TLR6 modulates first trimester trophoblast responses to peptidoglycan

Intrauterine bacterial infections are a well-established cause of pregnancy complications. One key observation in a number of abnormal pregnancies is that placental apoptosis is significantly elevated. First trimester trophoblast cells are known to express TLR1 and TLR2 and to undergo apoptosis following exposure to Gram-positive bacterial peptidoglycan (PDG). Thus, the objectives of this study were to determine whether PDG-induced pregnancy complications are associated with placental apoptosis and to characterize the cellular mechanisms involved. We have demonstrated, using an animal model, that delivery of PDG to pregnant mice early in gestation resulted in highly elevated placental apoptosis, evidenced by trophoblast M-30 and active caspase 3 immunostaining. Using an in vitro model of human first trimester trophoblasts, apoptosis induced by PDG was found to be mediated by both TLR1 and TLR2 and that this could be blocked by the presence of TLR6. Furthermore, in the presence of TLR6, exposure to PDG resulted in trophoblast NF-kappaB activation and triggered these cells to secrete IL-8 and IL-6. The findings of this study suggest that a Gram-positive bacterial infection, through TLR2 and TLR1, may directly promote the elevated trophoblast cell death and that this may be the underlying mechanism of pregnancy complications, such as preterm delivery. Furthermore, the expression of TLR6 may be a key factor in determining whether the response to PDG would be apoptosis or inflammation.     

Fetal cells in the maternal circulation during first trimester in pregnancies

To investigate the presence of fetal cells in the maternal circulation during early pregnancy, the polymerase chain reaction was used to test the presence of human Y chromosome-specific ZFY and SRY gene DNA sequences in maternal peripheral blood specimens from 19 women carrying male fetuses and 12 women carrying female fetuses. The presence of fetal cells was suggested as early as 6 weeks gestation in 1 of the 19 women bearing male fetuses. Fetal cells were present in the maternal circulation of 15 of the 19 women by 9 weeks gestation, and in only 1 of the 19 were fetal cells not detected until the 12th week after conception. These results suggest that identification of fetal cells in the maternal circulation is possible with a properly designed and executed polymerase chain reaction. However, there was considerable variation with respect to when these fetal cells first became detectable during pregnancy. These fetal cells are potentially a valuable source of material for biochemical and genetic studies of the fetuses.     

Effect of morphine on hCG release by first trimester human trophoblast in vitro

Opiate synthesis by human placental cells and the presence of kappa-type opiate binding sites in the syncytiotrophoblast brush border membrane may indicate the possible role of morphine-like substances in the autocrine regulation of trophoblast cell metabolism. This study was undertaken to examine the in vitro effect of morphine on hCG (human chorionic gonadotrophin) and hPL (human placental lactogen) release by 1st and 3rd trimester placental tissue explants. The results have shown that morphine (100 nM) significantly stimulated hCG secretion by 6-8 weeks old trophoblast and was without effect on hPL. Hormone secretion by term placental tissue explants was unaffected by morphine treatment. Based on these results we assume that opiates may have a role in the local (autocrine and/or paracrine) regulation of hCG secretion in early gestation.     

Prognosis of subsequent pregnancies after recurrent spontaneous abortion in first trimester.

The outcome of subsequent pregnancies was studied in 24 women with a history of three or more consecutive spontaneous abortions in the first trimester. Twenty one of the women agreed to forgo active treatment during the study period and three received progestogens or surgical intervention. As the outcome of their first pregnancy after recurrent abortion 18 of the 24 women delivered a liveborn infant after 28 weeks or more. Of all 49 pregnancies in the untreated group during the study, 10 ended in abortion. Only one woman in the study failed to achieve a live birth. Except in exceptional cases encouraging women with recurrent abortion to keep trying for a successful outcome is just as effective as currently recommended treatments.     

The regulation of arachidonic acid metabolism in human first trimester trophoblast by cyclic AMP

Human trophoblast cells are known to release a range of arachidonic acid metabolites into culture medium, including cyclo-oxygenase, lipoxygenase and epoxygenase products. In this study we investigated the effects of dibutyryl cyclic AMP (db cAMP) on arachidonic acid metabolism in human first trimester trophoblast cells, and also determined the distribution of metabolites between intracellular and extracellular compartments. db cAMP increased intracellular levels of radioactivity within 2 min, and extracellular levels of radioactivity were increased after 30 min. These changes were reflected in increased levels of arachidonic acid metabolites in both compartments, indicating that arachidonic acid was metabolised. db cAMP increased intracellular levels of 5,6-epoxyeicosatrienoic acid (5,6-EpETrE) within 2 min of addition to cultured cells. No changes were detected after 5-10 min, but substantial changes were found 30 min after the addition of db cAMP. The dihydroxyeicosatrienoic acid (DiHETrE) breakdown products also increased with similar kinetics. In contrast, levels of 14,15-EpETrE increased after 5-10 min.     

