Cells In Decidua Research Articles

Decidual vascularization during organogenesis after perigestational alcohol ingestion.

Perigestational alcohol consumption up to early organogenesis can produce abnormal maternal vascularization via altered decidual VEGF/receptor expression. CF-1 female mice were administered with 10% ethanol in drinking water for 17 days prior to and up to day 10 of gestation. Control females received water without ethanol. Treated females had reduced frequency of implantation sites with expanded vascular lumen (P < 0.05), α-SMA-immunoreactive spiral arteries in proximal mesometrial decidua, reduced PCNA-positive endothelial cells (P < 0.01) and diminished uterine NK cell numbers (P < 0.05) in proximal decidua compared to controls. The VEGF expression (laser capture microscopy, RT-PCR, western blot and immunohistochemistry) was reduced in decidual tissue after perigestational alcohol consumption (P < 0.05). The uNK-DBA+ cells of treated females had reduced VEGF immunoexpression compared to controls (P < 0.01). Very low decidual and endothelial cell KDR immunoreactivity and reduced decidual gene and protein KDR expression was found in treated females compared to controls (P < 0.001). Instead, strong FLT-1 immunoexpression was detected in decidual and uNK cells (P < 0.05) in the proximal decidua from treated females compared to controls. In conclusion, perigestational alcohol ingestion induces the reduction of lumen expansion of spiral arteries, concomitant with reduced endothelial cell proliferation and uNK cell population, and uncompleted remodeling of the artery smooth muscle. These effects were supported by low decidual VEGF and KDR gene and protein expression and increased FLT-1 expression, suggesting that VEGF and KDR reduction may contribute, in part, to mechanisms involved in deficient decidual angiogenesis after perigestational alcohol consumption in mouse.

PD-1 is expressed by and regulates human group 3 innate lymphoid cells in human decidua.

Group 3 innate lymphoid cells (ILC3) have been detected in both murine and human decidual tissues where they are thought to play a relevant role in the induction and maintenance of pregnancy. However, limited information exists on the molecular mechanisms that regulate these cells, including immune checkpoints. Here, we show that ILC3 express the inhibitory checkpoints programmed cell death (PD-1) and T cell immunoglobulin and mucin domain containing protein 3 (TIM-3) during the first trimester of pregnancy and that these receptors could regulate production of cytokines, including IL-22, IL-8, and TNF-α, induced by IL-23. We also show that the intermediate extravillous trophoblast (iEVT) expresses high levels of the PD-1-ligand PD-L1, suggesting that PD-1/PD-L1 interaction may regulate ILC3 function at the feto-maternal interface. Our present data provide the first evidence that human decidual ILC3 express a functional PD-1. It is possible that an altered expression or function of PD-1 may break the immune-tolerance resulting in pregnancy failure.

Heterogeneity of NK Cells and Other Innate Lymphoid Cells in Human and Murine Decidua.

Innate lymphoid cells (ILCs) represent a heterogeneous group of cells lacking genetically rearranged antigen receptors that derive from common lymphoid progenitors. Five major groups of ILCs have been defined based on their cytokine production pattern and developmental transcription factor requirements: namely, natural killer (NK) cells, ILC1s, ILC2s, ILC3s, and lymphoid tissue-inducer (LTi) cells. ILC1s, ILC2s, and ILC3s mirror the corresponding T helper subsets (Th1, Th2, and Th17, respectively) and produce cytokines involved in defense against pathogens, lymphoid organogenesis, and tissue remodeling. During the first trimester of pregnancy, decidual tissues contain high proportion of decidual NK (dNK) cells, representing up to 50% of decidual lymphocytes, and ILC3s. They release peculiar cytokines and chemokines that contribute to successful pregnancy. Recent studies revealed that ILCs display a high degree of plasticity allowing their prompt adaptation to environmental changes. Decidual NK cells may derive from peripheral blood NK cells migrated when pregnancy establishes or from in situ differentiation of hematopoietic precursors. Previous studies showed that human and murine decidua contain dNK cells, tissue resident NK cells, and ILC3s, all characterized by unique phenotypic and functional properties, most likely induced by decidual microenvironment to favor the establishment and the maintenance of pregnancy. Thus, during the early phase of pregnancy, the simultaneous presence of different ILC subsets further underscores the complexity of the cellular components of decidual tissues as well as the role of decidual microenvironment in shaping the plasticity and the function of ILCs.

