HLA-G expression in trophoblast cells is independent of embryonic development

HLA-G is a nonclassical major histocompatibility complex class I molecule that is selectively expressed on cytotrophoblasts at the feto-maternal interface where it may play a major role in maternal-fetal tolerance. In this study, we compared HLA-G expression in trophoblasts from normal and pathologic pregnancies by immunohistochemical analysis. First, we found a defective HLA-G expression in miscarriages associated with hypotrophic but normal eggs. Conversely, by studying molar pregnancies, we observed a high HLA-G expression in complete and partial hydatidiform moles. Finally, HLA-G expression could be visualized in extravillous trophoblasts that develop outside of their normal environment, as reported here in ectopic pregnancies. Taken together, these results suggest that HLA-G expression in extravillous trophoblasts is induced in an autonomous manner, independently of embryonic development, and may be an integral part of placental development allowing its tolerance from maternal immune system.     

HLA-G expression in extravillous trophoblasts is an intrinsic property of cell differentiation: a lesson learned from ectopic pregnancies

Human leukocyte antigen (HLA)-G is a major histocompatibility gene expressed almost exclusively in extravillous trophoblasts at the fetal-maternal interface. HLA-G may play a role in protecting the fetus from attack by the maternal natural killer cells. The extravillous trophoblasts invade the decidua and maternal spiral arteries. The factors which regulate the cell-specific expression of HLA-G are unknown. In this study we asked if HLA-G is expressed in extravillous trophoblasts that develop outside of their normal cellular environment, as in the case of ectopic pregnancies. Since all ectopic pregnancies implant in the absence of underlying decidua we also used a placenta accreta as an experimental control. We found that HLA-G mRNA and protein were expressed in the extravillous trophoblasts in the 13 ectopic specimens studied. In a case of placenta accreta (which develops without decidua basalis and is therefore adherent to the underlying myometrium), HLA-G mRNA and protein were also expressed. These results suggest that HLA-G expression is induced in a cell autonomous manner rather than determined by appropriate environmental cues.     

不同浓度Dlll4调节绒毛外滋养细胞的生物学功能

背景：早孕时期绒毛外滋养细胞功能的正常发挥对妊娠有着至关重要的影响,绒毛外滋养细胞功能异常时将导致子痫前期、胎儿生长受限及胎盘植入等多种妊娠期疾病。 目的：探讨Notch信号家族配体Dll4对绒毛外滋养细胞的生物学功能的影响。 方法：以不同质量浓度外源性Dll4(0,0.125,0.25,0.5,1.0 mg/L)处理人绒毛外滋养细胞系HTR-8/SVneo,以CCK-8试验检测细胞活性,Transwell实验检测细胞迁移、侵袭能力的改变。 结果与结论：Dll4可明显促进绒毛外滋养细胞迁移与侵袭能力,而对细胞活性不产生显著影响;Dlll4对绒毛外滋养细胞侵袭功能的影响具有浓度依赖性。结果提示Dll4可通过调节绒毛外滋养细胞生物学功能,在妊娠过程很中发挥重要作用。  中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Pre-term birth and severe pre-eclampsia are not associated with altered expression of HLA on human trophoblasts

PROBLEM: The unusual pattern of human leukocyte antigen (HLA) expression on human trophoblasts could play an important role in successful pregnancy outcome. To determine whether alterations in HLA expression are associated with pregnancy abnormalities we have investigated expression of these antigens on chorionic and extravillous cytotrophoblasts. METHODS: Frozen tissue sections of placenta and fetal membranes were collected after pre-term spontaneous delivery, severe pre-eclampsia pre-term Caesarean section, normal term delivery and term Caesarean section. HLA expression was analyzed by immunohistochemistry. RESULTS: We did not observe differences in the expression of HLA on chorionic and extravillous cytotrophoblasts in pregnancy abnormalities. However, we noted higher expression levels of HLA class Ia molecules in amnion epithelial cells in pre-term deliveries. Furthermore, in severe pre-eclampsia the number of extravillous cytotrophoblast islands were elevated when compared with pre-term deliveries. CONCLUSIONS: No alterations in expression of HLA class Ia, HLA-G and HLA class II on human trophoblasts in pregnancy abnormalities were seen.     

