Showing papers on "Decidual cells published in 2018"

Single-cell reconstruction of the early maternal–fetal interface in humans

Abstract: During early human pregnancy the uterine mucosa transforms into the decidua, into which the fetal placenta implants and where placental trophoblast cells intermingle and communicate with maternal cells. Trophoblast-decidual interactions underlie common diseases of pregnancy, including pre-eclampsia and stillbirth. Here we profile the transcriptomes of about 70,000 single cells from first-trimester placentas with matched maternal blood and decidual cells. The cellular composition of human decidua reveals subsets of perivascular and stromal cells that are located in distinct decidual layers. There are three major subsets of decidual natural killer cells that have distinctive immunomodulatory and chemokine profiles. We develop a repository of ligand-receptor complexes and a statistical tool to predict the cell-type specificity of cell-cell communication via these molecular interactions. Our data identify many regulatory interactions that prevent harmful innate or adaptive immune responses in this environment. Our single-cell atlas of the maternal-fetal interface reveals the cellular organization of the decidua and placenta, and the interactions that are critical for placentation and reproductive success.

The mammalian decidual cell evolved from a cellular stress response.

Abstract: Among animal species, cell types vary greatly in terms of number and kind. The number of cell types found within an organism differs considerably between species, and cell type diversity is a significant contributor to differences in organismal structure and function. These observations suggest that cell type origination is a significant source of evolutionary novelty. The molecular mechanisms that result in the evolution of novel cell types, however, are poorly understood. Here, we show that a novel cell type of eutherians mammals, the decidual stromal cell (DSC), evolved by rewiring an ancestral cellular stress response. We isolated the precursor cell type of DSCs, endometrial stromal fibroblasts (ESFs), from the opossum Monodelphis domestica. We show that, in opossum ESFs, the majority of decidual core regulatory genes respond to decidualizing signals but do not regulate decidual effector genes. Rather, in opossum ESFs, decidual transcription factors function in apoptotic and oxidative stress response. We propose that rewiring of cellular stress responses was an important mechanism for the evolution of the eutherian decidual cell type.

Zika Virus Replicates in Proliferating Cells in Explants From First-Trimester Human Placentas, Potential Sites for Dissemination of Infection.

Abstract: Background Maternal Zika virus (ZIKV) infection with prolonged viremia leads to fetal infection and congenital Zika syndrome. Previously, we reported that ZIKV infects primary cells from human placentas and fetal membranes. Here, we studied viral replication in numerous explants of anchoring villi and basal decidua from first-trimester human placentas and midgestation amniotic epithelial cells (AmEpCs). Methods Explants and AmEpCs were infected with American and African ZIKV strains at low multiplicities, and ZIKV proteins were visualized by immunofluorescence. Titers of infectious progeny, cell proliferation, and invasiveness were quantified. Results In anchoring villus, ZIKV replicated reproducibly in proliferating cytotrophoblasts in proximal cell columns, dividing Hofbauer cells in villus cores, and invasive cytotrophoblasts, but frequencies differed. Cytotrophoblasts in explants infected by Nicaraguan strains were invasive, whereas those infected by prototype MR766 largely remained in cell columns, and titers varied by donor and strain. In basal decidua, ZIKV replicated in glandular epithelium, decidual cells, and immune cells. ZIKV-infected AmEpCs frequently occurred in pairs and expressed Ki67 and phosphohistone H3, indicating replication in dividing cells. Conclusions ZIKV infection in early pregnancy could target proliferating cell column cytotrophoblasts and Hofbauer cells, amplifying infection in basal decidua and chorionic villi and enabling transplacental transmission.

Decidual cell regulation of trophoblast is altered in pregnancies at risk of pre-eclampsia.