Decidual RANKL/RANK interaction promotes the residence and polarization of TGF-\u03b21-producing regulatory \u03b3\u03b4 T cells

AbstactDecidual γδΤ (dγδΤ) cells play an essential role during successful pregnancy; however, the residence and polarization of γδΤ cells in decidua remain unclear. In this study, we observed higher levels of receptor activator for nuclear factor-κ B ligand (RANKL) on decidual stromal cells (DSCs), and its receptor RANK on dγδΤ cells in decidua from normal pregnancy compared with patients with recurrent spontaneous abortion (RSA). RANKL expressed by DSCs can induce the polarization of peripheral blood γδΤ (pγδΤ) and dγδΤ cells to Foxp3 + γδΤ cells, and upregulate the expression of transforming growth factor (TGF)-β1. This process is mediated through activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In addition, RANKL promotes the adhesion of dγδΤ cells to DSCs in vitro, which is associated with the upregulation of ICAM-1 and VCAM-1 on DSCs and integrins on dγδΤ cells. RANKL knockout leads to the decreased numbers of uterus total γδΤ cells, Foxp3+γδΤ cells and the expression of TGF-β1, and the increased pregnancy loss in mice. These results suggest that RANKL is a pivotal regulator of maternal-fetal tolerance by triggering the polarization and residence of TGF-β1-producing Foxp3+γδΤ cells in early pregnancy. The abnormal low level of RANKL/RANK results in pregnancy loss because of the dialogue disorder between DSCs and dγδΤ cells. This observation provides a scientific basis on which a potential marker can be detected to early warning of pregnancy loss.

Changes of γδT cell subtypes during pregnancy and their influences in spontaneous abortion

A successful pregnancy is a complicated process that involves the precisely timed regulation of endocrine as well as immune system. Despite increasing knowledge about immunology in gestation, the studies of immune cells in endometrium and decidua are still fragmented. Dynamic data is lacking on the transition of pre-pregnancy endometrial lymphocytes to initial pregnancy states as well as to second/third-trimester status. Here, we determined the composition of Gamma delta (γδ) T cells in endometrium and decidua from women with normal pregnancy and unexplained spontaneous abortion. We found that the frequency of γδT cells is fluctuating over the course of pregnancy, and these changes were regulated by progesterone. Different from peripheral blood, Vδ1+ γδT cells accounted for the majority in endometrium and early-pregnancy decidua of healthy women, and endometrial stromal cells (ESCs) may involve in Vδ1/ Vδ2 shift directly. Moreover, an increase in the percentage of γδT cells with Vδ2 subset predominant in early-pregnancy decidua was associated with unexplained spontaneous abortion. Our results unraveled the precise timing of γδT cells occurring during pregnancy and the close relationship among endocrine, immune cells and pregnancy, which can further help understand and solve the problem of infertility and unexplained spontaneous abortion.

The role of E2F8 in the human placenta.

Recent studies have reported that E2F transcription factor (E2F) 8, an atypical E2F transcription factor, serves a critical role in promoting the growth and development of the murine placenta. However, the function of E2F8 in the human placenta remains unknown. Invasion of extravillous trophoblasts (EVTs) into the maternal decidua is known to be important for the development of the human placenta. To investigate the role of E2F8 in human placental development, E2F8 localisation was examined in the human placenta and E2F8 mRNA expression was detected in primary cultured EVTs. The human EVT cell line, HTR-8/SVneo, was divided into two groups and treated separately, one with retrovirus expressing short hairpin (sh)-RNA against E2F8 (shE2F8 cells) and the other with non-target control shRNA (shControl cells). The cell functions, including cell cycle, proliferation, invasion and adhesion, were compared between the shE2F8 and shControl cells. A histological examination revealed that E2F8 was localised in the decidua cells, EVTs, and cytotrophoblasts in the placenta. E2F8 mRNA was confirmed to be expressed in cultured primary EVTs. No significant difference was observed in the cell cycle, proliferation or adhesion between the shE2F8 and shControl cells. The invasive ability was ~2-fold higher in the shE2F8 cells when compared with the shControl cells (P<0.01). Production of matrix metalloproteinase-1 was significantly increased in the shE2F8 cells when compared with the shControl cells (P<0.05). Taken together, E2F8 is present in the EVTs of the human placenta, but, unlike murine placenta, it may suppress the invasiveness of EVTs. E2F8 was also present in cytotrophoblasts in cell columns, which have no invasive ability and differentiate into EVTs. In conclusion, E2F8 also exists in the human placenta, and its function may be different from that in the murine placenta, although further investigation is required.

