scholarly journals Molecular Pathology Demonstration of SARS-CoV-2 in Cytotrophoblast from Placental Tissue with Chronic Histiocytic Intervillositis, Trophoblast Necrosis and COVID-19

2021 ◽  
Vol 9 (3) ◽  
pp. 33
Author(s):  
David A. Schwartz ◽  
Mattia Bugatti ◽  
Amerigo Santoro ◽  
Fabio Facchetti
Keyword(s):  
Molecular Pathology ◽  
Placental Tissue ◽  
Physical Contact ◽  
Positive Staining ◽  
Fibrin Deposition ◽  
Cell Type ◽  
Hofbauer Cells ◽  
Capillary Endothelial Cells ◽  
Rna In Situ Hybridization ◽  
Cytotrophoblast Cells

A subset of placentas from pregnant women having the SARS-CoV-2 infection have been found to be infected with the coronavirus using molecular pathology methods including immunohistochemistry and RNA in situ hybridization. These infected placentas can demonstrate several unusual findings which occur together—chronic histiocytic intervillositis, trophoblast necrosis and positive staining of the syncytiotrophoblast for SARS-CoV-2. They frequently also have increased fibrin deposition, which can be massive in some cases. Syncytiotrophoblast is the most frequent fetal-derived cell type to be positive for SARS-CoV-2. It has recently been shown that in a small number of infected placentas, villous stromal macrophages, termed Hofbauer cells, and villous capillary endothelial cells can also stain positive for SARS-CoV-2. This report describes a placenta from a pregnant woman with SARS-CoV-2 that had chronic histiocytic intervillositis, trophoblast necrosis, increased fibrin deposition and positive staining of the syncytiotrophoblast for SARS-CoV-2. In addition, molecular pathology testing including RNAscope and immunohistochemistry for SARS-CoV-2 and double-staining immunohistochemistry using antibodies to E-cadherin and GATA3 revealed that cytotrophoblast cells stained intensely for SARS-CoV-2. All of the cytotrophoblast cells that demonstrated positive staining for SARS-CoV-2 were in direct physical contact with overlying syncytiotrophoblast that also stained positive for the virus. The pattern of cytotrophoblast staining for SARS-CoV-2 was patchy, and there were chorionic villi having diffuse positive staining of the syncytiotrophoblast for SARS-CoV-2, but without staining of cytotrophoblast. This first detailed description of cytotrophoblast involvement by SARS-CoV-2 adds another fetal cell type from infected placentas that demonstrate viral staining.

scholarly journals Hofbauer cells and coronavirus disease 2019 (COVID-19) in pregnancy: Molecular pathology analysis of villous macrophages, endothelial cells, and placental findings from 22 placentas infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with and without fetal transmission

2021 ◽  
Author(s):  
David A. Schwartz ◽  
Marcella Baldewijns ◽  
Alexandra Benachi ◽  
Mattia Bugatti ◽  
Gaetano Bulfamante ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Severe Acute Respiratory Syndrome ◽  
Molecular Pathology ◽  
Capillary Endothelium ◽  
Cell Hyperplasia ◽  
Cell Staining ◽  
Hofbauer Cells ◽  
Capillary Endothelial Cells ◽  
Placental Macrophages ◽  
Maternal Fetal Transmission

Context.– Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can undergo maternal-fetal transmission, heightening interest in the placental pathology findings from this infection. Transplacental SARS-CoV-2 transmission is typically accompanied by chronic histiocytic intervillositis together with necrosis and positivity of syncytiotrophoblast for SARSCoV-2. Hofbauer cells are placental macrophages that have been involved in viral diseases including HIV and Zika virus, but their involvement in SARS-CoV-2 in unknown. Objective.– To determine whether SARS-CoV-2 can extend beyond the syncytiotrophoblast to enter Hofbauer cells, endothelium and other villous stromal cells in infected placentas of liveborn and stillborn infants. Design.– Case-based retrospective analysis by 29 perinatal and molecular pathology specialists of placental findings from a preselected cohort of 22 SARS-CoV-2-infected placentas delivered to pregnant women testing positive for SARS-CoV-2 from 7 countries. Molecular pathology methods were used to investigate viral involvement of Hofbauer cells, villous capillary endothelium, syncytiotrophoblast and other fetal-derived cells. Results.– Chronic histiocytic intervillositis and trophoblast necrosis was present in all 22 placentas (100%). SARS-CoV-2 was identified in Hofbauer cells from 4/22 placentas (18%). Villous capillary endothelial staining was positive in 2/22 cases (9%), both of which also had viral positivity in Hofbauer cells. Syncytiotrophoblast staining occurred in 21/22 placentas (95%). Hofbauer cell hyperplasia was present in 3/22 placentas (14%). In the 7 cases having documented transplacental infection of the fetus, 2 occurred in placentas with Hofbauer cell staining positive for SARS-CoV-2. Conclusions.– SARS-CoV-2 can extend beyond the trophoblast into the villous stroma, involving Hofbauer cells and capillary endothelial cells, in a small number of infected placentas. Most cases of SARS-CoV-2 transplacental fetal infection occur without Hofbauer cell involvement.