Identification of class I MHC mRNA in human first trimester trophoblast cells by in situ hybridization

Special gestation-related regulatory mechanisms for the expression of class I Ag by trophoblast cells directly exposed to maternal blood and tissues may be required for semiallogeneic pregnancy to be successful. Analysis of class I MHC mRNA by in situ hybridization and class I MHC Ag by immunohistology has revealed two phenotypically distinct subpopulations of trophoblast cells in term placentas and extraplacental membranes. Trophoblast cells external to the placenta are mRNA +/Ag+. They contain class I mRNA and express class I Ag that differ serologically from HLA-A,B,C. In contrast, trophoblast cells forming the syncytial layer of placental villi are mRNA-/Ag-. By immunohistology, trophoblast cells in 1st trimester placental tissues are similar to those in term tissues. In our study, in situ hybridization was used to determine if patterns of trophoblast cell class I mRNA were the same or different. Trophoblast cells external to the placental villi in 1st trimester tissues contained class I mRNA as would be predicted from the results with term tissues. Unexpectedly, class I mRNA was found in villous trophoblast cells. Thus, these studies identified an mRNA+/Ag- trophoblast cell subpopulation. The results suggest that tissue-specific mechanisms may interfere with translation of class I mRNA in 1st trimester villous trophoblast cells and/or that the protein products of the mRNA are not identified by available mAb.     

A role for TLRs in the regulation of immune cell migration by first trimester trophoblast cells

Normal pregnancy is characterized by the presence of innate immune cells at the maternal-fetal interface. Originally, it was postulated that the presence of these leukocytes was due to an immune response toward paternal Ags expressed by the invading trophoblasts. Instead, we and others postulate that these innate immune cells are necessary for successful implantation and pregnancy. However, elevated leukocyte infiltration may be an underlying cause of pregnancy complications, such as preterm labor or preeclampsia. Furthermore, such conditions have been attributed to an intrauterine infection. Therefore, we hypothesize that first trimester trophoblast cells, upon recognition of microbes through TLRs, may coordinate an immune response by recruiting cells of the innate immune system to the maternal-fetal interface. In this study, we have demonstrated that human first trimester trophoblast cells constitutively secrete the chemokines growth-related oncogene, growth-related oncogene alpha, IL-8, and MCP-1 and are able to recruit monocytes and NK cells, and to a lesser degree, neutrophils. Following the ligation of TLR-3 by the viral ligand, poly(I:C), or TLR-4 by bacterial LPS, trophoblast secretion of chemokines is significantly increased and this in turn results in elevated monocyte and neutrophil chemotaxis. In addition, TLR-3 stimulation also induces trophoblast cells to secrete RANTES. These results suggest a novel mechanism by which first trimester trophoblast cells may differentially modulate the maternal immune system during normal pregnancy and in the presence of an intrauterine infection. Such altered trophoblast cell responses might contribute to the pathogenesis of certain pregnancy complications.     

Modulation of the endocannabinoid system in viable and non-viable first trimester pregnancies by pregnancy-related hormones

Background

In early pregnancy, increased plasma levels of the endocannabinoid anandamide (AEA) are associated with miscarriage through mechanisms that might affect the developing placenta or maternal decidua.

Methods

In this study, we compare AEA levels in failed and viable pregnancies with the levels of the trophoblastic hormones (beta-human chorionic gonadotrophin (beta-hCG), progesterone (P4) and (pregnancy-associated placental protein-A (PAPP-A)) essential for early pregnancy success and relate that to the expression of the cannabinoid receptors and enzymes that modulate AEA levels.

Results

The median plasma AEA level in non-viable pregnancies (1.48 nM; n = 20) was higher than in viable pregnancies (1.21 nM; n = 25; P = 0.013), as were progesterone and beta-hCG levels (41.0 vs 51.5 ng/mL; P = 0.052 for P4 and 28,650 vs 6,560 mIU/L; P = 0.144 for beta-hCG, respectively, but were not statistically significant). Serum PAPP-A levels in the viable group were approximately 6.8 times lower than those in the non-viable group (1.82 vs 12.25 mg/L; P = 0.071), but again these differences were statistically insignificant. In the spontaneous miscarriage group, significant correlations between P4 and beta-hCG, P4 and PAPP-A and AEA and PAPP-A levels were observed. Simultaneously, immunohistochemical distributions of the two main cannabinoid receptors and the AEA-modifying enzymes, fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD), changed within both the decidua and trophoblast.