Expression of Epstein-Barr Virus-Induced Gene 3, an Interleukin-12 p40-Related Molecule, throughout Human Pregnancy : Involvement of Syncytiotrophoblasts and Extravillous Trophoblasts

In human pregnancy, trophoblasts are the only cells of fetal origin in direct contact with the maternal immune system: syncytiotrophoblasts are in contact with maternal blood, whereas extravillous trophoblasts are in contact with numerous maternal uterine natural killer (NK) cells. Therefore, trophoblasts are thought to play a key role in maternal tolerance to the semiallogeneic fetus, in part through cytokine production and NK cell interaction. Epstein-Barr virus-induced gene 3 (EBI3) encodes a soluble hematopoietin receptor related to the p40 subunit of interleukin-12. Previous studies indicated that EBI3 is expressed in the spleen and tonsils, and at high levels in full-term placenta. To investigate further EBI3 expression throughout human pregnancy, we generated monoclonal antibodies specific for EBI3 and developed an EBI3 enzyme-linked immunosorbent assay. Immunohistochemical experiments with EBI3 monoclonal antibody on first-, second-, and third-trimester placental tissues demonstrated that EBI3 was expressed throughout pregnancy by syncytiotrophoblasts and extravillous trophoblasts (cytotrophoblast cell columns, interstitial trophoblasts, multinucleated giant cells, and trophoblasts of the chorion laeve). EBI3 expression was also induced during in vitro differentiation of trophoblast cell lines. In addition, large amounts of secreted EBI3 were detected in explant cultures from first-trimester and term placentae. Consistent with these data, EBI3 levels were strongly up-regulated in sera from pregnant women and gradually increased with gestational age. These data, together with the finding that EBI3 peptide is presented by HLA-G, suggest that EBI3 is an important immunomodulator in the fetal-maternal relationship, possibly involved in NK cell regulation.     

Lack of human leukocyte antigen-G expression in extravillous trophoblasts is associated with pre-eclampsia

Pre-eclampsia, a common complication of first pregnancies, is thought to result from a poorly perfused placenta and may reflect an abnormal maternal immune reaction to the hemiallogenic fetus. Human leukocyte antigen (HLA)-G, a major histocompatibility tissue-specific antigen expressed in extravillous trophoblast cells (fetal-derived), may protect trophoblasts from maternal-fetal immune intolerance and allow these cells to invade the uterus. Through RNA in-situ hybridization analysis, we studied the expression pattern of HLA-G in normal placentae and placentae from pregnancies complicated by severe pre-eclampsia. In normal placenta we found HLA-G expression in the anchoring extravillous trophoblasts with an increasing gradient of expression in the more invasive cells. However, in nine out of 10 pre-eclamptic placentae HLA-G expression was absent or reduced. We conclude that HLA-G is normally expressed in invasive trophoblasts and HLA-G expression is defective in most pre-eclamptic placentae. We propose that trophoblasts lacking HLA-G are vulnerable to attack by the maternal immune system. These defective trophoblasts will be unable to invade the maternal spiral arteries effectively, thereby developing vessels which cannot adequately nourish the developing placenta. This poorly perfused placenta may initiate the systemic cascade of events associated with pre-eclampsia.     

Placental HLA-G protein expression in vivo: where and what for?

