Apoptosis in uterine epithelium and decidua in response to implantation: evidence for two different pathways

During the initial steps of implantation, the mouse uterine epithelium of the implantation chamber undergoes apoptosis in response to the interacting blastocyst. With progressing implantation, regression of the decidual cells allows a restricted and coordinated invasion of trophoblast cells into the maternal compartment. In order to investigate pathways of apoptosis in mouse uterine epithelium and decidua during early pregnancy (day 4.5–7.0 post coitum), we have investigated different proteins such as TNFalpha, TNF receptor1, Fas ligand, Fas receptor1, Bax and Bcl2 as well as caspase-9 and caspase-3 using immunohistochemistry. To detect cells undergoing apoptosis the Tunel assay was performed. Immunoreactivity for TNFalpha as well as for TNF receptor1 was observed exclusively in the epithelium of the implantation chamber and the adjacent luminal epithelium from day 4.5 post coitum onwards. In the developing decidua the Fas ligand, but not the Fas receptor, was expressed. Bax and Bcl2 revealed a complementary expression pattern with Bax in the primary and Bcl2 in the adjacent decidual zone. Strong immunolabelling for the initiator caspase-9 was restricted to the decidual compartment, whereas caspase-3 expression characterized the apoptotic uterine epithelium. Only some caspase-3 positive decidual cells were found around the embryo which correlated to the pattern of Tunel staining. Taken together, the apoptotic degeneration of the uterine epithelium seems to be mediated by TNF receptor1 followed by caspase-3, whereas the very moderate regression of the decidua did not show the investigated death receptor, but Bax and Blc2 instead and in addition caspase-9, which indicates a different regulation for epithelial versus decidual apoptosis.     

Decidual natural-killer-cell interaction with trophoblast: cytolysis or cytokine production?

At the implantation site, the uterine mucosa (decidua) is infiltrated by large numbers of natural killer (NK) cells. These NK cells are in close contact with the invading fetal trophoblast and we have proposed that they might be the effector cells that control the implantation of the allogeneic placenta. Recent characterization of NK cell receptors and their HLA class I ligands has suggested potential mechanisms by which NK cells might interact with trophoblast. However, what happens as a result of this interaction is not clear. The traditional method for investigating NK cell function in vitro is the protection from lysis of target cells by expression of HLA class I antigens. This might not be an accurate reflection of what happens in vivo. Another function of NK cells is the production of cytokines on contact with target cells. This could be an important outcome of the interaction between decidual NK cells and trophoblast. Decidual NK cells are known to produce a variety of cytokines; trophoblast cells express receptors for many of these cytokines, indicating that they can potentially respond. In this way, decidual NK cells have a significant influence on trophoblast behaviour during implantation.     

Murine T cell determination of pregnancy outcome.

At the fetomaternal interface, maternal effector cells come in intimate contact with fetal trophoblast cells which express paternal antigens. Failure of fetal trophoblast cells to activate maternal Th1 immune responses has been attributed in part to the absence of classical Class I and Class II major histocompatibilty complex (MHC) antigen expression and elaboration of factors which reduce TcR expression and shift any immune responses which may occur to Th2. Classical TcR alphabeta(+) T cells have not been found to be able to respond to trophoblasts. Recently, TcR gammadelta(+) T cells have been characterized in the low-abortion-rate pregnant C57Bl/10 mouse decidua, and the Vgamma1(+) subset may be able to respond to trophoblasts in a non-MHC-dependent manner. Trophoblast-recognizing T cells with Vgamma1 receptors are also present in the decidua of CBA/J mice pregnant by DBA/2, an abortion-prone mating combination. To test the role of the Vgamma1 subset of decidual gammadelta T cells in abortion-prone pregnancies, we altered this subset by injecting monoclonal anti-Vgamma1.1 antibody on gestation day 5.5, 1 day after implantation. This reduced detectability of a Vgammadelta subset producing TNF-alpha and reduced the abortion rate. Anti-Vgamma2, which reacts with a similar proportion of decidual gammadelta T cells as anti-Vgamma1.1, failed to prevent abortions. Vdelta6.3(+) cells are prominent at the fetomaternal interface, and anti-Vdelta6 antibody injected on day 5.5 prevented abortions. TGF-beta2(+) gammadelta cells first appear on day 8.5 of pregnancy; anti-Vgamma1.1 antibody injection on day 8.5 depleted these cells and boosted abortions; anti-Vdelta6.3 given on day 8.5 boosted abortions to the same level. These results suggest that two populations of Vgamma1.1(+)delta6.3(+) T cells may arise in the decidua: an early population that is Th1, abortogenic, and present during the time of implantation, and a Th2/3 cell subset that is present in the decidua later during pregnancy and which is pregnancy-protective.     

