Intrauterine growth restriction, human placental development and trophoblast cell death

Intrauterine growth restriction (IUGR) is a failure to achieve the growth potential of a fetus that is promised by the genetic constitution and environmental influences endogenous to the pregnancy. Optimal placental development and the ability of the placenta to compensate for stimulus-induced injury are central in promotion of normal fetal growth. In this review, we will overview placental development with a focus on how villous structure relates to function. We will also describe the differentiation and turnover of villous trophoblast while highlighting selected features of microscopic placental injury. Histopathological studies of the placenta in IUGR indicate that abnormalities of the maternal spiral arterioles, dysregulated villous vasculogenesis, and abundant fibrin deposition are characteristic of the injuries associated with this condition. We identify selected insults, including oxidative stress and complement activation, and key pathways that regulate apoptosis in villous trophoblast, including increased p53 activity, altered translation of AKT and mTOR proteins, and the stress response of the endoplasmic reticulum. We surmise that trophoblast dysregulation at a subcellular level and loss of functional mass of villous trophoblast via cell death pathways are key contributors to the suboptimal placental performance that yields IUGR. We predict that a better understanding of placental dysfunction in IUGR will lead to targeted therapeutic options for this important clinical condition.     

Distribution and characterization of anionic sites in trophoblast and capillary basal laminas of human placental villi

The distribution of anionic sites was studied in the trophoblastic and fetal capillary basal laminas of developing human placental villi with the cationic stain ruthenium red. At 7-12 weeks of gestation the trophoblastic basal lamina (TBL) contained ruthenium red-positive granules in a quasi-regular array throughout the lamina densa or sometimes concentrated at the interstitial surface of the lamina densa. The capillary basal lamina (CBL) (and anionic sites) were not present at this age. Anionic sites were also associated with collagen or reticular fibrils. At term, the TBL was largely devoid of anionic sites except for some distributed along its interstitial surface. The CBL was present in later gestation and sometimes had arrays of anionic sites. In order to characterize the anionic sites, minced pieces of villi were incubated in the presence or absence of either chondroitinase ABC, heparitinase, neuraminidase, or Streptomyces hyaluronidase in appropriate buffer systems. Incubation of early villi with heparitinase resulted in the disappearance of the TBL-associated sites. Chondroitinase ABC appeared to reduce staining of collagen-associated sites. In term villi, heparitinase removed those few sites still associated with the TBL but did not affect sites associated with the CBL or collagen. Chondroitinase ABC resulted in the disappearance of all anionic sites. In later gestation, a number of developmentally important macromolecules are transported across the trophoblast and enter the fetal capillaries. We conclude that the absence of an array of polyanionic sites from the term placenta TBL and the reduction in the amount of extracellular matrix intervening between the trophoblast and capillaries are adaptations to enhance the exchange of macromolecules across the placenta.     

Role of placental alkaline phosphatase in the interaction between human placental trophoblast and Trypanosoma cruzi.

Congenital Chagas disease, due to the intracellular parasite Trypanosoma cruzi, is associated with premature labor, miscarriage, and placentitis. Human enzyme placental alkaline phosphatase (PLAP) (EC 3.1.3.1.) is membrane-anchored through glycosylphosphatidylinositol (GPI). PLAP is present in plasma in late pregnancy, 36 to 40 weeks; there are lower levels in maternal Chagas disease. Infants born to such mothers may have congenital Chagas disease. Human placental villi (PV) were treated with phospholipase-C (PL-C) and then cultured with T. cruzi to determine the effect of the parasites on PLAP activity as an in vitro model. There is less PLAP activity after treatment by PL-C and during culture with T. cruzi. Pretreatment of PV with PL-C before culture with T. cruzi yielded essentially normal specific activity of PLAP and prevented or greatly reduced infective penetration of villi by parasites. The results are consistent with a pathogenetic role for placental alkaline phosphatase in congenital Chagas disease. Receptor activation of membrane attachment to PLAP may be a device used by T. cruzi to enable parasite invasion of human trophoblast.     

Cellular diversity of human placental stem villi: an ultrastructural and immunohistochemical study

The aim of the study was to investigate the distribution and differentiation of cell types in the stroma of human placental stem villi (SV). A total of 14 human term placental tissues were studied. Double immunolabeling was performed for desmin-vimentin, desmin--smooth actin and vimentin--smooth actin. Cytokeratin 7, proliferating cell nuclear antigen immunolabeling was also performed. Parallel tissue samples were examined by transmission electron microscopy. HSCORE was performed for the semi-quantitative analysis of distribution of cells in the stroma of SV. Vimentin-labeled cells were mostly distributed in the subtrophoblastic area. Desmin-vimentin double immunolabeling was mainly localized in the triangular area and to a lesser degree in the perivascular area and vessel walls (p=<0.001). However, desmin- smooth actin labeling was observed predominantly in the vessel wall and perivascular area. Vimentin- smooth actin immunoreactivity was significantly stronger in the triangular and perivascular areas compared to the vessel walls (p=0.003).