Innate Lymphoid Cells in the Maternal and Fetal Compartments.

Pregnancy success is orchestrated by the complex balance between the maternal and fetal immune systems. Herein, we summarize the potential role of innate lymphoid cells (ILCs) in the maternal and fetal compartments. We reviewed published literature describing different ILC subsets [ILC1s, ILC2s, ILC3s, and lymphoid tissue inducer (LTi) cells] in the uterus, decidua, fetal tissues [liver, secondary lymphoid organs (SLO), intestine, and lung] and amniotic cavity. ILC1s, ILC2s, and ILC3s are present in the murine uterus prior to and during pregnancy but have only been detected in the non-pregnant endometrium in humans. Specifically, ILC2s reside in the murine uterus from mid-pregnancy to term, ILC1s increase throughout gestation, and ILC3s remain constant. Yet, LTi cells have only been detected in the non-pregnant murine uterus. In the human decidua, ILC1s, ILC3s, and LTi-like cells are more abundant during early gestation, whereas ILC2s increase at the end of pregnancy. Decidual ILC1s were also detected during mid-gestation in mice. Interestingly, functional decidual ILC2s and ILC3s increased in women who underwent spontaneous preterm labor, indicating the involvement of such cells in this pregnancy complication. Fetal ILCs exist in the liver, SLO, intestine, lung, and amniotic cavity. The fetal liver is thought to be the source of ILC progenitors since the differentiation of these cells from hematopoietic stem cells occurs at this site, and mature ILC subsets can be found in this compartment as well. The interaction between LTi cells and specialized stromal cells is important during the formation of SLO. Mature ILCs are found at the mucosal surfaces of the lung and intestine, from where they can extravasate into the amniotic cavity. Amniotic fluid ILCs express high levels of RORγt, CD161, and CD103, hallmarks of ILC3s. Such cells are more abundant in the second trimester than later in gestation. Although amniotic fluid ILC3s produce IL-17A and TNFα, indicating their functionality, their numbers in patients with intra-amniotic infection/inflammation remain unchanged compared to those without this pregnancy complication. Collectively, these findings suggest that maternal (uterine and decidual) ILCs play central roles in both the initiation and maintenance of pregnancy, and fetal ILCs participate in the development of immunity.

Knockdown of activated Cdc42-associated kinase inhibits human extravillous trophoblast migration and invasion and decreases protein expression of pho-Akt and matrix metalloproteinase

Introduction: The sufficient invasion and migration of human extravillous trophoblast (EVTs) cells are crucial for placentation. Inadequate invasion of trophoblasts may correlate with the development of preeclampsia. Many studies have suggested that activated Cdc42-associated kinase (ACK1) is associated with tumor metastasis and invasion. This study investigated the ACK1 expression and its function in trophoblasts during placental development.Methods: ACK1 expression in human placentas was determined through immunofluorescence. We investigated the migration/invasion of the immortalized human first-trimester EVT cell line HTR8/SVneo. Hypoxia–reoxygenation (H/R) conditions were applied to mimic preeclampsia model in vitro. Lentiviral vector-based short-hairpin RNA directed against the sequence of ACK1 (ACK1 shRNA) was used to knock down ACK1 expression in HTR8/SVneo cells. Cell apoptosis and proliferation were determined through flow cytometry and cell counting Kit-8 (CCK-8) assays, respectively. The expression of matrix metalloproteinase (MMP) 2/9 and tissue inhibitors of metalloproteinase (TIMP) 1/2 was measured by western blotting.Results: ACK1 localized within trophoblasts of human placental villi, decidual cells in the maternal decidua. ACK1 levels in preeclampsia (PE) placentas were significantly lower than those in controls. ACK1 shRNA significantly inhibited HTR8/SVneo cells migration and invasion but did not affect their apoptosis and proliferation. ACK1 knockdown decreased MMP2/9 and increased TIMP1/2 expression, as well as downregulated the phosphorylation of AKt (p-Akt). In addition, ACK1 and MMP2/9 were downregulated following treatment with LY294002, whereas ACK1 shRNA had no effect on phosphorylation of PI3K(p-PI3K). After exposed in H/R condition, ACK1 expression, MMP2/9 protein, and p-Akt were also significantly decreased.Discussion and conclusions: ACK1 expression is lowered in preeclamptic placentas and promotes trophoblast cell invasion, migration. H/R conditions decrease ACK1 expression and appear to decouple the positive relationship between ACK1 expression and Akt activation.