Abstract: Successful implantation and placentation are dependent on the interaction between decidual stromal cells (DSC) and extravillous trophoblast (EVT) cells. The extent of trophoblast invasion relies on communication between the placenta and maternal decidua. The cyclical process of decidualisation induces a transformation of endometrial fibroblasts to secretory DSC; these secreted products have many functions including the control of trophoblast invasion. Inadequate trophoblast invasion and remodelling of the uterine vessels (the spiral arteries) are associated with pregnancy disorders such as pre-eclampsia. Uterine artery Doppler resistance index (RI) in the first trimester of pregnancy can be used as a proxy measure of remodelling. DSC were isolated from pregnancies with normal (normal RI) or impaired (high RI) spiral artery remodelling. Following isolation, DSC were re-decidualised using cAMP and MPA and secretion of the decidualisation markers IGFBP-1 and prolactin assessed. We examined the impact of DSC-secreted factors on trophoblast cell function, using the EVT cell line SGHPL-4. We demonstrated that DSC exposed to decidual factors were able to re-decidualise in vitro and that the chemoattraction of trophoblasts by DSC is impaired in pregnancies with high RI. This study provides new insights into the role that DSC play in regulating EVT functions during the first trimester of pregnancy. This is the first study to demonstrate that DSC from pregnancies with impaired vascular remodelling in the first trimester secrete factors that inhibit the directional movement of trophoblast cells. This finding may be important in understanding aberrant trophoblast invasion in pregnancies where vascular remodelling is impaired.

The Glycosyltransferase EOGT Regulates Adropin Expression in Decidualizing Human Endometrium.

Abstract: In pregnancy, resistance of endometrial decidual cells to stress signals is critical for the integrity of the feto-maternal interface and, by extension, survival of the conceptus. O-GlcNAcylation is an essential post-translational modification that links glucose sensing to cellular stress resistance. Unexpectedly, decidualization of primary endometrial stromal cells (EnSCs) was associated with a 60% reduction in O-GlcNAc modified proteins, reflecting downregulation of the enzyme that adds O-GlcNAc to substrates (O-GlcNAc transferase, OGT) but not the enzyme that removes the modification (O-GlcNAcase, OGA). Notably, EOGT, an endoplasmic reticulum-specific O-GlcNAc transferase that modifies a limited number of secreted and membrane proteins, was markedly induced in differentiating EnSCs. Knockdown of EOGT perturbed a network of decidual genes involved in multiple cellular functions. The most downregulated gene upon EOGT knockdown in decidualizing cells was ENHO, which encodes adropin, a metabolic hormone involved in energy homeostasis and glucose and fatty acid metabolism. Analysis of mid-luteal endometrial biopsies revealed an inverse correlation between endometrial EOGT and ENHO expression and body mass index. Taken together, our findings reveal that obesity impairs the EOGT-adropin axis in decidual cells, which in turn points towards a novel mechanistic link between metabolic disorders and adverse pregnancy outcome. [Abstract copyright: Copyright © 2017 Endocrine Society.]

Down-regulation of miR-378a-3p induces decidual cell apoptosis: a possible mechanism for early pregnancy loss.