Subtilisin proprotein convertase-6 expression in the mouse uterus during implantation and artificially induced decidualization

During implantation, a balance of factors regulates the invasive properties of the embryo and the anti-invasive properties of uterine decidua. Although antiproteinases such as the metalloproteinase inhibitor TIMP-3 are thought to play critical roles in preventing the overaggressive invasion of trophoblasts, the mechanism of antiproteinase regulation is unknown. Recently, the prohormone convertase SPC-6 has been found to be co-expressed in embryo-proximal decidua in association with TIMP-3. As members of this serine proteinase family are known to activate latent TGFbeta family members which regulate decidual TIMP-3 levels, we sought to characterize the expression of SPC-6 during pregnancy and artificial decidualization. In this study, we demonstrate that the zone of SPC-6 gene expression exhibits a great degree of temporal and spatial overlap with TIMP-3 gene expression in uterine decidua from E5.5 through to E8.5. Like TIMP-3, we demonstrate that SPC-6 expression is induced during the decidual cell response using an in vivo model of artificial decidualization. Both the secreted and membrane bound forms of SPC-6 are expressed throughout the period of decidualization, suggesting that SPC-6 may play multiple roles during this developmental period. This is confirmed by our observation of the movement of SPC-6 expression to the presumptive placental region, as TIMP-3 expression regresses at the implantation site.     

Induction of cytotoxic T-lymphocyte antigen-2beta, a cysteine protease inhibitor in decidua: a potential regulator of embryo implantation

During early pregnancy, the steroid hormone progesterone induces differentiation of uterine stroma to decidual cells, which regulate embryo-uterine interactions. The progesterone-induced signaling molecules that participate in the formation and function of decidua remain poorly understood. We recently utilized high-density oligonucleotide microarrays to identify several genes whose expression is markedly altered in pregnant uterus in response to RU486, a well characterized antagonist of the progesterone receptor (PR). Our study revealed that the gene encoding cytotoxic T-lymphocyte antigen-2beta (CTLA-2beta), a cysteine protease inhibitor, is expressed during PR-induced decidualization. The spatio-temporal expression of CTLA-2beta mRNA precisely overlapped with the decidual phase of pregnancy. Interestingly, administration of progesterone to estrogen-primed ovariectomized mice failed to induce CTLA-2beta expression. A concomitant artificial decidual stimulation was necessary to trigger this expression. Uteri of PR knockout mice failed to express this mRNA, even after a combined administration of steroid hormones and artificial stimulation. The uterine expression of CTLA-2beta was, therefore, dependent on PR as well as other unknown factor(s) associated with decidual response. To identify the molecular target(s) of CTLA-2beta,we analyzed its interaction with proteins present in soluble extracts prepared from day 7 pregnant uteri containing implanted embryos. A protein affinity strategy employing recombinant CTLA-2beta helped us to determine that cathepsin L, a cysteine protease, is one of its targets in the pregnant uterus. Consistent with this finding, expression of cathepsin L was detected in the giant trophoblast cells of the ectoplacental cone on day 7 of pregnancy. Collectively, our results support the hypothesis that expression of CTLA-2beta in the decidua may regulate implantation of the embryo by neutralizing the activities of one or more proteases generated by the proliferating trophoblast.     

Localization of transforming growth factor-beta at the human fetal-maternal interface: role in trophoblast growth and differentiation.