scholarly journals Molecular Pathology Analysis of SARS-CoV-2 in Syncytiotrophoblast and Hofbauer Cells in Placenta from a Pregnant Woman and Fetus with COVID-19

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 479
Author(s):  
Denise Morotti ◽  
Massimiliano Cadamuro ◽  
Elena Rigoli ◽  
Aurelio Sonzogni ◽  
Andrea Gianatti ◽  
...  
Keyword(s):  
Molecular Pathology ◽  
Nucleocapsid Protein ◽  
Positive Staining ◽  
Transplacental Transmission ◽  
Placental Pathology ◽  
Macrophage Marker ◽  
Immunodeficiency Virus ◽  
Hofbauer Cells ◽  
Maternal Fetal Transmission

A small number of neonates delivered to women with SARS-CoV-2 infection have been found to become infected through intrauterine transplacental transmission. These cases are associated with a group of unusual placental pathology abnormalities that include chronic histiocytic intervillositis, syncytiotrophoblast necrosis, and positivity of the syncytiotrophoblast for SARS-CoV-2 antigen or RNA. Hofbauer cells constitute a heterogeneous group of immunologically active macrophages that have been involved in transplacental infections that include such viral agents as Zika virus and human immunodeficiency virus. The role of Hofbauer cells in placental infection with SARS-CoV-2 and maternal-fetal transmission is unknown. This study uses molecular pathology techniques to evaluate the placenta from a neonate infected with SARS-CoV-2 via the transplacental route to determine whether Hofbauer cells have evidence of infection. We found that the placenta had chronic histiocytic intervillositis and syncytiotrophoblast necrosis, with the syncytiotrophoblast demonstrating intense positive staining for SARS-CoV-2. Immunohistochemistry using the macrophage marker CD163, SARS-CoV-2 nucleocapsid protein, and double staining for SARS-CoV-2 with RNAscope and anti-CD163 antibody, revealed that no demonstrable virus could be identified within Hofbauer cells, despite these cells closely approaching the basement membrane zone of the infected trophoblast. Unlike some other viruses, there was no evidence from this transmitting placenta for infection of Hofbauer cells with SARS-CoV-2.

scholarly journals Repeated social defeat exaggerates fibrin-rich clot formation in FeCl3-induced arterial thrombosis mouse model by enhancing NETs formation via modulation of neutrophil functional properties

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
T Sugimoto ◽  
H Yamada ◽  
H Kubota ◽  
D Miyawaki ◽  
M Saburi ◽  
...  
Keyword(s):  
Functional Properties ◽  
Arterial Thrombosis ◽  
Social Defeat ◽  
Arterial Injury ◽  
Physical Contact ◽  
Clot Formation ◽  
Positive Staining ◽  
Double Positive ◽  
The Impact