In contrast to HLA-A and -B class Ia genes that are down-regulated in human trophoblast cells, HLA-G class Ib molecules are expressed in the placenta throughout gestation. In addition to extravillous cytotrophoblast that invade the decidua basalis essentially, HLA-G was also observed in endothelial cells of fetal vessels in the chorionic villi as well as in amnion cells and amniotic fluid. Both membrane-bound and soluble HLA-G isoforms have been detected. In view of the recently published functional data showing that HLA-G: (i) has the capability to bind and present peptides; (ii) is recognized by at least three different killing inhibitory receptors; and (iii) is a regulator of HLA-E expression, we can predict that such functions are likely to be exerted by extravillous cytotrophoblast. Of particular importance will be the anti-viral function of HLA-G at this materno-fetal interface, knowing that HLA-G was shown to be expressed by thymic medullary epithelial cells. In addition to these immunological functions, due to its presence on chorionic fetal endothelial cells, we hypothesize that HLA-G could also be a regulator of chorionic villous angiogenesis. Finally, soluble HLA-G isoforms may act as specific immunosuppressors during pregnancy.     

Expression of matrix metalloproteinases 2 and 9 in human trophoblasts of normal and preeclamptic placentas: Preliminary findings

Objective: Here we test the hypothesis that “the expression of matrix metalloproteinase 2 and 9 proteins is altered in preeclamptic placentas compared to placentas of normal pregnancy.”Patients and methods: This case–control study includes preeclamptic placentas (40 women with preeclampsia) from a singleton pregnancy and placentas of normal pregnancies (control group, 40 women with uncomplicated pregnancy). The expression patterns of metalloproteinases 2 and 9 were examined using immunohistochemical staining methods.Results: Compared to uncomplicated pregnancy, the incidence of intrauterine growth restriction was high and the mean birth weight was markedly low in patients with preeclampsia. Both metalloproteinase 2 and 9 proteins were frequently and strongly expressed in the majority of placentas of uncomplicated pregnancies (control group). Metalloproteinase 9 expression was absent in the majority of the preeclamptic placentas. In the remaining cases of preeclamptic placentas, the expression of metalloproteinase 9 was weak. In contrast, a strong metalloproteinase 2 protein expression was seen in the majority of the preeclamptic placentas.Conclusions: These preliminary data demonstrate the expression of metalloproteinase 2 and 9 proteins in the placentas of uncomplicated pregnancies. The absence/reduced expression of metalloproteinase 9 in the preeclamptic placentas may be related to insufficient invasion of trophoblast, leading to superficial and unsuccessful placentation.     

Villous explant culture using early gestation tissue from ongoing pregnancies with known normal outcomes: the effect of oxygen on trophoblast outgrowth and migration

BACKGROUND: Early placental and embryo development occur in a physiologically low oxygen environment, with a rise in oxygen tension within the placenta towards the end of the first trimester. Oxygen is implicated in the regulation of trophoblast differentiation and invasion. This study examined the effects of oxygen tension on extravillous trophoblast outgrowth and migration from normal pregnancies free of significant pathology. METHODS: Early gestation villous tissue (11-14 weeks gestation), obtained by chorionic villus sampling, was cultured in 3 or 20% oxygen. Maternal and fetal outcomes were ascertained for all samples. The frequency and amount of trophoblast outgrowth and migration from villi were measured for up to 192 h. RESULTS: Significantly fewer explants produced outgrowths in 3% compared with 20% oxygen. The number of sites of trophoblast outgrowth and the extent of migration were also significantly less in 3% compared with 20% oxygen. In vitro hypoxia/reoxygenation further reduced trophoblast growth compared with 3% oxygen alone. HLA-G expression in extravillous trophoblasts was not affected by oxygen tension, with HLA-G positive extravillous trophoblasts being universally Ki67 negative. CONCLUSION: Human placental villi and extravillous trophoblasts in the late first trimester of pregnancy are sensitive to oxygen tension, with low oxygen inhibiting extravillous trophoblast outgrowth and migration.     