Ultrastructurally, cells in the stroma of SV were mesenchyme cells, reticulum cells, fibroblasts, myofibroblasts, smooth muscle cells, and Hofbauer cells, filamented and vacuolated cells. The differentiation of myofibroblasts in the triangular and perivascular areas may play a role in maturation of SV and villous contractility, modulation of the intervillous space and this may have effects on maternofetal placental circulation.     

Frequent apoptosis in placental villi from pregnancies complicated with intrauterine growth restriction and without maternal symptoms

The objective of this study was to examine the incidence of apoptosis in placentas of intrauterine growth restriction (IUGR) without maternal disease. We focused on cases of IUGR with both unknown causes and no maternal complications. In addition, apoptosis of IUGR placentas was correlated with the expression of proteins involved in apoptosis and cell turnover. The term placentas were obtained from 16 normal and 10 IUGR pregnancies without maternal symptoms. We analyzed the localization of apoptosis and counted >2000 nuclei by TUNEL assay and transmission electron microscopy in placentas from normal and IUGR cases. We found the incidence of apoptosis to be significantly higher in placentas with IUGR (2.98%) than in normal placentas (1.46%) by TUNEL assay (p=0.03). In addition, immunohistochemical analysis was performed to examine the expression of proteins related to apoptosis and general cell turnover, including the active form of caspase-3, Bax, p53, and Ki-67. Expression of the active form of caspase-3 was significantly higher in IUGR than in a normal pregnancy (p=0.03). These results suggest that enhanced execution of apoptosis through caspase-3 may play a role in IUGR without maternal symptoms.     

Hyaluronan, CD44 and its variant exons in human trophoblast invasion and placental angiogenesis

Both hyaluronan and one of its receptors, CD44, can be demonstratedin the early human conceptus and in placental stroma. The variantsof CD44 resulting from variable exon splicing are found in metastasizinghuman malignancies and are also involved in hyaluronan uptakeand degradation. The resulting hyaluronan fragments are knownto be highly angiogenic. We postulated that the self-limitedprocess of trophoblast invasion of the uterine decidua resultsin part from the strategy of alternative splicing of CD44, similarto that used by invasive cancer cells in the course of metastaticspread and possibly angiogenesis. Monoclonal antibodies specificfor CD44s and for an exon expressed during metastatic tumourprogression, CD44v7, were used to examine this hypothesis. Inthis study we found human trophoblasts, for the first time,to express CD44. Intermediate trophoblasts of first and secondtrimester exhibited the standard form of CD44 while extravilloustrophoblasts, which are responsible for the invading characteristicsof the placenta, were positive for the alternatively splicedform, the CD44v7–8. Moreover, in the case of placentaaccreta there was a prominent membrane staining of the trophoblaststhat were embedded in the fibrin layer over the myometrium.The highly metastatic choriocarcinoma cells also expressed CD44v7–8.We propose, therefore, that the invading trophoblasts utilizethe alternatively splicing machinery. These cells retain theirinvasive capabilities through the permissive ECM by carryingthe CD44v7–8 isoform, which binds weakly to hyaluronanand thus prevents it from being degraded by intracellular hyaluronidase. CD44/hyaluronan/invasion/placentaArophoblast     

Physiological concentrations of albumin stimulate chorionic gonadotrophin and placental lactogen release from human term placental explants

This study investigates whether albumin, a major plasma protein in direct contact with the trophoblast in vivo, can modulate human chorionic gonadotrophin (HCG) and human placental lactogen (HPL) releases from placental explants. Incubating explants with a near physiological, i.e. 5%, concentration of human or bovine albumin during 30 min increased HCG and HPL release by at least 150%. This albumin effect was not mediated by any difference in hormone adsorption onto glass surfaces. In contrast to the sustained stimulation of hormone releases elicited by the addition of 10 mmol/l extracellular calcium, the albumin-mediated secretory responses were transient. However, the albumin- and calcium-stimulatory effects were abolished at 4 degrees C, depressed by 0.36 mmol/l cycloheximide or 1 mmol/l colchicine and potentiated by 40 micromol/l cytochalasin B. Moreover, the stimulatory effect of albumin on the hormone releases was not modified in the absence of Ca(2+) or in the presence of 1 or 10 mmol/l Ca(2+) in the extracellular milieu. These data suggest that albumin is involved, at physiological concentration, in the secretion of HCG and HPL by human placenta. The cellular mechanism(s) underlying the albumin-mediated secretory responses may be partly different from those involved during the calcium-mediated stimulation.     