Abstract Background and objective Depression is an independent risk factor of cardiovascular disease (CVD). We have recently shown that repeated social defeat (RSD) precipitates depressive-like behaviors in apoE−/− mice and exaggerates atherosclerosis development by enhancing neutrophil extracellular traps (NETs) formation. Here, we investigated the impact of RSD on arterial thrombosis. Methods and results Eight-week-old male WT mice were exposed to RSD by housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction test to confirm depressive-like behaviors, defeated mice (19 of 31) and control mice (12 of 14) were underwent arterial injury at 10 wks of age. A filter paper saturated with 10% FeCl3 was applied on the adventitial surface of left carotid artery for 3 min and analyzed 3 hrs later. The volume of thrombi was comparable between the two groups. However, fibrinogen/fibrin-positive areas in immunofluorescent images significantly increased in defeated mice (27.8% vs. 48.8%, p<0.01). The number of Ly-6G-positive cells in thrombi was markedly higher in defeated mice (144/mm2 vs. 878/mm2, p<0.05). Further, Ly-6G-positive cells were almost accumulated at the inner surface of injured artery, which were co-localized with neutrophil elastase, Cit-H3, and CD41-positive staining. Treatment with DNase I completely diminished the exaggerated fibrin-rich clot formation in defeated mice to an extent similar to that in control mice (25.7% vs. 22.3%, p = ns), without affecting the volume of thrombi and accumulation of Ly-6G-positive cells. Given that platelet aggregations induced by ADP or collagen were comparable between the two groups, neutrophil functional properties primarily contribute to the exaggerated fibrin-rich clot formation in defeated mice. We then examined neutrophil subset and vulnerability to NETs formation. At 3 hrs after FeCl3 application, the numbers of immature neutrophils (Ly6Glo/+CXCR2-) were comparable between the two groups in both bone marrow (BM) and peripheral blood (PB). In contrast, the number of PB mature neutrophils (Ly6G+CXCR2+) was markedly higher in defeated mice than control mice (580±68 /μl vs. 1265±114, p<0.01). We next examined in vitro NETs formation upon PMA in BM mature neutrophils by FACS and nucleic acid staining. The percentage of double-positive cells (Cit-H3, MPO) was significantly higher in defeated mice (7.5% vs. 10.2%, p<0.05), as well as SYTOX green-positive cells expelling DNA fibers (8.1% vs. 11.8%, p<0.05). Conclusions Our findings demonstrate for the first time that repeated social defeat enhances fibrin-rich clot formation after arterial injury by enhancing NETs formation via modulation of neutrophil functional properties, suggesting that NETosis could be a new therapeutic target in depression-related CVD development. Funding Acknowledgement Type of funding source: None

Immunocytochemical localization of oestrogen receptors in the endometrium of the ewe

1991 ◽  
Vol 3 (3) ◽  
pp. 321 ◽  
Author(s):  
RA Cherny ◽  
LA Salamonsen ◽  
JK Findlay
Keyword(s):  
Stromal Cells ◽  
Cellular Response ◽  
Individual Cell ◽  
Cell Types ◽  
Staining Intensity ◽  
Steroid Treatment ◽  
Positive Staining ◽  
Cell Type ◽  
Peak Intensity

Immunocytochemistry with monoclonal antibodies to the oestrogen receptor (ER) was used to localize ERs in sections of endometrium obtained from cycling and pregnant Corriedale ewes. Representative tissue from Days 4, 10, 14, 15, 16 and 17 of the cycle (Day 0 = onset of oestrus) and Day 15 of pregnancy was used. ER localization was also examined in tissue obtained from ovariectomized (ovex) ewes with and without subcutaneous implants containing oestrogen, progesterone, or oestrogen and progesterone. ER distribution was examined in caruncular endometrium and intercaruncular endometrium. Staining intensity varied according to cell type, stage of the cycle, steroid treatment and pregnancy. No staining was observed in endothelial cells. In all cases, ER was localized within the nuclei of positive cells. Generally, ER levels were high on Day 4 and declined to negligible values by Day 10 (corresponding to peak progesterone values) except in the deep stroma of caruncular endometrium. Positive staining reappeared in stromal cells of caruncles on Day 13 and in the luminal epithelium of intercaruncular tissue on Day 14. Peak intensity was reached on Day 15 for caruncular tissue and Day 16 for intercaruncular tissue. Ovariectomy did not cause an overall reduction in ER levels, whereas treatment with oestrogen and progesterone had variable effects depending on cell type. Progesterone did not suppress overall ER. In Day 15 pregnant tissue, ER was undetectable in all compartments except deep stroma of caruncles, indicating that factors other than progesterone, perhaps embryonic in origin, were responsible. The observation that individual cell types display differential sensitivities to oestrogen and progesterone as regards their expression of ER is consistent with the role of cell-cell interactions as modulators of cellular response to steroids through the oestrous cycle and in pregnancy.

