostaglandin J2Receptor, Peroxisome Proliferator Activated Receptor-γ (PPARγ) is Expressed in Human Gestational Tissues and is Functionally Active in JEG3 Choriocarcinoma Cells

RNA was extracted from human gestational membranes and villous placental tissue following spontaneous delivery (n=15) or elective caesarean section (n=15) at term. The samples were subjected to Northern analysis, using a 2kb cDNA probe for peroxisome proliferator activated receptor (PPAR)-γ. The mRNA was detectable in all choriodecidual and villous placental samples, irrespective of mode of delivery, but was only rarely detectable in the amnion. The JEG3 choriocarcinoma cell line also expressed PPARγ. In order to evaluate PPAR mediated transcriptional activation in JEG3 cells, the cells were transfected with pTK-PPREx3-luc, a PPAR response element (PPRE) containing luciferase reporter construct. Subsequent treatment with 10μm 15-deoxy-Δ12,14prostaglandin J2(15dPGJ2) resulted in an eight-fold stimulation of luciferase production relative to controls transfected with the same construct lacking the PPRE. This stimulation was concentration-dependent. These results suggest roles for PPARγ and its ligand in lipid, steroid and inflammatory mediator homeostasis and in remodelling of gestational tissues.

First-trimester villous placenta has high prorenin and active renin concentrations

OBJECTIVE: Term villous placental concentrations of prorenin are known to be very low, whereas those of decidua and fetal membranes are high. It has been demonstrated that prorenin synthesis is modulated by hormones in othe reproductive tissues, thus suggesting a means for paracrine regulation in the placenta. This study was performed to test the hypothesis that placental tissue prorenin concentrations may be influenced by gestational age and are temporally related to alterations in the hormonal milieu. STUDY DESIGN: Decidua and villous placental tissue were obtained from term and first-trimester human pregnancies, and concentrations of prorenin, active renin, prolactin, and human chorionic gonadotropin were measured. Values were compared between gestational periods, and relationships between renin and hormone values were analyzed. RESULTS: Prorenin concentrations in first-trimester placenta were nearly 200-fold higher than at term. The proportion of active renin was higher with early gestation. Decidual prorenin and active renin concentrations were similar in both groups. Placental prorenin correlated with chorionic gonadotropin but not prolactin in both groups. CONCLUSIONS: This study demonstrates large differences in placenta prorenin and active renin in villous placental tissue between first-trimester and term gestation tissues, yet there differences were not observed in decidual tissues. The contrast in placental prorenin values observed at the extremes of pregnancy parallel those of placental human chorionic gonadotropin.

Effect of cholera toxin, oxygen and tissue submersion on prorenin and HCG secretion from human villous placental tissue in vitro

EFFECT OF CHOLERA TOXIN, OXYGEN AND TISSUE SUBMERSION ON PRORENIN AND HCG SECRETION FROM HUMAN VILLOUS PLACENTAL TISSUE IN VITRO. A.M. Poisner, G.J. Downing and R. Poisner, Department of Pharmacology, University of Kansas Medical Center, Kansas City, KS USA. Renin, mostly as prorenin (PR), is present in extremely low concentrations in the villous placenta. We now report that modified organ cultures of villous placenta, incubated for periods up to 72 hrs, show an increase in PR secretion and that this is greatly enhanced by treatment with cholera toxin (CTX), oxygen and tissue elevation. 1-3 mm pieces of villous placenta were placed in 24-well tissue culture plates and incubated in 2 ml of modified CMRL medium (either submerged or placed upon filters at the gas-liquid interface) under 5% CO 2 (in air or in 95% 02). PR was released at very low concentrations and this was greatly increased by CTX (100 ng/ml) and by 95% 02. PR secretion (uU/mg protein) increased with time. At 72 hrs, CTX increa/sed PR from 109 • 38 to 19,450 • 4,860 in air; and from 1,640 • 367 to 108,000 = 14,900 in 02. HCG secretion (ng/mg protein) was not stimulated in air (from 74.9 • 8.4 to 95.2 • 19.7) but was stimulated in O 2 (from 88.0 • 14.8 to 629 • 119). Elevation of the placental pieces enhanced the release of PR (20 fold in air; and 8 fold in Oz). There was a negligible effect on HCG secretion. The effects on placental PR secretion are far greater than any previously reported. These studies show differences in PR and HCG regulation and describe a new model for study of placental renin secretion. A.59 LONG-TERN ORGANOTYPIC CULTURE OF HUHAN PLACENTAL EXPLANTS: HORHONAL AND |Mt4UNOCYTOCHENICAL EVALUATIGN OF THE EXPERIMENTAL 14OOEL. 8rurm PotL iot t i 1, Scott Keesting 1, Mei Zhang 2, Eva Back61, Joseph Cabs 1, Francis Lee 1, David Schwartz 2, Naurice Psniget 1 and Andr~ Nahmias 1,2, Departments of Pediatr ics 1 and Pathology 2, Emery Universi ty, At lanta, CA, USA. The ef fects of d i f fe ren t infect ious agents on the various types of placental ce l ls require long-term ex vivo maintenance of placental tissue, pa r t i cu la r l y in case of viruses such as HIV and CNV = ui th long repl icat ion cycles. For th is purpose, we have studied several variables in placental t issue explants obtained a f ter cesarean section and incubated in RPH! supplemented with 104 FCS, with or without an t i b io t i c , for periods up to 13 days. The medium yes replaced every day and samples of the supernatsnt were frozen for later test ing of hPL release and glucose consumption. The t issue was also f ixed end embedded st d i f f e ren t periods for microscopic examinations. During the incubation period (11 to 13 days), the pH, pO 2 and 10(;02 were Bonitored and found to remain constant. The glucose u t i l i z a t i o n represented 25~ of the i n i t i a l concentration (1.74 g / l ) in the medium and was found to be the same for a l l the incubation in terva ls . Af ter a rapid decrease, hPL in the supernatant reached a basal level (2.77=1.51 ng/ml/mg/Z4hr.), which remained stable for mere than 10 days. Af ter 13 days, the chorlonir v i l l i trophobtast was s t i l t h i s to log ica l l y recognizable as a dist inguishable layer with an apical brush border. Dense secretory granules of hPL, characterized by i ~ c y t o c h e m i s t r y , were local ized in the syncytiotrophoblast layer. P ro l i fe ra t ion marker (Ki67), detected by the HIB1 monoctona{ antibody, was also observed in some residual cytotrophoblast ce l ls . At the end of the cul ture, blood capi l la r ies were no longer observed in the connective t issue stroma. Long-term organotypic cultures should help to understand better the pathogenesis of infect ious agents in the placenta, in the present model, the placental t issue can be incubated ex vivo for prolonged periods, without necessari ly the addit ion of any an t ib io t i c which could in ter fe re in studies of some infect ious agents. Horeover, unl ike many other orgsnotypic cul ture procedures, th is method allows extensive washing of the explant in order to el iminate the remaining infect ious inocutum and to measure the release of placental products in cul ture supernatants.