Conclusions

The association of higher AEA levels with early pregnancy failure and with beta-hCG and PAPP-A, but not with progesterone concentrations suggest that plasma AEA levels and pregnancy failure are linked via a mechanism that may involve trophoblastic beta-hCG, and PAPP-A, but not, progesterone production. Although the trophoblast, decidua and embryo contain receptors for AEA, the main AEA target in early pregnancy failure remains unknown.     

Proteolytic activity of first trimester human placenta: localization of interstitial collagenase in villous and extravillous trophoblast

In human placentation, events of implantation and early blastocyst development are mediated by fetal trophoblastic cells which penetrate into the maternal endometrium and myometrium. Although highly regulated in its biological behavior, trophoblast simulates a malignant neoplasm by virtue of invading the uterine wall and uterine spiral arteries and by embolizing throughout the systemic circulation. This process is at least in part dependant on the regulated production of proteolytic enzymes to degrade extracellular matrix. The most abundant extracellular protein is connective tissue type (interstitial) collagen. The uterine remodeling during the establishment of the embryo requires collagenase which catalyzes the initial step in the breakdown of collagen. This study demonstrates the presence of interstitial collagenase in villous and extravillous trophoblast of first trimester placenta using immunocytochemical methods on light microscopic and ultrastructural levels. Intracytoplasmic staining for interstitial collagenase was present in cyto- and syncytiotrophoblast covering the chorionic villi as well as in extravillous intermediate trophoblast invading spiral arteries in the placental bed. Furthermore, outgrowth cultures of chorionic villi were studied with the immunogold method. Gold labelling was associated with the cell surface of trophoblastic cells as well as with fibrillary collagen like proteins of newly synthesized extracellular matrix. We speculate that interstitial collagenase plays a role in the degradation of uterine collagen within the developing human placenta.     

Proteolytic activity of first trimester human placenta: localization of interstitial collagenase in villous and extravillous trophoblast

Summary In human placentation, events of implantation and early blastocyst development are mediated by fetal trophoblastic cells which penetrate into the maternal endometrium and myometrium. Although highly regulated in its biological behavior, trophoblast simulates a malignant neoplasm by virtue of invading the uterine wall and uterine spiral arteries and by embolizing throughout the systemic circulation. This process is at least in part dependant on the regulated production of proteolytic enzymes to degrade extracellular matrix. The most abundant extracellular protein is connective tissue type (interstitial) collagen. The uterine remodeling during the establishment of the embryo requires collagenase which catalyzes the intial step in the breakdown of collagen. This study demonstrates the presence of interstitial collagenase in villous and extravillous trophoblast of first trimester placenta using immunocytochemical methods on light microscopic and ultrastructural levels. Intracytoplasmic staining for interstitial collagenase was present in cyto- and syncytiotrophoblast covering the chorionic villi as well as in extravillous intermediate trophoblast invading spiral arteries in the placental bed. Furthermore, outgrowth cultures of chorionic villi were studied with the immunogold method. Gold labelling was associated with the cell surface of trophoblastic cells as well as with fibrillary collagen like proteins of newly synthesized extracellular matrix. We speculate that interstitial collagenase plays a role in the degradation of uterine collagen within the developing human placenta.     

Fatty acid-induced angiogenesis in first trimester placental trophoblast cells: Possible roles of cellular fatty acid-binding proteins

Angiogenesis is involved in the growth of new blood vessels from the existing one. Consequently, angiogenesis plays an indispensable role in tissue growth and repair including early placentation processes. Besides angiogenic growth factors (vascular endothelial growth factor (VEGF), angiopoietin-like 4 (ANGPTL4), placental growth factor (PlGF), platelet derived growth factor (PDGF), fibroblast growth factors (FGF)), dietary fatty acids (c>16) also directly or indirectly modulate angiogenic processes in tumors and other cell systems. Usually n − 3 fatty acids inhibit whereas n − 6 fatty acids stimulate angiogenesis in tumors and other cells. Contrary to this, docosahexaenoic acid, 22:6n − 3 (DHA) and other fatty acids including conjugated linoleic acid stimulate angiogenesis in placental first trimester cells. In addition to the stimulation of expression of major angiogenic factors such as VEGF and ANGPTL4, fatty acids also stimulate expression of intracellular fatty acid-binding proteins (FABPs) FABP-4 and FABP-3 those are known to directly modulate angiogenesis. Emerging data indicate that FABPs may be involved in the angiogenesis process. This paper reviews the fatty acid mediated angiogenesis process and the involvement of their binding proteins in these processes.     