Abstract: Study question Do microRNAs (miRNAs) contribute to human early pregnancy loss (EPL)? Summary answer miR-378a-3p expression is regulated by progesterone and is down-regulated in ducidua of EPL patients which may contribute to decidual apoptosis through Caspase-3 activation. What is known already A variety of miRNAs have been demonstrated to be associated with the development of decidualization and placental formation. However, little has been reported on the roles of miRNA in the pathogenesis of EPL. Study design, size, duration Normal and EPL decidual tissues were collected from patients with normal pregnancies undergoing elective termination of gestation, and from patients with EPL, respectively. Participants/materials, setting, methods miRNA microarrays were used to identify the differentially expressed miRNAs between normal and EPL decidua, and miRNA expression was confirmed by qRT-PCR, qRT-PCR, western blotting and luciferase reporter assays were employed to validate the downstream targets of miR-378a-3p. The effects of miR-378a-3p were evaluated using miR-378a-3p-transfected decidual cells. Main results and the role of chance Of note, 32 up-regulated miRNAs and 38 down-regulated miRNAs were identified by microarray analysis when comparing EPL to normal decidua. MiR-378a-3p was significantly down-regulated in the EPL decidua and was found to inversely regulate the expression of Caspase-3 by directly binding to its 3'-UTRs. In decidual cells, transfection of miR-378a-3p mimics resulted in the inhibition of cell apoptosis and in the increase of cell proliferation through Caspase-3 suppression. Moreover, we found that progesterone could induce the expression of miR-378a-3p in decidual cells. Limitations, reasons for caution This study focused on the function of miR-378a-3p and its target Caspase-3, however, numerous other targets and miRNAs may also be responsible for the pathogenesis of EPL. Therefore, further studies are required to elucidate the role of miRNAs in EPL. Wider implications of the findings Our findings indicate that miR-378a-3p may contribute to the development of EPL, and that it could serve as a new potential predictive and therapeutic target of progesterone-treatment for EPL. Study funding/competing interest This study was supported by National Basic Research Program of China (No.2012CB944900); National Science Foundation of China (No.31471405 and 81490742, No.81361120246); The National Science and Technology Support Program (No.2012BA132B00). Authors declare no competing interests.

Decreased autophagy was implicated in the decreased apoptosis during decidualization in early pregnant mice

Abstract: Folate deficiency is a major risk factor of birth defects. Mechanistic studies on folate deficiency resulting in birth defects have mainly focused on fetal development. There have been few studies on folate deficiency from the point of view of the mother's uterus. In our previous study, we demonstrated that folate deficiency inhibits apoptosis of decidual cells, thereby restraining decidualization of the endometrium and impairing pregnancy. In this study, we further investigated the potential mechanism by which folate deficiency decreases endometrial apoptosis during decidualization. To investigate whether endometrium autophagy was inhibited under folate deficiency during decidualization, we performed real-time PCR for endometrial LC3 and P62 on day 6 (D6) to D8 of pregnancy in mice, and both were significantly changed compared to non-folate-deficient mice. Western blots showed that LC3-II and P62 were also changed in folate-deficient mice. Compared with control mice, a few punctuate LC3-II structures were detected in the folate deficiency group by immunofluorescence. Transmission electron micrographs of decidual cells on D8 showed that there were no evident autophagosomes in the folate deficiency group. In addition, apoptosis-related protein analysis by western blotting, TUNEL staining and flow cytometry showed that decreased endometrial apoptosis on D8 of pregnancy under folate deficiency was reversed after treatment with rapamycin, an autophagy inducer. ROS measurement showed that the endometrium ROS level was reduced by folate deficiency and that rapamycin reversed this effect on day 8 of pregnancy. All the results suggest that inhibiting endometrial autophagy may be implicated in the decreased endometrial apoptosis under folate deficiency during decidualization.

Dysfunction of DNA damage-inducible transcript 4 in the decidua is relevant to the pathogenesis of preeclampsia.

Abstract: Preeclampsia (PE) is a pregnancy-related disorder that occurs after 20 weeks of gestation and affects 3-5% of all human pregnancies worldwide. However, the pathogenesis of PE still remains poorly understood. A deficiency in decidualization is considered a contributing factor to the development of PE. The DNA damage inducible transcript 4 (DDIT4) gene encodes a protein whose main function is inhibiting mammalian target of rapamycin (mTOR) under stress, and several studies have demonstrated that its expression promotes tumor cell apoptosis. Our previous RNA-Seq results showed that DDIT4 is significantly decreased in the decidua of PE women. Here, we aimed to define the role of DDIT4 in human decidualization and its relationship with PE. The results indicated that DDIT4 was markedly decreased in the decidua of severe PE compared with those from uncomplicated pregnancies. The expression of DDIT4 in human endometrial stromal cell (hESC) line and primary hESCs was up-regulated during decidualization. Knockdown DDIT4 in hESCs and primary hESCs caused a significant reduction in the transcription of decidualization markers, insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL). In addition, silencing DDIT4 caused up-regulated p-mTOR and p-p70s6k and reduced apoptosis, whereas rapamycin, an inhibitor of mTOR, reversed the result of apoptosis. Moreover, the expression of cleaved-caspase 3 in severe PE was significantly lower than that of uncomplicated pregnancies, which was unfavorable for trophoblast invasion. Our data suggest that DDIT4 is critical for normal decidualization and the apoptosis of decidual cells. DDIT4 deficiency is likely involved in the development of PE.