Histological evaluation of vitamin-D deficiency on the endometrial decidualization in early pregnancy in rat model

ABSTRACTIntroduction: Vitamin-D is a pleiotropic secosteroid hormone that exerts classical effect in calcium uptake and bonemetabolism. In addition, it has non-classical effects on variety of organs. Endometrial decidualization, placentation, and thedevelopment of the embryo itself have all pivotal roles for normal pregnancy outcomes after embryo implantation. Earlymaternal decidua and fetal trophoblast cells illustrate expression of vitamin-D receptor and 1α-hydroxylase enzyme thatcatalyzes vitamin-D into the active metabolite, calcitriol.Aim: To record the histological effect of vitamin-D deficiency on endometrial decidualization during early pregnancy on day8 post-coitum.Materials and Methods: Thirty female albino rats were divided into 2 groups: control group (GpI) and vitamin-D deficientgroup (GpII). After establishment of pregnancy, the animals were sacrificed on the 8th gestational day. The bicornuate pregnantuteri were processed for histological analysis using H&E and PAS stains in addition to immunohistochemical staining forosteopontin (OSP), as a marker for decidualization, and for CD34 to assess the decidual angiogenesis. Morphometric andstatistical studies were performed.Results: Substantial decrease in the number of implantation sites in vitamin-D deficient rats was noted by gross examination.Histological examination revealed significant decrease in endometrial thickness at the antimesometrial region with remarkablyimpaired stromal cells differentiation. PAS staining illustrated fewer PAS positively stained uterine natural killer cells comparedto control. Weak cytoplasmic OSP immunostaining was observed in the luminal as well as glandular epithelium, vascularendothelium and few stromal cells in mesometrial region. CD34 immunostaining demonstrated impaired angiogenesis in thelateral decidua as compared to control. These findings were confirmed by morphometric and statistical analysis.Conclusion: Vitamin-D deficiency impairs the uterine stromal cells differentiation, affects the immunity tolerance andweakens decidual angiogenesis during early pregnancy in rat model.

Progesterone and cyclic adenosine monophosphate down-regulate CD90 in the stromal cells of human decidua. In vitro evidence and in situ findings.

CD90 is a glycoprotein involved in leukocyte relocation and cell differentiation. CD90 is expressed in endothelial and stromal cells in human endometrium; however, its role in the remodeling of the decidual tissue during pregnancy is poorly understood. Here, we investigate how CD90 expression in decidual stromal cells (DSCs) is regulated. The native CD90 receptor in stromal cells in decidua was investigated via histology. We further develop in vitro culture of DSCs which allows us to test the effects of hormones and paracrine signals on CD90 expression. Stromal cells in first-trimester human decidua display heterogeneous levels of CD90 expression. In vitro analyses reveal that progesterone, a factor normally secreted by trophoblast cells in the placenta, and extracellular cyclic adenosine monophosphate, a known downstream signaling messenger of progesterone, reduce CD90 expression in DSCs by ~30%. This reduction in CD90 expression correlates with a change toward a more highly differentiated cell state. DSCs in early pregnancy show different levels of CD90 expression, suggesting different DSC differentiation and selective interactions with cells during decidual morphogenesis.

Natural Cytotoxicity Receptors in Decidua Natural Killer Cells of Term Normal Pregnancy.