We examined the localization of transforming growth factor (TGF)-beta in first-trimester and term human decidua and chorionic villi and explored the role of this factor on the proliferation and differentiation of cultured trophoblast cells. Two antibodies, 1D11.16.8, a mouse monoclonal neutralizing antibody capable of recognizing both TGF-beta 1 and TGF-beta 2 and CL-B1/29, a rabbit polyclonal antibody capable of recognizing TGF-beta 2, were used to immunolocalize TGF-beta in fixed, paraffin-embedded, or fixed, frozen sections of placenta and decidua, providing similar results. Intense labeling was observed in the extracellular matrix (ECM) of the first-trimester decidua and cytoplasm of term decidual cells. Syncytiotrophoblast cell cytoplasm as well as the ECM in the core of the chorionic villi of both first-trimester and term placentas exhibited a moderate degree of labeling. Strong cytoplasmic labeling was observed in the cytotrophoblastic shell of the term placenta. To examine the role of TGF-beta on trophoblast proliferation and differentiation, early passage cultures of first-trimester and primary cultures of term trophoblast cells were established and characterized on the basis of numerous immunocytochemical and functional markers. These cells expressed cytokeratin, placental alkaline phosphatase, urokinase-type plasminogen activator, and pregnancy-specific beta glycoprotein, but not factor VIII or 63D3; they also produced hCG and collagenase type IV. Exposure of first-trimester trophoblast cultures to TGF-beta 1 significantly inhibited proliferation in a dose-dependent manner. An antiproliferative effect was also noted in the presence of TGF-beta 2. These effects were abrogated in the presence of the neutralizing anti-TGF-beta antibody (1D11.16.8) in a concentration-dependent manner. In a 3-day culture, exogenous TGF-beta 1 stimulated formation of multinucleated cells by the first trimester as well as term trophoblast cells. Addition of neutralizing anti-TGF-beta antibody to first-trimester trophoblast cells stimulated proliferation beyond control levels in a 24-h culture and reduced formation of multinucleated cells in a 3-day culture, indicating the presence of endogenous TGF-beta activity. These results indicate that TGF-beta produced at the human fetal-maternal interface plays a major regulatory role in the proliferation and differentiation of the trophoblast.     

Localization of type I interferon in murine trophoblast and decidua during decidual formation.

Mouse trophoblast and decidua were examined by means of immunohistochemistry to define the localization of type I interferon. The decidua were stained for type I interferon at the time of implantation. The strong reaction was first observed in the primary decidual zone on day 5 and subsequently in the secondary decidual zone on day 6. After day 10, the decidua basalis and decidua capsularis showed a strong reaction. At the one-cell stage, embryos were weakly labelled, but a positive reaction was recognized in compacted morulae. Blastocysts on days 3 and 4 were positive in trophoblast and inner cell mass and a strong reaction was observed in the primitive endoderm on day 4. The visceral endoderm on day 5 and the trophoblast on day 6 were positive. After day 10, the trophoblast giant cells, labyrinth, visceral yolk sac and fetal blood cells gave a positive reaction. This study is the first demonstration of type I interferon localization in situ in mouse trophoblast and decidua during decidual formation.     

Dendritic cells in the human decidua

Dendritic cells (DCs) in the pregnant human uterine mucosa have been poorly characterized, although they are likely to regulate immune responses to both placental trophoblast cells and uterine infections. In this study an HLA-DR+, CD11c+ lin- (CD3-, CD19-, CD56-, CD14-) population has been identified by three-color flow cytometry. The cell isolates were prepared either by collagenase digestion or mechanically from first-trimester decidual tissue. The decidual DCs comprised approximately 1.7% of CD45+ cells in the isolates and had the phenotype of immature myeloid DCs. No CD1a+ Langerhans cells or CD123+ plasmacytoid DCs were detected. The decidual DCs were DC-SIGN-, DEC-205+, CD40+. Two subsets could be distinguished on the basis of relative expression of HLA-DR, which also differed in expression of DC-activation markers. The DCs were identified in situ by immunohistology by DEC-205 staining. Cells with dendritic processes were found scattered through both the decidua basalis (in which trophoblast cells are infiltrating) and the decidua parietalis. They were also visible in endothelial-lined spaces. This is the first study to identify and describe the phenotype and distribution of human decidual DCs.     