Granulysin-mediated apoptosis of trophoblasts in blighted ovum and missed abortion.

Abstract: Problem Granulysin (GNLY) is a cytotoxic molecule mostly present in decidual natural killer (NK) cells. Blighted ovum (BO) and missed abortion (MA) represent the early pathological pregnancies with hindered development of the embryoblast or a dead embryo. We investigated the GNLY-mediated apoptotic mechanism potentially responsible for delayed termination of pregnancy. Method of study We performed immunohistological and immunofluorescence labeling of decidual tissues (GNLY, Apaf-1, NF-κB). NKG2A expression was analyzed by flow cytometry and GNLY mRNA by RT-qPCR. Results The GNLY labeling intensity (H score) was lower in the nuclei of trophoblast cells in BO and MA. GNLY gene levels were inversely detected in BO and MA. A decreased decidual NK cell percentage was found in MA. NK cells from pathological pregnancies expressed lower NKG2A levels. The highest frequency of Apaf-1 was found in trophoblast cells of MA. NF-kB was highly expressed in decidual cells of BO. Conclusion The reduced activation of GNLY-mediated killing might be implicated in the slower rejection of trophoblast cells in BO and MA. A decreased authentic decidual NK cell number could be responsible for low cytotoxicity against trophoblast cells in MA. In BO, trophoblast cells have a higher survival potential due to increased NF-kB expression.

High Mobility Group Box 1 Regulates Uterine Decidualization through Bone Morphogenetic Protein 2 and Plays a Role in Kruppel-Like Factor 5-Induced Stromal Differentiation.

Abstract: Background/aims High mobility group box 1 (Hmgb1) is associated with a variety of physiological processes including embryonic development, cell proliferation and differentiation, but little information is available regarding its biological role in decidualization. Methods In situ hybridization, real-time PCR, RNA interference, gene overexpression and MTS assay were used to analyze the spatiotemporal expression of Hmgb1 in mouse uterus during the pre-implantation period, and explore its function and regulatory mechanisms during uterine decidualization. Results Hmgb1 mRNA was obviously observed in uterine epithelium on day 2 and 3 of pregnancy, but its expression was scarcely detected on day 4 of pregnancy. With the onset of embryo implantation, abundant Hmgb1 expression was noted in the subluminal stromal cells around the implanting blastocyst at implantation sites. Meanwhile, the accumulation of Hmgb1 mRNA was visualized in the decidual cells. Hmgb1 advanced the proliferation of uterine stromal cells and induced the expression of prolactin family 8, subfamily a, member 2 (Prl8a2), a reliable differentiation marker for decidualization. In uterine stromal cells, cAMP analogue 8-Br-cAMP up-regulated the expression of Hmgb1, but the up-regulation was abrogated by protein kinase A (PKA) inhibitor H89. Silencing of Hmgb1 by specific siRNA impeded the induction of 8-Br-cAMP on Prl8a2. Further analysis evidenced that Hmgb1 was a critical mediator of Kruppel-like factor 5 (Klf5) function in stromal differentiation. Knockdown of bone morphogenetic protein 2 (Bmp2) prevented the up-regulation of Prl8a2 elicited by Hmgb1 overexpression, whereas addition of exogenous recombinant Bmp2 protein (rBmp2) reversed the repression of Hmgb1 siRNA on Prl8a2 expression. Conclusion Hmgb1 may play an important role during mouse uterine decidualization.