Differential expression of LIGHT and its receptors in human placental villi and amniochorion membranes

mRNA encoding LIGHT (homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes), a member of the tumor necrosis factor superfamily of ligands, as well as mRNAs encoding LIGHT receptors [HVEM, LTbetaR, and TR6 (DcR3)] are present in placentas and cytotrophoblast cells at term. To establish translation of these messages and determine directions for functional studies, term placentas, amniochorion membranes, and purified cytotrophoblast cells were evaluated by immunoblotting and immunohistochemistry. Ligand and receptor proteins were identified in lysates from all three sources although the soluble receptor, TR6, was scarce in placentas and all receptors were in low abundance in cytotrophoblast cells. These results were confirmed and cell type-specific expression was documented by immunohistochemistry. Ligand and receptor proteins were differentially expressed according to cell type. For example, HVEM was identified on syncytiotrophoblast but not in villous mesenchymal cells; amnion epithelial cells were positive for all proteins whereas chorion membrane cytotrophoblasts exhibited none. Because LIGHT is a powerful cytokine that can alter gene expression and promote apoptosis, these experiments suggest that ligand-receptor interactions may critically influence structural and functional aspects of human placentas through as yet undefined autocrine/paracrine pathways.     

Immunoelectron microscope observations on secretion of human placental lactogen (hPL) in the human chorionic villi

Immunoelectron microscope localization of human placental lactogen (hPL) was investigated in the chorionic villi from week 7 to full-term gestation with the protein A-gold technique. With specific antiserum against hPL, immuno-reactive gold particles were found to be preferentially located in Golgi-derived, electron-dense small granules of 80-180 nm in the syncytiotrophoblast. Our electron micrographs indicate that these small granules increase in number in the course of gestation and are released by exocytosis from the apical cell surface. The present study reveals that hPL is segregated from the Golgi apparatus, stored in the syncytiotrophoblast as secretory granules, and released into the maternal blood.     

Development of the human fetal insular cortex: study of the gyration from 13 to 28 gestational weeks

To describe the morphological stages of insular sulci and gyri development we carried out a macroscopical study on 21 human fetal brains, showing no anomalies, from 13 to 28 gestational weeks (GWs). Particular focus was given to morphological appearance during the development of insular and periinsular structures, especially the gyration and sulcation of the insula, central cerebral region and opercula, as well as the vascularization of these regions. The periinsular sulci and the central (insular and cerebral) sulci were the first macroscopical structures identified on the lateral surface of the human fetal cerebral hemisphere with earlier development on the right hemisphere. Here we describe five stages of insular gyral and sulcal development closely related to gestational age: stage 1: appearance of the first sulcus at 13-17 GWs, stage 2: development of the periinsular sulci at 18–19 GWs, stage 3: central sulci and opercularization of the insula at 20–22 GWs, stage 4: covering of the posterior insula at 24–26 GWs, stage 5: closure of the sylvian fissure at 27–28 GWs. We provide evidence that cortical maturation (sulcation and gyration) and vascularization of the lateral surface of the brain starts with the insular region, suggesting that this region is a central area of cortical development.     

Production of interferons in human placental trophoblast subpopulations and their possible roles in pregnancy.

