Placental villous mesenchymal cells trigger trophoblast invasion

The successful transformation of uterine spiral arteries by invasion trophoblasts is critical for the formation of the human hemochorial placenta. Placental trophoblast migration and invasion are well regulated by various autocrine/paracrine factors at maternal–fetal interface. Human placental multipotent mesenchymal stromal cells (hPMSCs) are a subpopulation of villous mesenchymal cells and have been shown to produce a wide array of soluble cytokines and growth factors including HGF (hepatocyte growth factor). The function of hPMSCs in placental villous microenvironment has not been explored. The interaction between hPMSCs and trophoblasts was proposed in vitro in a recent article. HGF produced by hPMSCs was able to engage c-Met receptor on trophoblast and induced the trophoblast cAMP expression. The cAMP activated PKA, which in turn, signaled to Rap1 and led to integrin β1 activation. The total integrin β1 protein expression by trophoblasts was not affected by HGF stimulation. Hypoxia downregulated HGF expression by hPMSCs. HGF and PKA activator 6-Bnz-cAMP increased trophoblast adhesion and migration that were inhibited by PKA inhibitor H89 or Rap1 siRNA. Thus, hPMSCs-derived paracrine HGF can regulate trophoblast migration during placentation. These findings provided insight revealing at least one mechanism by which hPMSCs implicated in the development of preeclampsia.     

Placental villous mesenchymal cells trigger trophoblast invasion

The successful transformation of uterine spiral arteries by invasion trophoblasts is critical for the formation of the human hemochorial placenta. Placental trophoblast migration and invasion are well regulated by various autocrine/paracrine factors at maternal–fetal interface. Human placental multipotent mesenchymal stromal cells (hPMSCs) are a subpopulation of villous mesenchymal cells and have been shown to produce a wide array of soluble cytokines and growth factors including HGF (hepatocyte growth factor). The function of hPMSCs in placental villous microenvironment has not been explored. The interaction between hPMSCs and trophoblasts was proposed in vitro in a recent article. HGF produced by hPMSCs was able to engage c-Met receptor on trophoblast and induced the trophoblast cAMP expression. The cAMP activated PKA, which in turn, signaled to Rap1 and led to integrin β1 activation. The total integrin β1 protein expression by trophoblasts was not affected by HGF stimulation. Hypoxia downregulated HGF expression by hPMSCs. HGF and PKA activator 6-Bnz-cAMP increased trophoblast adhesion and migration that were inhibited by PKA inhibitor H89 or Rap1 siRNA. Thus, hPMSCs-derived paracrine HGF can regulate trophoblast migration during placentation. These findings provided insight revealing at least one mechanism by which hPMSCs implicated in the development of preeclampsia.     

Ultrastructural development of chorioallantoic placenta in the Indian Miniopterus bat, Miniopterus schreibersii fuliginosus (Hodgson).

The chorioallantoic placental interhemal membrane of Miniopterus schreibersii fuliginosus has been described electron-microscopically. Morphologically there are three main types of placentae which develop in chronological sequence. They are (1) primary placenta, (2) secondary placenta and (3) tertiary placenta. In neural groove and limb-bud embryos the primary placenta consists of the following elements which separate the maternal and fetal circulations: (1) a continuous ectoplasmic layer, (2) intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The primary placenta degenerates until term when it consists of a thin syncytiotrophoblastic layer resting on basal lamina. Mesenchyme does not show the presence of fetal capillaries. The secondary placenta is formed in early limb-bud embryos. The electron microscope has revealed that the placenta is of the endotheliomonochorial type and (1) consists of a well-developed maternal endothelium, (2) the trophoblast surrounding the maternal blood tubule is cellular, not syncytial as previously thought and the apical plasma membrane of these trophoblastic cells is in direct contact with the discontinuous interstitial membrane, (3) basal lamina, (4) mesenchyme and (5) fetal endothelium. Tertiary placenta at full term stage is of the hemodichorial type having the following elements: (1) thin ectoplasmic layer, (2) a thick intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The definitive chorioallantoic placental barrier in this bat thus differs from the organization earlier proposed by Chari and Gopalakrishna [Proc. Indian Acad. Sci. 93: 463-483, 1984] on the basis of light-microscopic observations: (1) the absence of maternal endothelium in the primary placenta from the neural groove and early limb-bud embryos, (2) the existence of only cellular trophoblast in the secondary placenta throughout the gestation and (3) the presence of well-developed hemodichorial tertiary placenta is the unique feature of the interhemal membrane in higher Chiroptera.     

Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.

The purpose of this study was to examine the expression of hemeoxygenases HO-1 and HO-2, which are responsible for the production of carbon monoxide (CO), in the human placenta and placental bed and to determine the role of inhibitors of HO on placental perfusion pressure. We hypothesized that HO is expressed within the placenta and that invading cytotrophoblast cells (CTB) express HO isoforms. The expression of HO-1 and HO-2 was studied on placenta and placental bed biopsies, obtained using a transcervical sampling technique, from normal human pregnancies between 8 and 19 wk gestation and at term. In the placenta, HO-2 immunostaining was prominent in syncytiotrophoblast in the first trimester and reduced toward term (P<0.0005). HO-2 endothelial immunostaining was weak in the first trimester, but increased by term (P<0.0005). Within the placental bed, HO-2 was expressed by CTB in cell columns, the cytotrophoblast shell, and cell islands. Both intravascular CTB and interstitial CTB expressed HO-2. HO-1 immunostaining was low in the placenta but intense on the CTB within the placental bed. A striking feature was the absence of HO-1 from the proximal layers of cell columns, with strong expression on the more distal CTB layers of the cell columns. In placental perfusion studies, a significant dose-dependent increase in perfusion pressure was observed in the presence of zinc protoporphyrin, an inhibitor of HO. These results suggest a role for CO in placental function, trophoblast invasion, and spiral artery transformation. Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.     

Regulation of pericellular proteolysis by hepatocyte growth factor activator inhibitor type 1 (HAI-1) in trophoblast cells

Hepatocyte growth factor activator inhibitor type 1/serine protease inhibitor Kunitz type 1 (HAI-1/SPINT1) is a membrane-bound Kunitz-type serine protease inhibitor that is abundantly expressed on the surface of cytotrophoblasts, and is critically required for the formation of the placenta labyrinth in mice. HAI-1/SPINT1 regulates several membrane-associated cell surface serine proteases, with matriptase being the most cognate target. Matriptase degrades extracellular matrix protein such as laminin and activates other cell surface proteases including prostasin. This study aimed to analyze the role of HAI-1/SPINT1 in pericellular proteolysis of trophoblasts. In HAI-1/SPINT1-deficient mouse placenta, laminin immunoreactivity around trophoblasts was irregular and occasionally showed an intense punctate pattern, which differed significantly from the linear distribution along the basement membrane observed in wild-type placenta. To explore the molecular mechanism underlying this observation, we analyzed the effect of HAI-1/SPINT1 knock down (KD) on pericellular proteolysis in the human trophoblast cell line, BeWo. HAI-1/SPINT1-KD BeWo cells had increased amounts of cellular laminin protein and decreased laminin degradation activity in the culture supernatant. Subsequent analysis indicated that cell-associated matriptase was significantly decreased in KD cells whereas its mRNA level was not altered, suggesting an enhanced release and/or dislocation of matriptase in the absence of HAI-1/SPINT1. Moreover, prostasin activation and pericellular total serine protease activities were significantly suppressed by HAI-1/SPINT1 KD. These observations suggest that HAI-1/SPINT1 is critically required for the cell surface localization of matriptase in trophoblasts, and, in the absence of HAI-1/SPINT1, physiological activation of prostasin and other protease(s) initiated by cell surface matriptase may be impaired.     

Identification of antibodies against HAI-1 and integrin alpha6beta4 as immunohistochemical markers of human villous cytotrophoblast.

Syncytiotrophoblast and invasive extravillous trophoblast arise from a common stem cell, namely villous cytotrophoblast, but have very different characteristics. The study of the differentiation process relies on the availability of suitable markers for these different cell types of developing placenta. In this work, we have produced monoclonal antibodies that are specific to human villous cytotrophoblast. Monoclonal antibody (MAb) MG2 was specific to villous cytotrophoblast across gestation, and recognizes hepatocyte growth factor activator inhibitor type 1. MAb MD10 stained villous cytotrophoblast across gestation and also some endothelial cells, particularly in the second or third trimester. MAb MD10 recognizes human integrin alpha6beta4. As a test for specificity, the novel MAbs were also used for staining of frozen tissue from human colon carcinoma. The results show that the two antibodies can be used as tools to study human villous cytotrophoblasts and also human tumors. The MG2 antibody seems most specific and promising for the study of various aspects of human villous cytotrophoblast.     