Amino acid profiles in first trimester amniotic fluids of healthy bovine cloned pregnancies are similar to those of IVF pregnancies,but not nonviable cloned pregnancies

Somatic cell nuclear transfer (SCNT), or cloning, is one of the assisted reproductive technologies currently used in agriculture. Commercial applications of SCNT are presently limited to the production of animals of high genetic merit or the production of the most elite show cattle owing to its relatively low efficiency. In current practice, 20% to 40% of SCNT pregnancies do not result in viable offspring. In an effort to better understand some of the anomalies associated with SCNT pregnancies, we investigated amino acid compositions of first trimester amniotic fluid. In this retrospective study, amniotic fluids were collected from SCNT and control IVF pregnancies at Day 75 of gestation and grouped according to the pregnancy results: control IVF (IVF), viable SCNT pregnancies that resulted in live healthy calves (SCNT-HL), nonviable SCNT pregnancies that were aborted before Day 150 (SCNT-ED), and nonviable SCNT pregnancies that were aborted after Day 150 or produced deceased calves (SCNT-LD). High-performance liquid chromatography (HPLC) was used to analyze the concentrations of 22 amino acids (AAs) in the amniotic fluid samples. There were no differences in average AA concentrations between IVF and SCNT-HL groups, whereas SCNT-LD and SCNT-ED had higher levels of total AA concentrations. Concentrations of asparagine, citruline, arginine, and valine were significantly higher in the SCNT-LD group. Both SCNT-LD and SCNT-ED groups had relatively large intragroup variances in AA concentrations. Urea concentration was also measured in the SCNT amniotic fluid samples. No correlations between urea concentrations and arginine concentrations or pregnancy outcomes were found. The findings in this study not only deepen the understanding on SCNT pregnancy anomalies, but also provide a potentially useful screening tool for assessing viable and nonviable SCNT pregnancies.     

Serum-dependent effects of IGF-I and insulin on proliferation and invasion of human first trimester trophoblast cell models

Extravillous cytotrophoblasts are specialised epithelial cells of the placenta that proliferate or invade the maternal decidua. Little is known about the mechanisms that regulate these processes. Here the effects of several insulin and insulin-like growth factor-I (IGF-I) doses, either singly or in synergy with serum, on human chorionic gonadotropin-beta (hCG-beta) secretion (RIA), proliferation (cell counting, cyclin B(1) levels) and invasion [Matrigel invasion assay, secretion of matrix metalloproteinases (MMP) 2 and 9] were investigated. The choriocarcinoma cell lines BeWo, JAR and JEG-3 served as models for first trimester human trophoblasts. Both growth factors altered hCG-beta secretion and proliferation dependent on the cell line. Insulin stimulated proliferation in JAR cells and, to a lesser extent, in JEG-3 cells, and when cultured in serum-free medium, BeWo was not affected. Invasion was not affected although proMMP-2 levels in culture medium were altered under some conditions. A strong synergistic effect with serum was noted. In the presence of serum both growth factors reduced proliferation and invasion in a similar fashion. Since the cell models differ by their degree of differentiation, the data demonstrate that the effects of insulin and IGF-I strongly depend on serum and the degree of differentiation. It can be speculated that IGF-I can take on tasks of insulin in the regulation of trophoblast functions under conditions of insulinopenia.     

ADAM12 and PAPP-A: Candidate regulators of trophoblast invasion and first trimester markers of healthy trophoblasts

ABSTRACT

Proper placental development and function is crucial for a healthy pregnancy, and there has been substantial research to identify markers of placental dysfunction for the early detection of pregnancy complications. Low first-trimester levels of a disintegrin and metalloproteinase 12 (ADAM12) and pregnancy-associated plasma protein-A (PAPP-A) have been consistently associated with the subsequent development of preeclampsia and fetal growth restriction. These molecules are both metalloproteinases secreted by the placenta that cleave insulin-like growth factor binding proteins (IGFBPs), although ADAM12 also has numerous other substrates. Recent work has identified ADAM12, and particularly its shorter variant, ADAM12S, as a regulator of the migration and invasion of trophoblasts into the lining of the uterus, a critical step in normal placental development. While the mechanisms underlying this regulation are not yet clear, they may involve the liberation of heparin-binding EGF-like growth factor (HB-EGF) and/or IGFs from IGFBPs. In contrast, there has been relatively little functional work examining PAPP-A or the IGFBP substrates of ADAM12 and PAPP-A. Understanding the functions of these markers and the mechanisms underlying their association with disease could improve screening strategies and enable the development of new therapeutic interventions.