The human cytotrophoblasts are the first fetal cells to arise during embryogenesis and are the progenitor cells to villous (noninvasive), syncytiotrophoblast (noninvasive), "intermediate" extravillous (invasive), and "anchoring" extravillous (invasive) trophoblast subpopulations. These trophoblast subpopulations were isolated from first- and third-trimester placentae and were stimulated with Sendai virus, granulocyte-macrophage colony-stimulating factors (GM-CSF), and platelet-derived growth factor (PDGF) to produce interferons (IFNs). GM-CSF and PDGF induced very low levels of IFN in first-trimester extravillous and villous trophoblast subpopulations. Highly proliferating and invasive intermediate extravillous trophoblast cultures produced five- to eightfold more IFNs than villous trophoblast cultures and two- to fivefold more IFN than the syncytiotrophoblast cultures when stimulated with Sendai virus. Syncytiotrophoblast cultures produced higher levels of IFNs (up to twofold) than villous trophoblast cultures when stimulated with the same virus. Pretreatment of first-trimester extravillous and villous trophoblast cultures with GM-CSF and PDGF followed by infection with Sendai virus resulted in greater IFN production than when the cultures were stimulated with virus alone. The levels of IFN produced were dependent on the type of trophoblast, the type of inducer, and the stage of differentiation of the trophoblasts. The purified trophoblast IFNs have potent antiviral activities when assayed on human amniotic WISH cells, and they inhibited proliferation of normal trophoblasts and trophoblast-derived malignant cells in vitro without any toxicity. Furthermore, the trophoblast IFNs activated NK cell activity and suppressed mitogen-stimulated lymphocyte proliferation at concentrations of between 10 and 1,000 IU/ml. The possible functions of the trophoblast IFNs during pregnancy are discussed with respect to human placental and fetal protection and development.     

Immunolocalisation of nucleoside transporters in human placental trophoblast and endothelial cells: evidence for multiple transporter isoforms

Polyclonal antibodies raised against the human erythrocyte nucleoside transporter were used to investigate the distribution of the nucleoside transporters in the placenta. Immunoblots of brush-border membranes isolated from the human syncytiotrophoblast revealed a cross-reactive species that co-migrated with the erythrocyte nucleoside transporter as a broad band of apparent M _r 55,000. In contrast, no labelling was detected in basal membranes containing a similar number of equilibrative nucleoside transporters as assessed by nitrobenzylthioinosine (NBMPR)-binding. The absence of cross-reactive epitopes in basal membranes and their presence in brush-border membranes was confirmed by confocal immunofluorescence microscopy. These results suggest that at least two isoforms of the NBMPR-sensitive nucleoside transporter are present in the human placenta. The lumenal surfaces of fetal capillaries, small placental vessels and umbilical vein were also strongly labelled by the antibody, a finding that suggests that the high fetal-placental adenosine uptake previously reported is due to endothelial transporters.Abbreviations NT The human erythrocyte nucleoside transporter - Anti-NT Polyclonal antibody against the nucleoside transporter - NBMPR Nitrobenzylthioinosine {6-[(4-nitrobenzyl)thio]-9--D-ribofuranosylpurine}. - GLUTl The human erythrocyte glucose transporter - PBS Phosphate-buffered saline - TBS TRIS-buffered saline - TTBS TRIS-buffered saline containing 0.2% Tween-20     

A database to support the interpretation of human mismatch repair gene variants

Germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2 can cause Lynch syndrome. This syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC), is an autosomal dominantly-inherited disorder predominantly characterized by colorectal and endometrial cancer. Truncating MMR gene mutations generally offer a clear handle for genetic counseling and allow for presymptomatic testing. In contrast, the clinical implications of most missense mutations and small in-frame deletions detected in patients suspected of having Lynch syndrome are unclear. We have constructed an online database, the Mismatch Repair Gene Unclassified Variants Database (www.mmruv.info), for information on the results of functional assays and other findings that may help in classifying these MMR gene variants. Ideally, such mutations should be clinically classified by a broad expert panel rather than by the individual database curators. In addition, the different MMR gene mutation databases could be interlinked or combined to increase user-friendliness and avoid unnecessary overlap between them. Both activities are presently being organized by the International Society for Gastrointestinal Hereditary Tumours (InSiGHT; www.insight-group.org).     

The distribution and mobility of anionic sites on the surface of human placental syncytial trophoblast

The purpose of this study was to examine the distribution and mobility of anionic sites on the surface of fetal trophoblast in contact with maternal blood using polycationic ferritin (PCF) as a probe. Pieces of human placental villi were washed to remove maternal blood, and fresh, unfixed tissue was expose to PCF for varying times, concentrations, and temperatures to determine the effects on labeling patterns. The major findings were: (1) anionic sites were localized almost exclusively on the microvillous portion of the trophoblast surface; intermicrovillous regions of the surface, including the coated pits, were generally not labeled with PCF; (2) PCF binding was present as small clusters on the microvilli. This pattern was observed in tissue incubated 5–10 sec at 4°C and 23°C. The size of the cluster was increased with increased incubation time, suggesting some aggregation or patching can occur; (3) following the fomation of patches, the anionic sites showed no evidence of being cleared from the membrane by endocytosis during incubation subsequent to labeling; (4) the binding of PCF to the surface was reduced by pretreatment of the tissue with neuraminidase. Tissue fixed in glutaraldehyde prior to PCF exposure showed both clustered and more dispersed labeling. The results indicate that anionic sites on human trophoblast surface have a non-random distribution and have restricted mobility on the surface. This may be indicative of a segregation of different membrane proteins and functions within different structural regions of the placental cell surface.     