203. A differential pattern of follistatin expression in the placenta between spontaneous, induced and non-labouring patient groups

2005 ◽  
Vol 17 (9) ◽  
pp. 77
Author(s):  
K. M. Rae ◽  
K. G. Hollebone ◽  
L. Meng ◽  
D. C. Clausen ◽  
J. R. McFarlane
Keyword(s):  
Vascular Endothelial Cells ◽  
Placental Tissue ◽  
Cell Types ◽  
Cross Sectional Study ◽  
Polyclonal Antiserum ◽  
Antigen Retrieval ◽  
Positive Staining ◽  
Cross Sectional ◽  
Chorionic Villi ◽  
Patient Groups

Follistatin has been identified in human placenta, fetal membranes and fluids, with serum follistatin concentrations rising during pregnancy, particularly near term. Our laboratory has shown follistatin concentrations rise across labour in spontaneous but not induced women.1 As the placenta is a source of follistatin, this study examined placental tissues using immunohistochemistry to determine differences in follistatin localization between groups. Placental tissue was collected immediately following delivery from three groups of women at term, spontaneous onset (n = 4), induction (n = 4) and non-labouring caesarian (n = 4), and immediately formalin fixed. Antigen-retrieval immunohistochemistry using a specific chicken polyclonal antiserum (CK20) raised against a follistatin peptide (AA 121-133) or pre-immune chicken serum was performed. Positive staining of syncytiotrophoblast cells of the chorionic villi was seen in patients undergoing spontaneous labour but not in the induced and caesarian delivery group. The two labouring groups (spontaneous and induced) both showed positive staining for the vascular endothelial cells within the chorionic villi and the stratum basale, whilst the caesarian delivery group was negative for any staining within these vessels. Positive staining of Hofbauer cells was observed in both labouring groups; however, the caesarian group showed infrequent positive staining of these cell types. The differences in expression pattern in the two labouring groups (spontaneous v. induced) may be due to variations in labour lengths (6.5 v. 4.5 h, respectively); however, we would have expected a lower level of expression in the same cell types rather than the complete absence of staining. The positive follistatin staining in the syncytiotrophoblast of spontaneous patients suggests this may be the source of the rising plasma follistatin seen in this group. These differences in staining support our hypothesis that an earlier endocrine signal is absent in the induced and caesarian patient groups. (1)Rae K, Hollebone K, Clausen DC, Chetty V, McFarlane JR. (2004). A Cross-Sectional Study of Follistatin During Labour in Women. The Endocrine Societies 86th Annual Meeting, New Orleans, 2004.

scholarly journals A Novel 3D Model for Visualization and Tracking of Fibroblast-Guided Directional Cancer Cell Migration

Biology ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 328
Author(s):  
Yihe Zhang ◽  
Bingjie Jiang ◽  
Meng Huee Lee
Keyword(s):  
Cell Migration ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Molecular Mechanisms ◽  
Cell Mass ◽  
Physical Contact ◽  
Physical Interaction ◽  
Matrix Remodeling ◽  
Cancer Cell Migration ◽  
Cell Type

Stromal fibroblasts surrounding cancer cells are a major and important constituent of the tumor microenvironment not least because they contain cancer-associated fibroblasts, a unique fibroblastic cell type that promotes tumorigenicity through extracellular matrix remodeling and secretion of soluble factors that stimulate cell differentiation and invasion. Despite much progress made in understanding the molecular mechanisms that underpin fibroblast–tumor cross-talk, relatively little is known about the way the two cell types interact from a physical contact perspective. In this study, we report a novel three-dimensional dumbbell model that would allow the physical interaction between the fibroblasts and cancer cells to be visualized and monitored by microscopy. To achieve the effect, the fibroblasts and cancer cells in 50% Matrigel suspension were seeded as independent droplets in separation from each other. To allow for cell migration and interaction, a narrow passage of Matrigel causeway was constructed in between the droplets, effectively molding the gel into the shape of a dumbbell. Under time-lapse microscopy, we were able to visualize and image the entire process of fibroblast-guided cancer cell migration event, from initial vessel-like structure formation by the fibroblasts to their subsequent invasion across the causeway, attracting and trapping the cancer cells in the process. Upon prolonged culture, the entire population of fibroblasts eventually infiltrated across the passage and condensed into a spheroid-like cell mass, encapsulating the bulk of the cancer cell population within. Suitable for almost every cell type, our model has the potential for a wider application as it can be adapted for use in drug screening and the study of cellular factors involved in cell–cell attraction.

scholarly journals Evidence of extrahepatic replication of hepatitis E virus in human placenta