Reconstruction of the Decidual Pathways in Human Endometrial Cells Using Single-Cell RNA-Seq

Abstract: The fate of the human endometrium is determined during the mid-luteal window of implantation, coinciding with differentiation of endometrial stromal cells (EnSCs) into specialized decidual cells. In response to successful embryo implantation, differentiating EnSCs transform the endometrium into a decidua that maintains the placenta throughout gestation; whereas falling progesterone levels in the absence of pregnancy lead to tissue destruction and menstrual shedding. We used single-cell RNA sequencing (scRNA-seq) to map the temporal transcriptomic changes in cultured EnSCs along a decidual time-course and in response to withdrawal of differentiation signals. We demonstrate that decidual transformation of EnSCs first involves a continuous and largely synchronous transition through intermediate states before emerging as divergent subpopulations, representing mature decidual cells and stressed cells. Subsequent withdrawal of decidual signals imposes a second branching event, driven primarily by de-differentiation of a subset of cells. Further, scRNA-seq analysis of timed biopsies highlighted endometrial cellular complexity, confirmed the preponderance of different EnSC subpopulations upon progression from mid- to late-luteal phase, and uncovered genes with conserved branching dynamics in vivo and in vitro. Taken together, our single-cell analysis indicates that multiple EnSC states and dynamic subpopulations of decidual cells underpin endometrial fate decisions during the window of implantation.

Evolution of embryo implantation was enabled by the origin of decidual cells in eutherian mammals

Abstract: Embryo implantation is the first step in the establishment of pregnancy in eutherian (Placental) mammals. Although viviparity evolved prior to the common ancestor of marsupials and eutherian mammals (therian ancestor), implantation is unique to eutherians. The ancestral therian pregnancy likely involved a short phase of attachment between the fetal and maternal tissues followed by parturition rather than implantation, similar to the mode of pregnancy found in marsupials such as the opossum. Embryo implantation in eutherian mammals as well as embryo attachment in opossum, induce a homologous inflammatory response in the uterus. Here, we elucidate the evolutionary mechanism by which the ancestral inflammatory fetal-maternal attachment was transformed into the process of implantation. We performed a comparative transcriptomic and immunohistochemical study of the gravid and non-gravid uteri of two eutherian mammals, armadillo ( Dasypus novemcinctus ) and hyrax ( Procavia capensis ); a marsupial outgroup, opossum ( Monodelphis domestica ); and compared it to previously published data on rabbit ( Oryctolagus cuniculus ). This taxon sampling allows inference of the eutherian ancestral state. Our results show that in the eutherian lineage, the ancestral inflammatory response was domesticated by suppressing a detrimental component viz. signaling by the cytokine IL17A, while retaining components that are beneficial to placentation, viz. angiogenesis, vascular permeability, remodeling of extracellular matrix. IL17A mediates recruitment of neutrophils to inflamed mucosal tissues, which, if unchecked, can damage the uterus as well as the embryo and lead to expulsion of the fetus. We hypothesized that the uterine decidual stromal cells, which evolved coincidentally with embryo implantation, evolved, in part, to prevent IL17A-mediated neutrophil infiltration. We tested a prediction of this hypothesis in vitro , and showed that decidual stromal cells can suppress differentiation of human naive T cells into IL17A-producing Th17 cells. Together, these results provide a mechanistic understanding of early stages of the evolution of the eutherian mode of pregnancy, and also identify a potentially ancestral function of an evolutionary novelty, the decidual stromal cell-type.

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae.