Aim To investigate the changes in the maternal immune system at term pregnancy, we studied the expression of natural cytotoxicity receptors (NCRs) and the cytokine production of NK cells in term placenta decidua and peripheral blood. Methods Term decidua and peripheral blood were taken from patients undergoing elective cesarean section. The lymphocytes were separated using density gradient centrifugation (DGC) from peripheral blood and were separated from decidua using DGC after enzyme digestion. These cells were stained with FITC anti-CD56 and Per-CP anti-CD3 monoclonal antibodies, and the NCRs were stained with PE-conjugated anti-NKG2D, NKp46, NKp30, and NKp44 monoclonal antibodies. Cytokines, including IFN-γ, TNF-α, IL-10, and TGF-β, were stained and then analyzed by flow cytometry. Results There were fewer cells positive for NKG2D, NKp46, and NKp30 among CD56+CD3- cells in deciduas than in peripheral blood, but the percentages of NKp44-positive cells in CD56+CD3- lymphocytes in deciduas tended to be higher. Conclusion The decreased expression of some NCRs in deciduas may be related to decreased cytotoxicity at term pregnancy, but the increased expression of NKp44 may affect the increased cytokine production in the decidua. Similarly, the expression of NCRs in the decidua may be connected to the maintenance of pregnancy at term.

Unique Cytokine Profile of Novel Innate Lymphoid Cells in Human Decidua

Abstract Immune cells at the maternal-fetal interface play a complex role in regulation of vascular remodeling, fetal tolerance and protection from infection. We have previously presented the identification of two novel CD56highNKs based on Eomes expression (Eomeslo/hi). Activation of these subsets revealed preferential production of IL-8 and IL-17A by decidual innate lymphoid cells (ILCs). Decidual specimens were dissected from term placentas and mononuclear cells (MCs) were isolated by mechanical (GentleMACS) and enzymatic (Collagenase, DNAse) disruption. MCs were then simulated with PMA/ionomycin or IL-23, then labeled by flurochrome-conjugate antibodies against surface CD3, 14, 16, 19, 34, 45, 49a, 56, 94, 117, 127, 335 and intracellular Granzyme B, IL-8, IL-17A, IL-22, INFγ, TNFα. Data acquisition was performed using BD Fortessa flow cytometer in a 5 laser, 18 detector configuration and data analysis was performed using FlowJo 10.2. Activation of decidual ILCs revealed a unique milieu of cytokine production. The novel subsets, CD56high Eomeslo/hi, preferentially produced IL-8 and IL-17A, while decreasing production of INFγ, Granzyme B, and TNFα. In addition, as expected, ILC3s and LTi-like cells produced IL-17A and IL-22. Furthermore, differences in response were observed between decidual basalis and decidual parietalis ILCs, with decidual parietalis ILCs being more responsive to stimulation. Preliminary results suggest that both CD56high Eomeslo/hi subsets demonstrate a similar altered cytokine profile that is conducive to decidual remodeling. Further gene expression analysis will reveal if these novel subsets represent a truly unique population or are members of the expanding tissue-resident NK cell family.

Induction of Zika specific CD8 T cell responses in the placenta after heat shock protein gp96-Ig vaccination

Abstract Heat shock protein (HSP) gp96 is a biological adjuvant that activates antigen-presenting cells and simultaneously delivers antigen specific peptides to MHC I for CD8 T cell activation. Here, we have transformed human embryonic kidney cell line (HEK-293) into powerful miniature osmotic pumps that continually secrete engineered form of gp96, gp96-Ig, along with its chaperoned antigens (ZIKV envelope immunogenic peptides) to induce ZIKV specific CD8+ cytotoxic T cell immune responses. B6 females were mated with B6 or BALB/c males and gestation day (GD) 0.5 was determined by the presence of vaginal plug. At GD 7.5 one million of irradiated 293-gp96-Ig secreting cells were injected subcutaneously (SC). Mice were followed up until the end of pregnancy (GD 19.5–21.5) or humanely euthanized 5 days after vaccination (GD 12.5). Frequency of CD8+ T cells was determent by flow cytometry, immunohistochemistry and antigen, ZIKV envelope specificity was confirmed by in vitro ZIKV peptide stimulation and by intracellular cytokine staining. We found that vaccinated mice have the same number of live-born pups as control mice without pathological changes in placenta or in fetus. This is the first finding that confirms the presence of gp96-Ig-ZIKV-induced CD8 T cells around arteries and larger blood vessels in the maternal part of the placenta, decidua. Furthermore, secreted gp96 vaccine induces very high frequency of ZIKV specific CD8 T cells in the maternal decidua (15%) without compromising placental or fetal viability. In summary, we have succeeded in generating vaccine that is expressing gp96-Ig and ZIKV envelope protein and also we confirmed that vaccination with secreted gp96-Ig-ZIKA is safe and induces CD8 T cell responses in maternal decidua.