2014 ◽  
Vol 95 (6) ◽  
pp. 1266-1271 ◽  
Author(s):  
Purabi Deka Bose ◽  
Bhudev Chandra Das ◽  
Rajib Kishore Hazam ◽  
Ashok Kumar ◽  
Subhash Medhi ◽  
...  
Keyword(s):  
Human Placenta ◽  
Hepatitis E Virus ◽  
Placental Tissue ◽  
Hepatitis E ◽  
Fetal Mortality ◽  
Positive Staining ◽  
Structural Protein ◽  
Tissue Sections ◽  
Extrahepatic Replication ◽  
Immunohistochemical Studies

The incidence and severity of hepatitis E virus (HEV) infection in pregnant women is high in developing countries. Transplacental transmission of HEV in the third trimester of pregnancy has been found to be associated with high fetal mortality. Based on this evidence and in the absence of reports on HEV replication in extrahepatic sites, this study was carried out to investigate if HEV replication occurs in the placenta of infected mothers. The study included 68 acute viral hepatitis (AVH) and 22 acute liver failure (ALF) pregnant patients. Viral RNA was extracted from blood and placenta. HEV replication in placenta was confirmed by a replicative negative-strand-specific reverse transcriptase PCR. Viral load was estimated by real-time PCR. Immunohistochemical studies were also carried out for in situ detection of HEV in placental tissue sections. Replicative HEV RNA was detectable only in the placenta in ALF and AVH cases and not in blood samples. Positive staining of placental tissue sections with HEV antibody against the viral structural protein ORF3 was observed. HEV replication in placenta also correlated with fetal and maternal mortality in ALF patients. It is demonstrated for the first time that HEV replication occurs in human placenta and that placenta may be a site of extrahepatic replication of HEV in humans.

scholarly journals 4 Placental Hofbauer Cells As a Proxy Cell Type for Fetal Brain Microglia

2021 ◽  
Vol 224 (2) ◽  
pp. S2-S3
Author(s):  
Rose De Guzman ◽  
Rebecca Batorsky ◽  
Sezen Kislal ◽  
Sara Brigida ◽  
Staci Bilbo ◽  
...  
Keyword(s):  
Fetal Brain ◽  
Cell Type ◽  
Hofbauer Cells

Co-Inheritance of FV Leiden and High Levels of FIX Results in Novel Models for Thrombophilia and Spontaneous Fetal Loss.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1946-1946
Author(s):  
Christian Furlan Freguia ◽  
Joerg Schuettrumpf ◽  
Stefano Baila ◽  
Jianhua Liu ◽  
Ralph Bunte ◽  
...  
Keyword(s):  
Gene Dosage ◽  
Fetal Loss ◽  
Placental Tissue ◽  
High Rate ◽  
Fibrin Deposition ◽  
Additional Risk ◽  
Maternal Genotype ◽  
Fv Leiden ◽  
Common Risk Factors ◽  
Risk For Thrombosis