基质金属蛋白酶-9和血管内皮生长因子在子痫前期患者胎盘组织中的表达

目的 检测子痫前期患者胎盘组织中RECK、基质金属蛋白酶-9（MMP-9）、血管内皮生长因子（VEGF）的基因表达,探讨它们与胎盘滋养细胞浸润过程的调控关系。 方法 采用RT-PCR、Western blotting、免疫组织化学检测120例妊娠足月剖宫产（正常妊娠、轻度子痫前期、中度子痫前期、重度子痫前期各30例）胎盘组织中RECK、MMP-9、VEGF的基因表达。结果 3组子痫前期患者胎盘组织中MMP-9及VEGF的mRNA表达水平均显著低于正常妊娠组（P<0.05）;重度子痫前期组中RECK mRNA的表达显著高于正常妊娠组（P<0.05）;3组子痫前期患者胎盘组织中MMP-9及VEGF蛋白表达均显著低于正常妊娠组（P<0.05）,中度和重度子痫前期组中RECK蛋白表达显著高于正常妊娠组（P<0.05）。结论 子痫前期患者胎盘中RECK与MMP-9、VEGF之间存在负相关性,它们可能参与了胎盘滋养细胞浅浸润过程的调控。     

The expression of human leukocyte antigen-G on trophoblasts abolishes the growth-suppressing effect of interleukin-2 towards them

PROBLEM: We have shown the attenuated human leukocyte antigen (HLA)-G expression on trophoblasts and an aberrant expression of interleukin (IL)-2, a cytotoxic cytokine, in decidual tissue in preeclampsia, where deteriorated trophoblastic invasion into decidual layers may constitute a crucial pathogenesis. We hypothesized that the absence of HLA-G might make trophoblasts susceptible to compromise by IL-2. METHOD OF STUDY: We analyzed the growth of HLA-G-negative and positive cell lines, all of which possessed IL-2 receptors, in the culture with or without IL-2 supplementation. RESULTS: The proliferation of HLA-G positive trophoblastic cell lines (BeWo and JEG-3) was not influenced by the addition of IL-2, whereas a HLA-G-negative trophoblastic cell line (JAR) exhibited significantly decreased proliferation when cultured with IL-2. Interestingly, the transfection of JAR cells with HLA-G completely eliminates the growth-inhibitory effect of IL-2. CONCLUSION: The expression of HLA-G may commit trophoblasts to evade cell damage by IL-2, which may be relevant to maternal tolerance of the fetus during pregnancy and its derangement as exemplified by preeclampsia.     

HLA-G genotype is associated with fetoplacental growth

The human leukocyte antigen (HLA)-G is expressed by extravillous cytotrophoblast cells in the feto-maternal contact zone. Polymorphisms have been described in the HLA-G gene and have been linked with differences in HLA-G mRNA alternative splicing patterns and protein expression. Differences in the isoform profile or the degree of HLA-G expression may influence cytokine production and, thereby, placental and fetal growth. Associations between a 14 bp deletion polymorphism in the 3'UTR part of the HLA-G gene and birth weight in relation to gestational age and placental weight were studied in 47 pregnancies complicated with preeclampsia and 87 with no preeclampsia. An HLA-G genotype homozygous for the presence of the 14 bp sequence polymorphism was significantly associated with increased birth weight in relation to gestational age (one-way analysis of variance; 2 degrees of freedom: p = 0.02) and with placental weight at birth (>38 weeks of gestation; +14 bp/+14 bp vs others; unpaired t-test: p = 0.03). There was also a slightly higher placental ratio in the offspring with the +14 bp/+14 bp genotype. The implications of these findings are discussed in relation to certain complications of pregnancy and in an evolutionary perspective.     

Histochemical Demonstration of Interleukin-2 in Decidua Cells of Patients With Preeclampsia

PROBLEM : The objective of this study was to gain insight into the immunological aspects of preeclampsia. METHOD : The presence of interleukin-2 (IL-2) protein in the decidua was examined in five preeclamptic patients and seven normal pregnant women employing the immunohistochemistry. RESULTS : The decidua cells were strongly stained for IL-2 in four out of five preeclamptic patients, while only very weak, if any, staining was observed in all the uncomplicated pregnant women. CONCLUSIONS : IL-2 was clearly present in decidua cells in the preeclamptic setting, suggesting the possible involvement of immune-activation in the phathophysiology of preeclampsia.     