Infection-induced thrombin production: a potential novel mechanism for preterm premature rupture of membranes (PPROM)

Preterm premature rupture of membranes is a leading contributor to maternal and neonatal morbidity and death. Epidemiologic and experimental studies have demonstrated that thrombin causes fetal membrane weakening and subsequently preterm premature rupture of membranes. Although blood is suspected to be the likely source of thrombin in fetal membranes and amniotic fluid of patients with preterm premature rupture of membranes, this has not been proved. Ureaplasma parvum is emerging as a pathogen involved in prematurity, which includes preterm premature rupture of membranes; however, until now, prothrombin production that has been induced directly by bacteria in fetal membranes has not been described. This study was designed to investigate whether Ureaplasma parvum exposure can induce prothrombin production in fetal membranes cells. Primary fetal membrane cells (amnion epithelial, chorion trophoblast, and decidua stromal) or full-thickness fetal membrane tissue explants from elective, term, uncomplicated cesarean deliveries were harvested. Cells or tissue explants were infected with live Ureaplasma parvum (1×105, 1×106 or 1×107 colony-forming units per milliliter) or lipopolysaccharide (Escherichia coli J5, L-5014; Sigma Chemical Company, St. Louis, MO; 100 ng/mL or 1000 ng/mL) for 24 hours. Tissue explants were fixed for immunohistochemistry staining of thrombin/prothrombin. Fetal membrane cells were fixed for confocal immunofluorescent staining of the biomarkers of fetal membrane cell types and thrombin/prothrombin. Protein and messenger RNA were harvested from the cells and tissue explants for Western blot or quantitative reverse transcription polymerase chain reaction to quantify thrombin/prothrombin protein or messenger RNA production, respectively. Data are presented as mean values ±standard errors of mean. Data were analyzed using 1-way analysis of variance with post hoc Dunnett's test. Prothrombin production and localization were confirmed by Western blot and immunostainings in all primary fetal membrane cells and tissue explants. Immunofluorescence observations revealed a perinuclear localization of prothrombin in amnion epithelial cells. Localization of prothrombin in chorion and decidua cells was perinuclear and cytoplasmic. Prothrombin messenger RNA and protein expression in fetal membranes were increased significantly by Ureaplasma parvum, but not lipopolysaccharide, treatments in a dose-dependent manner. Specifically, Ureaplasma parvum at a dose of 1×107 colony-forming units/mL significantly increased both prothrombin messenger RNA (fold changes in amnion: 4.1±1.9; chorion: 5.7±4.2; decidua: 10.0±5.4; fetal membrane: 9.2±3.0) and protein expression (fold changes in amnion: 138.0±44.0; chorion: 139.6±15.1; decidua: 56.9±29.1; fetal membrane: 133.1±40.0) compared with untreated control subjects. Ureaplasma parvum at a dose of 1×106 colony-forming units/mL significantly up-regulated prothrombin protein expression in chorion cells (fold change: 54.9±5.3) and prothrombin messenger RNA expression in decidua cells (fold change: 4.4±1.9). Our results demonstrate that prothrombin can be produced directly by fetal membrane amnion, chorion, and decidua cells. Further, prothrombin production can be stimulated by Ureaplasma parvum exposure in fetal membranes. These findings represent a potential novel underlying mechanism of Ureaplasma parvum-induced rupture of fetal membranes.

Assessing whether progesterone-matured dendritic cells are responsible for retention of fertilization products in missed abortion

We hypothesize that progesterone causes tolerogenic maturation of myeloid dendritic cells (DCs) in human decidua of threatened miscarriage or missed abortion characterized by a distinct phenotype and cytokine production, including reduction of the main NK cell proliferation and cytotoxic factor interleukin (IL)-15. During DC/NK cell interaction, progesterone-shaped DCs cannot efficiently multiply or equip NK cells with the cytotoxic mediators peforin and granulysin, which might harm trophoblasts and induce abortion. We propose that the presence, and maturation stage of decidual myeloid DCs be investigated using semi-quantitative immunohistological analyses and/or double-color immuno-fluorescent labeling of DC lineage and activation markers. The spatial arrangement of granulysin+ cells, NKp46+ NK cells, DCs, and trophoblasts might provide information about their mutual interactions in vivo. Multiple flow cytometry analyses of NK-receptors would provide insight into NK cell activation status. NK cell activation status could be also assessed by cytotoxicity assays against trophoblast cell lines, or isolated cognate extra-villous trophoblast cells. A correlation between decidual progesterone concentration or IL-15 expression, and the degree of DC maturation or the frequency of granulysin+ cells, might help to elucidate the mechanism of abortion retention in utero.