Abstract High levels of FIX is emerging as a risk factor for spontaneous venous thrombosis, affecting ~20% of unselect patients and increasing rates of recurrent thromboembolic events. We have generated mice expressing supraphysiological levels of human FIX (up to 400% of normal) but these animals did not develop thrombosis. Therefore, we decided to test whether an additional risk for thrombosis would trigger a prothrombotic phenotype in these mice. Because the FVL mutation is also present in 20% of thrombophilia subjects, we chose FVL mice as model. Breedings included both parents being heterozygous FVL(+/−) but only one was transgenic for FIX (tFIX: 4–8μg/ml plus the endogenous murine FIX). All mice were on C57Bl/6 background. More than 200 newborns were obtained but surprisingly no animal of FVL(+/+)/tFIX genotype was identified. When pregnancies were interrupted at embryonic age of E9.5 to E16.5, no deviation from the expected/observed embryos genotypes was observed. However, the number of reabsorbed embryos increased significantly from 13% (E9.5) to 33% (E.16.5), p<0.02. All FVL(+/+)/tFIX embryos exhibited extensive bruises and hemorrhagic areas which colocalize with markedly fibrin deposition, findings consistent with disseminated intravascular coagulation. Placental analysis (n=18) of FVL(+/+)/tFIX embryos harvested at E12.5 or later demonstrated abnormalities such as excessive fibrin deposition and innumerous apoptotic cells, determined by TUNEL assay, mainly in the labyrinthine trophoblast zone. The maternal genotype clearly influenced the pregnancy outcome since the number of newborns per litter was 2-fold lower when the mother carried tFIX compared with father tFIX (average 4.3 vs. 8.3, respectively; p<0.02). No mice of other genotypes analyzed (n=112) presented similar abnormalities neither in embryos nor in placental tissue. Mice that achieve adulthood developed normally. However, FVL(+/−)tFIX mice (>7 months of age) presented high rate of thrombotic episodes (3/40; 7.5%) followed by 5.2% FVL(−/ −)/tFIX mice, whereas no other age-matched mice (n=74) developed such complication. Monitoring for coagulation activation by serial aPTT, PT, thrombin-antithrombin (TAT) and D-Dimers demonstrated genotype-dependent procoagulant activity. In FVL(+/−)/tFIX or FVL(−/ −)/tFIX mice, levels of TAT (70ng/ml, n=28/group) and D-dimers (230ng/ml, n=17/group) were ~ 2-fold higher (P<0.04), when compared to mice (TAT: 30ng/ml and D-Dimer: 100ng/ml) of other genotypes. Furthermore, FVL(+/−)/tFIX mice presented the highest fibrin deposition determined by Western blot analysis of tissues harvested, followed by FVL(−/ −)/tFIX mice when compared to the other animals (P<0.05). To exclude the possibility of abnormal interaction of human (h) FIX with murine antithrombin (AT), we compared the rate of inactivation of hFIXa by murine or human AT using purified proteins. The results showed that murine AT efficiently inactivated hFIXa in a similar fashion of that determined for human AT. Moreover, early work showed that hFIX efficiently corrected the phenotype of FIX-deficient mice. Therefore, these data revealed that gene-dosage dependent interaction of FVL and high levels of FIX in mice increase rates of spontaneous fetal loss and adulthood thrombosis. These models provide opportunities to better understand and to test therapeutics for thrombophilia-related complications due to common risk factors identified in humans.

scholarly journals Spatiotemporal Expression of C-CAM in the Rat Placenta

1997 ◽  
Vol 45 (7) ◽  
pp. 1021-1034 ◽  
Author(s):  
Hiroki Sawa ◽  
Hiroyuki Ukita ◽  
Minoru Fukuda ◽  
Hajime Kamada ◽  
Isamu Saito ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Broad Band ◽  
Placental Tissue ◽  
Maternal Blood ◽  
Immunoglobulin Superfamily ◽  
Pcr Analysis ◽  
Spatiotemporal Pattern ◽  
Positive Staining ◽  
Placental Development ◽  
Embryonic Vessels

We investigated the expression of the immunoglobulin superfamily cell adhesion molecule, C-CAM, in developing and mature rat placenta. By immunohistochemical staining at the light microscopic level, no C-CAM-expression was seen before Day 9 of gestation, when it appeared in the trophoblasts of ectoplacental cones. On Day 10.5, spongiotrophoblasts and invasive trophoblasts around the maternal vessels of the decidua basalis were stained positively. On Day 12.5, C-CAM was detected in the spongiotrophoblasts of the junctional layer, but labyrinth trophoblasts and secondary giant trophoblasts were not stained. On Day 17.5, C-CAM was found only in the labyrinth and lacunae of the junctional layer. At this stage, both the labyrinth cytotrophoblasts of the maternal blood vessels and the endothelial cells of the embryonic capillaries were strongly stained. Placental tissues from gestational Days 12.5 and 17.5 were analyzed by immunoelectron microscopy to determine the location of C-CAM at the subcellular level. On Day 12.5, positive staining of the spongiotrophoblasts was observed, mainly on surface membranes and microvilli between loosely associated cells. On Day 17.5, staining was found primarily on the microvilli of the maternal luminal surfaces of the labyrinth cytotrophoblasts, and both on the luminal surface and in the cytoplasm of endothelial cells of the embryonic vessels. RT-PCR analysis and Southern blotting of the PCR products revealed expression of mRNA species for both of the major isoforms, C-CAM1 and C-CAM2. Immunoblotting analysis of C-CAM isolated from 12.5-day and 14.5-day placentae showed that it appeared as a broad band with an apparent molecular mass of 110–170 kD. In summary, C-CAM was strongly expressed in a specific spatiotemporal pattern in trophoblasts actively involved in formation of the placental tissue, suggesting an important role in placental development. In the mature placenta, C-CAM expression was confined to the trophoblastic and endothelial cells lining the maternal and embryonic vessels, respectively, suggesting important functions in placental physiology.

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