Nitric oxide synthase activity in human trophoblast, term placenta and pregnant myometrium

To investigate the possible role of nitric oxide (NO) produced locally or intramurally in the quiescence of the pregnant myometrium, nitric oxide synthase (NOS) activity was measured in samples from first trimester (villous, and non villous-trophoblast), term placenta and pregnant myometrium. Trophoblast tissue was obtained from psychosocial termination of pregnancy (9 – 12 weeks' gestation) whereas placenta and myometrium, from the same patient, at deliveries by Caesarean section. NOS activity was measured in both cytosolic and particulate fractions by the formation of ¹⁴C-citrulline from ¹⁴C-arginine. Western immunoblotting was used to identify the endothelial NOS (eNOS) and neuronal (nNOS) isoforms. The activity of NOS in particulate fractions from all preparations was considerably higher than the cytosolic fractions. Activity in all fractions except the myometrium was highly Ca-dependent. More than 50% of particulate NOS from the myometrium was Ca-independent. NOS activity was highest in the villous trophoblast and there was a significant difference between the villous and non-villous trophoblast. In placenta and myometrium, NOS was 2–4 fold and 20–28-fold lower than the villous trophoblast, respectively. Western blot analysis showed clearly eNOS in the particulate fraction and a weak eNOS band in the cytosolic fractions, whereas nNOS was not detectable in any of the fractions. In view of the marginal activity of NOS in the myometrium, NO produced by the trophoblast and placenta could play a significant role in maintaining uterine quiescence by paracrine effect.     

Integrin-linked kinase (ILK) is highly expressed in first trimester human chorionic villi and regulates migration of a human cytotrophoblast-derived cell line

The placenta represents a critically important fetal-maternal interaction. Trophoblast migration and invasion into the uterine wall is a precisely controlled process and aberrations in these processes are implicated in diseases such as preeclampsia. Integrin-linked kinase (ILK) is a multifunctional, cytoplasmic, serine/threonine kinase that has been implicated in regulating processes such as cell proliferation, survival, migration, and invasion; yet the temporal and spatial pattern of expression of ILK in human chorionic villi and its role in early human placental development are completely unknown. We hypothesized that ILK would be expressed in trophoblast subtypes of human chorionic villi during early placental development and that it would regulate trophoblast migration. Immunoblot analysis revealed that ILK protein was highly detectable in placental tissue samples throughout gestation. In floating branches of chorionic villi, from 6 to 15 wk of gestation immunofluorescence analysis of ILK expression in placental tissue sections demonstrated that ILK was highly detectable in the cytoplasm and membranes of villous cytotrophoblast cells and in stromal mesenchyme, whereas it was barely detectable in the syncytiotrophoblast layer. In anchoring branches of villi, ILK was highly localized to plasma membranes of extravillous trophoblast cells. Transient expression of dominant negative E359K-ILK in the villous explant-derived trophoblast cell line HTR8-SVneo dramatically reduced migration into wounds compared to cells expressing wild-type ILK or empty vector. Therefore, our work has demonstrated that ILK is highly expressed in trophoblast subtypes of human chorionic villi during the first trimester of pregnancy and is a likely mediator of trophoblast migration during this period of development.     

Quantitative characteristics of components of the placental barrier of women with uncomplicated full-term pregnancies]

Placentas from four healthy women have been studied electron microscopically after urgent spontaneous deliveries. In every placenta 130-200 resorptive villi of the chorion were photographed. The results on morphometric analysis of 1,205 electronograms of the placental barrier are presented. At full-term uncomplicated pregnancies all components of the placental barrier (syncytium, cytotrophoblast, basal membrane of the trophoblast, stromal connective-tissue layer, basal membrane and endothelium of fetal capillaries) have been stated to demonstrate rather stable quantitative characteristics (see Table in the text). The quantitative data obtained can serve for electron microscopic investigation of placenta at different pathologic states of the mother and the fetus, make it possible to differentiate more precisely physiological and pathological changes in placental ultrastructure.     