GPR18 Distribution and Expression in Human Placenta

BackgroundOptimally functioning inflammation resolution systems during pregnancy are essential in regulating a series of complex processes that could degenerate into persistent inflammation and result in poor maternal‐child outcomes. Infants developing in a pro‐inflammatory intrauterine environment are at significantly higher risk for severe complications than infants born without pregnancy inflammatory risk factors.Specialized pro‐resolving lipid mediators (SPM) are chemical structures derived from polyunsaturated fatty acids. Resolution of acute inflammation is an active process regulated by SPM, including resolvin D2 (RvD2), a potent docosahexaenoic acid‐derived resolution‐phase mediator, which activates a cell surface G protein–coupled receptor, GPR18/RvD2.Preliminary data from our group indicate that the SPM RvD2 is significantly increased in maternal plasma taken at the time of delivery compared to cord blood plasma (3,450 vs. 689 pg/mL for maternal vs. cord, respectively, p&lt;0.001), with further increases in the maternal plasma of cases with adverse maternal‐fetal outcomes, including NICU admission. Based on this data, we hypothesize that the placenta is a target site of action of SPM during perinatal growth and development. We identified the expression of the RvD2 receptor, GPR18 by immunostain on decidual cells, smooth muscle of the maternal and fetal arteries and syncytiotrophoblast, cytotrophoblast and macrophages of chorionic villi. Since the decidual cells showed significant expression of GRP18 therefore, the objective of this study was to analyze and categorize the different levels of staining intensity in this cell type.MethodsTen cross‐sections of placental tissues containing both maternal and fetal sides were stained for SPM‐binding receptor, GPR18 immunostaining, and digitized at 40× magnification using VENTANA iScan HT scanner. Our novel approach including Definiens Tissue Studio® software was used to quantify the cellular analyses of decidual cells in these digitized images that quantify low, medium, and high expressions for GPR18.ResultsThe majority of decidual cells showed medium or high GPR18 membranous and cytoplasmic expression (Figure 1). 20% of decidua cells had no areas of low expression. Seven out of ten samples had areas of medium expression that were greater than 50%. Areas of low expression ranged from 0–31%, while areas of medium expression ranged from 11–81%, and high expression ranged from 5–75%.ConclusionsThe decidual cells of the placenta have medium/high membranous and cytoplasmic expression of GPR18. SPM may be an important component of the inflammation physiology in the placental tissue. The expression of GPR18 may play a protective role in pregnancy and perinatal disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Downregulated N-acetylglucosaminyltransferase III is involved in attenuating trophoblast migration and invasion under hypoxia–reoxygenation condition

Objective: Many studies have confirmed that N-acetylglucosaminyltransferase III (GnT-III) is correlated with tumor invasion and metastasis. However, the expression of GnT-III and its role in normal pregnancy and preeclampsia (PE) has not been reached. So the primary objective of this study is to determine GnT-III expression in normal pregnancy and whether its expression is vulnerable to oxidative stress in the trophoblast cells.Methods: Human first trimester villous tissues from normal pregnancies and third trimester placentas from pregnancies with or without preeclampsia (PE) were used for the detection of GnT-III expression. Human first trimester extravillous trophoblast cell line (HTR8/SVneo) exposed to hypoxia/reoxygenation (H/R) condition was employed as an oxidative stress model in vitro to investigate the expression of GnT-III.Results: GnT-III was strongly expressed in cytotrophoblast (CTBs), syncytiotrophoblast (STBs) and the trophoblast columns (TCs) of human placental villi, and decidual cells in the maternal decidua. The expression of GnT-III was decreased in PE placentas compared with the normal control placentas. In addition, GnT-III was found to have decreased expression in H/R-exposed HTR8/SVneo cells, and the invasive and migratory abilities of HTR8/SVneo cells were attenuated, too.Conclusions: These findings suggest that GnT-III is an important regulator at the maternal-fetal interface during early pregnancy. Excessive oxidative stress can decrease GnT-III expression in trophoblast and the decreased expression of GnT-III may be involved in the development of preeclampsia.