Structural identification and characterization of arteries and veins in the placental stem villi

The vascular wall structure in the human full-term placental villi of normal pregnancy was studied by means of light and electron microscopy with an improved technique of perfusion fixation and tissue preparation. We observed 81 sections of stem villi that showed cross-sectional profiles of paired vessels in their center. Both vascular walls contained a large amount of extracellular matrix and no elastic lamina between smooth muscle cells of the media, making identification of the artery and the vein quite difficult at first sight. We then noted that the density of the smooth muscle cell population was always considerably higher in one than the other, and identified the former as artery and the latter as vein on the basis of their connection with larger arteries and veins running on the chorionic plate. Between the paired vessels, the artery had a smaller caliber than the vein, and the ratio of venous to arterial caliber was distributed from 1.0 to 2.5. The thickness of media was usually thicker in the vein than in the artery. Clusters of elastic fibers were found occasionally in the media of arteries and veins, and basement membrane-like materials were associated frequently with the elastic fibers and were distributed widely in the media as well as in the adventitia. In the veins, the smooth muscle cells of the most superficial part of the media contained well-developed rough endoplasmic reticulum and Golgi apparatus, indicating differentiation to secrete extracellular matrices. The present study revealed the difference of wall structure between arteries and veins in the placental stem villi for the first time at the ultrastructural level, and suggested differentiation of venous smooth muscle cells, possibly by some influence from the luminal side.     

Surface and branching of placental villi in early abortion: relationship to karyotype Scanning electron microscopic study

Summary The placental villi of 61 early abortions with known karyotype and 7 legally induced abortions were investigated by scanning electron microscopy and documented in standardised enlargements. Five groups were established from the findings: uniformly branched villi with a velvety surface (group A) were found in 4 of the 7 induced abortions, abundant syncytial sprouts (group B) in 4 of the 6 cases with monosomy X; all 5 cases of triploidy were classified in the group bulbous or spherical villi (group C); 13 out of 25 cases of trisomy were found to have little branching and a surface densely covered with microvilli (group D), while 14 out of the 25 cases of euploidy belonged in the group with slender villi and surface with focal areas of denudation (group E). Forty of the 68 cases were properly assignable to the correct groups (58.8%). The non-uniformity of the villous morphology in the case of induced abortions shows that there is no uniform development of the (early) placenta. The variable morphology seen in abortions with euploidy reflects the various mechanisms of abortion applicable to this group.     

Participation of collagen types I, III, IV, V, and fibronectin in the formation of villi fibrosis in human term placenta.

The indirect immunofluorescence method was used to study the human term placenta in pathological pregnancy for the distribution of collagen types I, III, IV, V, and fibronectin in fibrosis stromatis villi. All collagen types and fibronectin were shown to participate in fibrosis villorum formation. Fibronectin was also detected in the fibrinoid that surrounded villi at stroma. The presence of free cytotrophoblast cells in the fibrinoid was accompanied by a noticeable increase in fibronectin fluorescence. A significant amount of collagen types IV and V and a less amount of collagen types I and III were identified.     

IL-10在反复自然流产人绒毛组织的表达及意义

目的:通过研究白细胞介素-10 (IL-10)在反复自然流产和正常流产人绒毛组织和血清中的表达比较来探讨IL-10与不明原因反复自然流产的相关性及其临床意义.方法:采用免疫组织化学技术和逆转录-聚合酶链反应技术检测35例反复自然流产者(实验组)和35例正常流产者(对照组)人绒毛组织中IL-10的细胞学定位以及在mRNA水平的表达;ELISA法定量分析IL-10在两组血清中的表达.结果:免疫组织化学检测结果显示两组组织中均有IL-10阳性表达,实验组IL-10在人绒毛组织中的阳性表达率低于对照组(P＜0.05).RT-PCR结果显示实验组和对照组均有IL-10表达,对比显示实验组IL-10表达低于对照组.ELISA测定结果显示IL-10在实验组的表达低于对照组.结论:IL-10的表达在实验组早孕绒毛组织细胞学定位及mRNA水平检测均较对照组降低,在血清中的表达量也是实验组低于对照组,提示自然流产妊娠妇女存在IL-10细胞因子表达异常,IL-10可能在维持正常妊娠过程中起到重要作用.