Abstract: The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

The mammalian decidual cell evolved from a cellular stress response

Abstract: Among animal species, cell types vary greatly in terms of number and kind. The broad range of number of cell types among species suggests that cell type origination is a significant source of evolutionary novelty. The molecular mechanisms giving rise to novel cell types, however, are poorly understood. Here we show that a novel cell type of eutherian mammals, the decidual stromal cell (DSC), evolved by rewiring an ancestral cellular stress response. We isolated the precursor cell type of DSCs, endometrial stromal fibroblasts (ESFs), from the opossum Monodelphis domestica. We show that,in opossum ESF, the majority of decidual core regulatory genes respond to decidualizing signals, but do not regulate decidual effector genes. Rather, in opossum ESF, decidual transcription factors function in apoptotic and oxidative stress response. We propose that the rewiring of cellular stress responses could be a general mechanism for the evolution of novel cell types.

Egr2 mediates the differentiation of mouse uterine stromal cells responsiveness to HB-EGF during decidualization.

Abstract: Although Egr2 is involved in regulating the folliculogenesis and ovulation, there is almost no data describing its physiological function in embryo implantation and decidualization. Here, we showed that Egr2 mRNA was distinctly accumulated in subluminal stromal cells around implanting blastocyst on day 5 of pregnancy as well as in estrogen-activated implantation uterus. Estrogen induced the expression of Egr2 in uterine epithelia. Elevated expression of Egr2 mRNA was also observed in the decidual cells. Silencing of Egr2 by specific siRNA weakened the proliferation of uterine stromal cells and reduced the expression of Ccnd1, Ccnd3, Cdk4, and Cdk6. Furthermore, Egr2 advanced the expression of Prl8a2, Prl3c1, and Pgr, the well-established differentiation markers for decidualization. Administration of exogenous recombinant heparin-binding EGF-like growth factor (rHB-EGF) to uterine stromal cells resulted in an increase in the level of Egr2 mRNA. Moreover, siRNA-mediated attenuation of Egr2 impeded the stimulation of HB-EGF on stromal cell differentiation. Knockdown of Egr2 led to a reduction in the expression of Cox-2, mPGES-1, Vegf, Trp53, and Mmp2. Further analysis found that Egr2 may serve as an intermediate to mediate the regulation of HB-EGF on Cox-2, mPGES-1, Vegf, Trp53, Mmp2, and Ccnd3. Collectively, Egr2 may play an important role during embryo implantation and decidualization.

Decidualization modulates a signal transduction system via protease‐activated receptor‐1 in endometrial stromal cells

Abstract: Problem Decidual cells are thought to be involved in the maintenance of pregnancy. We conducted this study to evaluate the cellular function of endometrial stromal cells (ESCs) transitioning to decidualization. Methods of study Normal endometrial specimens were obtained from premenopausal patients who had undergone hysterectomies for subserosal leiomyomas. Decidualization of the ESCs (DSCs) was induced by incubating subconfluent cells in media containing medroxyprogesterone acetate and dibutyryl-cyclic adenosine monophosphate. We first analyzed the expression profile of protease-activated receptor-1 (PAR-1) between ESCs and DSCs. To investigate the intracellular signal transduction system in the DSCs, we incubated cells with thrombin receptor activator peptide 6 (TRAP-6). The levels of IL-8, monocyte chemo-attractant protein-1, matrix metalloproteinase (MMP)-1, and vascular endothelial growth factor in the culture medium were measured by enzyme-linked immunosorbent assays. The activation of the MAP kinase signaling pathway was detected by a Western blot analysis. The activation was evaluated for the expression of p21. Results PAR-1 receptor expression is upregulated in DSCs. The productions of chemokine and MMP-1 increased in the DSCs with the addition of TRAP-6. The activity of both the ERK-1 and ERK-2 isoforms was increased by 5-15 minute after TRAP-6 treatment. p70 S6 kinase showed the strongest expression after 1 hour. p21 was strongly observed in ESCs compared to the DSCs. Conclusion Our results suggest that cell function is changed by decidualization in association with increasing PAR-1 expression. The upregulation of PAR-1 may have some influence on pregnancy in the decidua.