Maternal pentraxin 3 deficiency compromises implantation in mice

Reduced litter sizes in mice missing pentraxin 3 (Ptx3) have been attributed to fertilization failure. However, our global gene expression studies showed high uterine Ptx3 expression at the implantation site in mice, suggesting its role in blastocyst implantation. We initiated molecular and genetic studies in mice to explore the importance of uterine Ptx3 in this process. We found that Ptx3 is expressed in a unique and transient fashion at implantation sites. With the initiation of implantation on midnight of Day 4 of pregnancy, Ptx3 is expressed exclusively in stromal cells at the site of blastocysts. On Day 5, its expression is more intense in decidualizing stromal cells, but it disappears on Day 6. The expression again becomes evident in the deciduum on Day 7, followed by a more robust expression on Day 8, particularly at the antimesometrial pole. From Day 9, with the initiation of placentation, Ptx3 expression becomes undetectable. These results suggest a role for PTX3 in implantation and decidualization. Indeed, deletion of Ptx3 results in both compromised implantation and decidualization. Interleukin 1B (IL1B), a known inducer of Ptx3, is also transiently expressed in stromal cells at the implantation site, suggesting that IL1B is an inducer of uterine Ptx3 expression. In fact, uterine Ptx3 expression follows that of Il1b induced by lipopolysaccharide treatment on Day 7 of pregnancy. Collectively, these findings provide evidence for an important role for PTX3 in implantation and decidualization. This study has clinical implications, since PTX3 is expressed in the receptive endometrium, and trophoblast cells influence decidual Ptx3 expression in humans.     

Loss of collagen type VI from rat endometrial stroma during decidualization.

The expression of collagen type VI in the extracellular matrix of rat uterine endometrial stroma after a decidual stimulus was examined by immunolocalization and immunoblotting. The intermediate filament protein, desmin, was used as a marker to identify decidual cells. Tissue was examined from pregnant animals and from ovariectomized, hormone-treated rats in which decidualization had been induced artificially. In undifferentiated tissue from both groups of animals, collagen type VI was abundant, and desmin was present only in vascular smooth muscle cells. By 72 h after a decidual stimulus, however, collagen type VI had essentially disappeared from the matrix of the antimesometrial stromal compartment, and desmin was highly expressed in the decidualizing cells. During regression of the decidual tissue, collagen type VI began to reappear in the stromal matrix, whereas desmin expression declined as decidual cells degenerated. These results indicate that remodeling of the uterine extracellular matrix in response to embryo implantation is a function of the differentiating decidual cell.     

Interaction of mouse ectoplacental cone trophoblast and uterine decidua in vitro

Summary During the peri-implantation stages of mouse development, the secondary trophoblast invades into the uterine decidua. This uniquely controlled invasive process results in the formation of the placenta. We have analyzed this process in vitro using cultures of decidua and microdissected ectoplacental cones from Day 7 embryos. The results showed that the interaction between these two cell types is comparable to that seen in vivo. Morphologically, the decidua maintained close contact with the spreading trophoblast, limiting its invasion and producing a multilayered trophoblast outgrowth. Attachment to the decidua was not mediated through cell-matrix binding, but the subsequent invasion into the decidua was dependent on normal matrix interactions. Secretion of proteinases by the trophoblast also seemed to be a requirement for successful invasion, but not attachment.     

Decidual cells produce a heparin-binding prolactin family cytokine with putative intrauterine regulatory actions

Pregnancy in mice and rats is associated with the production of a large family of hormones/cytokines related to prolactin (PRL). The hormones/cytokines are hypothesized to coordinate maternal and fetal adaptations to pregnancy. In this study, PRL-like protein-J (PLP-J, also known as PRL family 3, subfamily c, member 1 (Prl3c1)) is shown to be a product of the uterine decidua and a regulator of postimplantation intrauterine events. PLP-J-specific antibodies and a series of recombinant PLP-J proteins were generated and used to investigate PLP-J expression and as ligands for investigating biological targets. Decidual PLP-J migrates as a 29-kDa protein and localizes to a band of decidual cells surrounding the trophoblast cell layer on gestation day 8.5. PLP-J ligands specifically bound in situ to the surrounding uterine stromal cells and vasculature within the decidua of gestation day 8.5 implantation sites. We then investigated the in vitro actions of PLP-J on uterine stromal cells and endothelial cells. PLP-J specifically interacted with both cell populations. PLP-J promoted uterine stromal cell proliferation and inhibited endothelial cell proliferation. We determined that PLP-J does not interact with PRL receptors. Instead, PLP-J interacts with heparin-containing molecules, including syndecan-1, which is expressed in gestation day 8.5 pregnant uteri, as well as in uterine stromal cells and endothelial cells. The restricted expression of PLP-J and its specific interactions with uterine stromal cells and endothelial cells suggests that it acts locally and regulates decidual cell development and the endometrial vasculature.     