Isolation and characterization of human trophoblast side-population (SP) cells in primary villous cytotrophoblasts and HTR-8/SVneo cell line

Recently, numerous studies have identified that immature cell populations including stem cells and progenitor cells can be found among "side-population" (SP) cells. Although SP cells isolated from some adult tissues have been reported elsewhere, isolation and characterization of human trophoblast SP remained to be reported. In this study, HTR-8/SVneo cells and human primary villous cytotrophoblasts (vCTBs) were stained with Hoechst 33342 and SP and non-SP (NSP) fractions were isolated using a cell sorter. A small population of SP cells was identified in HTR-8/SVneo cells and in vCTBs. SP cells expressed several vCTB-specific markers and failed to express syncytiotrophoblast (STB) or extravillous cytotrophopblast (EVT)-specific differentiation markers. SP cells formed colonies and proliferated on mouse embryonic fibroblast (MEF) feeder cells or in MEF conditioned medium supplemented with heparin/FGF2, and they also showed long-term repopulating property. SP cells could differentiate into both STB and EVT cell lineages and expressed several differentiation markers. Microarray analysis revealed that IL7R and IL1R2 were exclusively expressed in SP cells and not in NSP cells. vCTB cells sorted as positive for both IL7R and IL1R2 failed to express trophoblast differentiation markers and spontaneously differentiated into both STB and EVT in basal medium. These features shown by the SP cells suggested that IL7R and IL1R2 are available as markers to detect the SP cells and that vCTB progenitor cells and trophoblast stem cells were involved in the SP cell population.     

Chorioallantoic placenta formation in the rat: I. Luminal epithelial cell death and extracellular matrix modifications in the mesometrial region of implantation chambers.

On days 7 and 8 of pregnancy, mesometrial regions of rat gestation sites were examined by light microscopy and transmission electron microscopy to determine what changes occur before the chorioallantoic placenta forms in that region. By day 7, gestation sites contained a uterine lumen mesometrially and an antimesometrial extension of the uterine lumen, the implantation chamber. The implantation chamber consisted of a mesometrial chamber between the uterine lumen and the conceptus, an antimesometrial chamber that contained the conceptus, and a decidual crypt antimesometrial to the conceptus. Stromal cells that formed the walls of the implantation chamber were closely packed decidual cells, while those that surrounded the uterine lumen were loosely arranged. Late on day 7, a portion of the epithelium lining the mesometrial chamber was degenerating, but this area of initial degeneration was never adjacent to the antimesometrial chamber. By early day 8, most of the epithelial cells lining the mesometrial chamber were degenerating and were being sloughed into the chamber lumen. Although degeneration of these epithelial cells morphologically resembled necrosis, it was precisely controlled, since adjacent epithelial cells lining the uterine lumen remained healthy. The space that separated the denuded luminal surface of the mesometrial chamber from underlying decidual cells became wider and was occupied by an extracellular matrix rich in cross-banded collagen fibrils. Decidual cell processes, that earlier had penetrated the basal lamina beneath healthy epithelial cells, protruded into this matrix and penetrated the basal lamina at the luminal surface. By late day 8, large areas of denuded chamber wall were covered with decidual cell processes, little remained of the basal lamina, and cross-banded collagen fibrils were scarce in the area occupied by decidual cell processes. During the times studied, uterine tissues that formed the walls of the mesometrial chamber were not in direct contact with the conceptus. This study indicates that trophoblast does not play a direct role in epithelial degeneration, basal lamina penetration, or extracellular matrix modifications in the mesometrial region of implantation chambers where part of the chorioallantoic placenta forms, although trophoblast may be required to trigger or modulate some of the changes.     