The effects of oxygen concentration and gestational age on extravillous trophoblast outgrowth in a human first trimester villous explant model

BACKGROUND: In the first trimester of human pregnancy, extravillous trophoblasts from placental villi invade the decidua temporarily occluding the spiral arteries, preventing maternal blood flow and creating a low-oxygen environment, which is believed to play an important role in the regulation of extravillous trophoblast outgrowth. This work aimed to quantify the effects of gestational age and oxygen concentration on extravillous trophoblast outgrowth. METHODS: A quantitative first trimester villous explant model was used to measure the frequency and area of extravillous trophoblast outgrowths from villi grown in 1.5 or 8% oxygen. RESULTS: The percentage of explants producing outgrowth declined independently of oxygen concentration as gestation increased from 8 to 12 weeks. Culture in 1.5% oxygen significantly reduced the frequency and area of outgrowths in comparison with 8% oxygen. HLA-G and alpha1 integrin were both expressed throughout outgrowths, with no difference in the expression between oxygen concentrations. Gestation influenced the response of explants to oxygen, with a significant differential response to oxygen concentration in placentae under 11 weeks of gestation, whereas in villi from placentae of 11 or 12 weeks, no differential response was observed. CONCLUSIONS: In the first trimester, oxygen and gestational age both regulate extravillous trophoblast outgrowth.     

Necrotic feature of the trophoblasts lacking HLA-G expression in normal and pre-eclamptic placentas

PROBLEM: Human leukocyte antigen (HLA)-G is thought to be expressed in all placental extravillous trophoblasts (EXTs). In pre-eclamptic placentas, a lack of HLA-G expression on EXTs had been found, and deduced as a possible cause of pre-eclampsia. However, a subset of EXTs lacking expression of HLA-G can also be found in normal placenta. Therefore, we sought to compare these cells in normal and pre-eclamptic placentas. METHODS OF STUDY: Frozen sections of normal and pre-eclamptic placentas were examined by immunohistochemical staining using HLA-G monoclonal antibody 87G, histochemical enzymatic analysis of succinate dehydrogenase (SDH) and ultrastructural analysis. RESULTS: A subset of EXTs lacking HLA-G expression was found in both normal and pre-eclamptic placentas. These cells showed necrotic features such as the swelling of cells, eosin-achromatophilia, the loss of SDH activity and swelling mitochondria. Cells from both tissues were identical with regard to these features. CONCLUSION: The features of the EXTs lacking HLA-G expression indicated they had undergone necrosis and thus could not express HLA-G protein. Therefore, an alternative interpretation to the lack of HLA-G expression in pre-eclamptic placentas is that it is the result of cell death and not the cause.     

Expression of TAP1 by human trophoblast

Successful placentation in the human is dependent on the trophoblast evading recognition and destruction by the maternal immune system. However, invasive cytotrophoblast express HLA-G which may be able to present peptide to T cells. Transporter proteins are essential for peptide presentation and major histocompatibility complex (MHC) class I assembly. We have determined their expression by trophoblast in relation to HLA-G, using immunohistochemistry. Antitransporter protein antibody (TAP1) labeling closely paralleled that of MHC class I, but the intensity of its expression was much greater on the HLA-G⁺ extravillous cytotrophoblast than any other fetal or maternal tissue in the first trimester and at term. This suggests that the extravillous cytotrophoblast are very actively assembling MHC class I antigens with peptides. However, expression of MHC class I by the cytotrophoblast was not correspondingly elevated. This pattern could result from HLA-G being shed from the surface of the trophoblast, a process which may play a central role in protecting the fetus from maternal immune attack.