606: Placental alkaline phosphatase: Is it placenta-specific?

Placental alkaline phosphatase (PLAP), biochemically and genetically distinct from other human tissue alkaline phosphatases, is expressed by trophoblast cells and increases throughout gestation. Recently, PLAP has been used extensively to isolate placenta-derived exosomes from biological specimens and quantitative differences of PLAP-positive exosomes have been associated with various adverse pregnancy outcomes. By examining exosomes from other uterine tissues and immunostaining of various feto-maternal tissues, we tested the placental specificity of PLAP. Exosomes from amnion, decidua, and myometrial cell cultures were isolated by differential centrifugation and characterized by size and exosome markers. PLAP expression in exosomes was tested by western blot using a PLAP-specific antibody (ab133602, Abcam). Additionally, amniochorion with decidua, and myometrium were collected from normal term pregnancies and PLAP localization was performed using immunohistochemistry (IHC) using the same antibody. Exosomes isolated from BeWo (trophoblast cell line) were used as positive control. Exosomes from various cell types were round with a size of 150nm and positive for exosomal proteins CD81, CD9 and CD63. Reactivity to PLAP was seen in exosomes isolated from all cell types (amnion, decidua and myometrium) (Figure 1A). As expected, BeWo cells were positive for PLAP. This was further confirmed by IHC were PLAP was localized in fetal (amnion, chorion, and cells of the extracellular matrix) and maternal uterine tissues (decidua and myometrium) confirming PLAP production by these cell types (Figure 1B). Chorion had the highest intensity staining and was weaker in decidua and myometrium. PLAP is produced by fetal membranes, uterine decidua and myometrial tissues and packaged in their respective exosomes. This led us to speculate that PLAP-containing exosomes isolated from maternal biological samples throughout pregnancy are not necessarily trophoblast in origin, but represent all gestational tissues. PLAP-specific exosome based biomarker studies should be cautious in interpreting their data as those exosomes are not likely indicators of placental function alone but overall gestational tissue functions.

Effect of Total Flavones from Cuscuta Chinensis on Anti-Abortion via the MAPK Signaling Pathway.

For centuries, the Chinese herb Cuscuta chinensis has been applied clinically for abortion prevention in traditional Chinese medicine (TCM). Total flavones extracted from Cuscuta chinensis (TFCC) are one of the active components in the herb and also display anti-abortion effect similar to the unprocessed material. However, how TFCC exerts the anti-abortion effect remains largely unknown. In this study, we aim at characterizing the anti-abortion effects of TFCC and its underlying molecular mechanism in vitro and in vivo using human primary decidua cells and a mifepristone-induced abortion model in rat, respectively. The damage to the decidua caused by mifepristone in vivo was reversed by TFCC treatment in a dosage-dependent manner. High dosage of TFCC significantly upregulated the expression of estrogen receptor (ER), progesterone receptor (PR), and prolactin receptor (PRLR) in decidua tissue but downregulated the expression of p-ERK. Furthermore, we detected higher level of p-ERK and p-p38 in primary decidua cells from spontaneous abortion while treatment by TFCC downregulated their expression. Our results suggest TFCC mediates its anti-abortion effect by interfering with MAPK signaling pathway.

The role of indoleamine-2,3-dioxygenase in normal and pathological pregnancies.

The survival of allogeneic fetus during pregnancy contradicts the laws of immune responses. Behind this paradoxical phenomenon, the mechanism is quite complex. Indoleamine-2,3-dioxygenase (IDO) is the first and rate-limiting enzyme of tryptophan catabolism. Emerging evidence shows that IDO is expressed at the maternal-fetal interface, including trophoblast cells, decidual stroma cells, decidual immune cells (eg, natural killer cells, T cells, and macrophages), and vascular endothelial cells of decidua and chorion. Moreover, the expression and activity of IDO are different among non-pregnant, normal pregnant, and pathological pregnant conditions. IDO plays important roles in normal pregnancy through immune suppression and regulation of fetal invasion and circulation. However, the abnormal expression and dysfunction of IDO are associated with some pathological pregnancies (including recurrent spontaneous abortion, preeclampsia, preterm labor, and fetal growth restriction).