Up‐regulation of SPINT2/HAI‐2 by Azacytidine in bone marrow mesenchymal stromal cells affects leukemic stem cell survival and adhesion

The role of tumour microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stromal cell (BMMSC) contribution to disease progression remains poorly explored. We previously reported that the expression of serine protease inhibitor kunitz‐type2 (SPINT2/HAI‐2), an inhibitor of hepatocyte growth factor (HGF) activation, is significantly lower in BMMSC from myelodysplastic syndromes (MDS) patients compared to healthy donors (HD). Thus, to investigate whether this loss of expression was due to SPINT2/HAI‐2 methylation, BMMSC from MDS and de novo acute myeloid leukaemia (de novo AML) patients were treated with 5‐Azacitidine (Aza), a DNA methyltransferase inhibitor. In MDS‐ and de novo AML‐BMMSC, Aza treatment resulted in a pronounced SPINT2/HAI‐2 levels up‐regulation. Moreover, Aza treatment of HD‐BMMSC did not improve SPINT2/HAI‐2 levels. To understand the role of SPINT2/HAI‐2 down‐regulation in BMMSC physiology, SPINT2/HAI‐2 expression was inhibited by lentivirus. SPINT2 underexpression resulted in an increased production of HGF by HS‐5 stromal cells and improved survival of CD34⁺ de novo AML cells. We also observed an increased adhesion of de novo AML hematopoietic cells to SPINT2/HAI‐2 silenced cells. Interestingly, BMMSC isolated from MDS and de novo AML patients had increased expression of the integrins CD49b, CD49d, and CD49e. Thus, SPINT2/HAI‐2 may contribute to functional and morphological abnormalities of the microenvironment niche and to stem/progenitor cancer cell progression. Hence, down‐regulation in SPINT2/HAI‐2 gene expression, due to methylation in MDS‐BMMSC and de novo AML‐BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment.     

Involvement of calcitonin gene-related peptide in control of human fetoplacental vascular tone

Calcitonin gene-related peptide (CGRP), one of the most potent endogenous vasodilators known, has been implicated in vascular adaptations and placental functions during pregnancy. The present study was designed to examine the existence of CGRP-A receptor components, the calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein 1 (RAMP1), in the human placenta and the vasoactivity of CGRP in the fetoplacental circulation. Immunofluorescent staining of the human placenta in term labor using polyclonal anti-CRLR and RAMP1 antibodies revealed that labeling specifically concentrated in the vascular endothelium and the underlying smooth muscle cells in the umbilical artery/vein, chorionic artery/vein, and stem villous vessels as well as in the trophoblast layer of the placental villi. In vitro isometric force measurement showed that CGRP dose dependently relaxes the umbilical artery/vein, chorionic artery/vein, and stem villous vessels. Furthermore, CGRP-induced relaxation of placental vessels are inhibited by a CGRP receptor antagonist (CGRP8-37), ATP-sensitive potassium (KATP) channel blocker (glybenclamide), and cAMP-dependent protein kinase A inhibitor (Rp-cAMPS) and partially inhibited by a nitric oxide inhibitor (Nomega-nitro-l-arginine methyl ester). We propose that CGRP may play a role in the control of human fetoplacental vascular tone, and the vascular dilations in response to CGRP may involve activation of KATP channels, cAMP, and a nitric oxide pathway.     

Proliferation of villous trophoblast of the human placenta in normal and abnormal pregnancies.

The proliferation of villous trophoblast in the human placenta was estimated throughout normal gestation and in term placentae from preeclamptic and smoking mothers by two different methods. These were: 1) labeling of DNA producing cells by bromodeoxyuridine (BrdU) followed by immunohistochemistry using a monoclonal anti-BrdU antibody, and 2) immunohistochemical identification of all proliferating cells by the monoclonal antibody Ki67. Both methods revealed comparable results. In uncomplicated pregnancies there was a remarkable decrease in the labeling indices from early gestation to term. This was the result of a diminution of the number of Langhans' cells, although the cell division rate within the Langhans' cell layer remained nearly constant throughout gestation. A prolongation of the cell cycle in the cytotrophoblast cells at term was indicated by an increase in the Ki67/BrdU ratio. Compared with normal term placentae, there was an increase in the trophoblast proliferation rate in preeclampsia, but not in placentae from smoking mothers. Moreover, the number of Langhans' cells was diminished in placentae from smokers. The results indicate that there are different pathogenetic mechanisms of placental impairment in preeclampsia and in maternal smoking. In preeclampsia an injury to the syncytiotrophoblast seems to lead to a repair hyperplasia of the cytotrophoblast, whereas in maternal smoking, there seems to be a direct toxic effect on the cytotrophoblastic cells.     