Downregulation of SPARC Expression Inhibits the Invasion of Human Trophoblast Cells In Vitro

Successful pregnancy depends on the precise regulation of extravilloustrophoblast (EVT) invasion into the uterine decidua. SPARC (secreted protein acidic and rich in cysteine) is a matricellular glycoprotein that plays critical roles in the pathologies associated with obesity and diabetes, as well as tumorigenesis. The objective of this study was to investigate the role of SPARC in the process of trophoblast invasion which shares many similarities with tumor cell invasion. By Western blot, higher expression of SPARC was observed in mouse brain, ovary and uterus compared to other mouse tissues. Immunohistochemistry analysis revealed a spatio-temporal expression of SPARC in mouse uterus in the periimplantation period. At the implantation site of d8 pregnancy, SPARC mainly accumulated in the secondary decidua zone (SDZ), trophoblast cells and blastocyst. The expression of SPARC was also detected in human placental villi and trophoblast cell lines. In a Matrigel invasion assay, we found SPARC-specific RNA interference significantly reduced the invasion of human extravilloustrophoblast HTR8/SVneo cells. Microarray analysis revealed that SPARC depletion upregulated the expression of interleukin 11 (IL11), KISS1, insulin-like growth factor binding protein 4 (IGFBP4), collagen type I alpha 1 (COLIA1), matrix metallopeptidase 9 (MMP9), and downregulated the expression of the alpha polypeptide of chorionic gonadotropin (CGA), MMP1, gap junction protein alpha 1 (GJA1), et al. The gene array result was further validated by qRT-PCR and Western blot. The present data indicate that SPARC may play an important role in the regulation of normal placentation by promoting the invasion of trophoblast cells into the uterine decidua.     

Zonula occludens-1 and E-cadherin are coordinately expressed in the mouse uterus with the initiation of implantation and decidualization

Two-way interactions between the blastocyst trophectoderm and the uterine luminal epithelium are essential for implantation. The key events of this process are cell-cell contact of trophectoderm cells with uterine luminal epithelial cells, controlled invasion of trophoblast cells through the luminal epithelium and the basement membrane, transformation of uterine stromal cells surrounding the blastocyst into decidual cells, and protection of the "semiallogenic" embryo from the mother's immunological responses. Because cell-cell contact between the trophectoderm epithelium and the luminal epithelium is essential for implantation, we investigated the expression of zonula occludens-1 (ZO-1) and E-cadherin, two molecules associated with epithelial cell junctions, in the mouse uterus during the periimplantation period. Preimplantation uterine epithelial cells express both ZO-1 and E-cadherin. With the initiation and progression of implantation, ZO-1 and E-cadherin are expressed in stromal cells of the primary decidual zone (PDZ). As trophoblast invasion progresses, these two molecules are expressed in stroma in advance of the invading trophoblast cells. These results suggest that expression of these adherence and tight junctions molecules in the PDZ serves to function as a permeability barrier to regulate access of immunologically competent maternal cells and/or molecules to the embryo and provide homotypic guidance of trophoblast cells in the endometrium.     

Disordered IL-33/ST2 Activation in Decidualizing Stromal Cells Prolongs Uterine Receptivity in Women with Recurrent Pregnancy Loss

Decidualization renders the endometrium transiently receptive to an implanting blastocyst although the underlying mechanisms remain incompletely understood. Here we show that human endometrial stromal cells (HESCs) rapidly release IL-33, a key regulator of innate immune responses, upon decidualization. In parallel, differentiating HESCs upregulate the IL-33 transmembrane receptor ST2L and other pro-inflammatory mediators before mounting a profound anti-inflammatory response that includes downregulation of ST2L and increased expression of the soluble decoy receptor sST2. We demonstrate that HESCs secrete factors permissive of embryo implantation in mice only during the pro-inflammatory phase of the decidual process. IL-33 knockdown in undifferentiated HESCs was sufficient to abrogate this pro-inflammatory decidual response. Further, sequential activation of the IL-33/ST2L/sST2 axis was disordered in decidualizing HESCs from women with recurrent pregnancy loss. Signals from these cultures prolonged the implantation window but also caused subsequent pregnancy failure in mice. Thus, Il-33/ST2 activation in HESCS drives an autoinflammatory response that controls the temporal expression of receptivity genes. Failure to constrain this response predisposes to miscarriage by allowing out-of-phase implantation in an unsupportive uterine environment.