Alpha-1-Antitrypsin: A Novel Human High Temperature Requirement Protease A1 (HTRA1) Substrate in Human Placental Tissue

The human serine protease high temperature requirement A1 (HTRA1) is highly expressed in the placental tissue, especially in the last trimester of gestation. This suggests that HTRA1 is involved in placental formation and function. With the aim of a better understanding of the role of HTRA1 in the placenta, candidate substrates were screened in a placenta protein extract using a gel-based mass spectrometric approach. Protease inhibitor alpha-1-antitrypsin, actin cytoplasmic 1, tropomyosin beta chain and ten further proteins were identified as candidate substrates of HTRA1. Among the identified candidate substrates, alpha-1-antitrypsin (A1AT) was considered to be of particular interest because of its important role as protease inhibitor. For investigation of alpha-1-antitrypsin as substrate of HTRA1 synthetic peptides covering parts of the sequence of alpha-1-antitrypsin were incubated with HTRA1. By mass spectrometry a specific cleavage site was identified after met-382 (AIPM³⁸²↓³⁸³SIPP) within the reactive centre loop of alpha-1-antitrypsin, resulting in a C-terminal peptide comprising 36 amino acids. Proteolytic removal of this peptide from alpha-1-antitrypsin results in a loss of its inhibitor function. Beside placental alpha-1-antitrypsin the circulating form in human plasma was also significantly degraded by HTRA1. Taken together, our data suggest a link between the candidate substrates alpha-1-antitrypsin and the function of HTRA1 in the placenta in the syncytiotrophoblast, the cell layer attending to maternal blood in the villous tree of the human placenta. Data deposition: Mass spectrometry (MS) data have been deposited to the ProteomeXchange with identifier PXD000473.     

滋养层细胞侵袭相关基因表达谱分析

分离收集正常妊娠第8～12周的细胞滋养层细胞和绒毛外滋养层细胞,提取细胞总RNA,制备cRNA探针并与AffymetrixU133plus2.0基因芯片进行杂交,获得正常细胞滋养层细胞和绒毛外滋养层细胞基因表达谱芯片。经计算机分析共筛选到1318个差异表达基因,其中上调基因813个,下调505个。所有差异表达基因按GeneOntoloty功能分类标准进行了功能检索。为胚胎发育早期绒毛外滋养层细胞侵袭的基因调控机制的研究提供了实验基础。     

Dysferlin is expressed in human placenta but does not associate with caveolin

A proteomics screen of human placental microvillous syncytiotrophoblasts (STBs) revealed the expression of dysferlin (DYSF), a plasma membrane repair protein associated with certain muscular dystrophies. This was unexpected given that previous studies of DYSF have been restricted to skeletal muscle. Within the placenta, DYSF localized to the STB and, with the exception of variable labeling in the fetal placental endothelium, none of the other cell types expressed detectable levels of DYSF. Such restricted expression was recapitulated using primary trophoblast cell cultures, because the syncytia expressed DYSF, but not the prefusion mononuclear cells. The apical plasma membrane of the STB contained approximately 4-fold more DYSF than the basal membrane, suggesting polarized trafficking. Unlike skeletal muscle, DYSF in the STB is localized to the plasma membrane in the absence of caveolin. DYSF expression in the STB was developmentally regulated, because first-trimester placentas expressed approximately 3-fold more DYSF than term placentas. As the current literature indicates that few cell types express DYSF, it is of interest that the two major syncytial structures in the human body, skeletal muscle and the STB, express this protein.     

Ultrastructural observations on the chorioallantoic placenta of the golden-rumped elephant shrew, Rhynchocyon chrysopygus

The chorioallantoic placenta of the elephant shrew (Rhynchocyon) was studied at the ultrastructural level. The placenta is haemonochorial with an appparent lack of microvilli on the syncytiotrophoblastic surface. There is, however, an unusual development of subsurface bays which are covered only by an ectoplasmic layer of trophoblast. The contents of the bays is PAS-negative and is similar in both texture and appearance to the maternal blood plasma. The basal trophoblastic cells are exceptionally tall and are held together by numerous desmosomes. Their basal lamina, which is on the trophospongial side, it unusually thick. At the fetomaternal junction the basal trophoblastic cells are coated with a finely granular electron-dense precipitate of unknown nature. Electron-dense particles resembling iron deposits are demonstrable in the fetal mesenchyme of the labyrinthine zone.