A light and electron microscopical study of the Tragulid (mouse deer) placenta

The Tragulidae are the living relics of the basal ruminant stock. They have a diffuse placenta, with no aggregations of the placental villi into localised placentomes characteristic of all other ruminants. Despite this difference, this ultrastructural and immunocytochemical investigation demonstrates that in Tragulus the trophoblast binucleate cell (BNC) plays the same central role in development and structure as in all other ruminants. It shows an identical development and ultrastructure, produces granules reactive with bovine placental lactogen and pregnancy associated glycoprotein antibodies, and migrates when mature through the trophoblast tight junction to fuse into a mosaic of syncytial plaques from which the granules are released to the mother and which have replaced the uterine epithelium. Unlike the persistent plaques in the sheep and goat placenta, in Tragulus they are transient, dying by apoptosis with the fragments phagocytosed by the trophoblast. This brings the trophoblast into direct endotheliochorial apposition to maternal tissue until BNC migration and fusion replace the dead plaque. This intimate fetomaternal confrontation has not been shown in any other ruminant, and could be a relic of the evolutionary development of the synepitheliochorial from the original basic eutherian endo- or hemo-chorial placenta.     

Basal membrane localization of MRP1 in human placental trophoblast

The placental trophoblast is considered to act as a barrier between mother and fetus, mediating the exchange of various materials across the placenta. ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp) and multidrug-resistance protein (MRP) are expressed in the placenta and function as efflux transport systems for xenobiotics. In the present study, we aimed to determine the localization of MRP1 in the human placenta in comparison with that of P-gp. Western blotting analysis with human placental membrane vesicles indicated that P-gp and MRP1 are localized on the brush-border membranes and basal membranes, respectively. Immunohistochemical analysis with human normal full-term placenta showed that anti-P-gp monoclonal antibody F4 stained the brush-border side of the trophoblast cells, whereas anti-MRP1 monoclonal antibody MRPr1 stained the basal side. These results confirm that P-gp and MRP1 are located on the brush-border membranes and basal membranes, respectively, of human full-term placental trophoblast. MRP1 was also detected on the abluminal side of blood vessels in the villi. Accordingly, MRP1 may play a role distinct from that of P-gp, which is considered to restrict the influx of xenobiotics into the fetus.     

Antibodies to trophoblast antigens HLA-G, placenta growth factor, and neuroD2 do not improve detection of circulating trophoblast cells in maternal blood

OBJECTIVES: Non-invasive prenatal diagnosis using circulating fetal trophoblast cells has been challenging due to lack of a reproducible trophoblast-specific antibody. We investigated the use of three trophoblast cell-specific antibodies, HLA-G, placenta growth factor, and neuroD2, for the isolation of trophoblast cells from the maternal circulation. METHODS: Trophoblast cells were isolated by density centrifugation from maternal blood samples (gestational age 10-20 weeks, n = 9). All women were carrying a male fetus. Following immunocytochemical staining with the trophoblast-specific antibodies, fluorescent in situ hybridization was performed, to verify whether any stained cells were indeed fetal. RESULTS: The HLA-G antibody had a ubiquitous staining pattern, which was not specific for trophoblast cells. Neither the placenta growth factor nor the neuroD2 antibodies were able to identify any trophoblast cells. Following fluorescent in situ hybridization, no male cells were detected on any of the slides. CONCLUSION: The antibodies used in this study were unable to improve detection of trophoblast cells in the maternal circulation.     

Characterization of a sheep trophoblast-derived antigen first appearing at implantation

A monoclonal antibody designated SBU-3 was produced by the fusion of mouse NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with sheep trophoblast microvilli. Lee et al (1985) have reported the immunohistological staining of sheep trophoblast with SBU-3 showing that, as early as 21 days of gestation, the monoclonal antibody recognizes an antigen restricted to the binucleate cells of the trophoblast which are located only at sites of invasion of the underlying uterine tissue. Subsequently the antigen appears in the maternal syncytial layer. Immunoprecipitation of 125I-labelled microvilli by SBU-3, characterization of the antigen on immunoblots, and biochemical analysis all suggest that this monoclonal antibody specifically recognizes a carbohydrate epitope on a series of glycoproteins of molecular weights between 30 000 and 200 000. SBU-3 antigen is present in allantoic fluid but is not detectable in any fetal or adult tissue studied, including maternal and fetal sera. It is suggested that this antigen may have a role in the placentation process.     

The evolving placenta: convergent evolution of variations in the endotheliochorial relationship

Endotheliochorial placentas occur in orders from all four major clades of eutherian mammal. Species with this type of placenta include one of the smallest (pygmy shrew) and largest (African elephant) land mammals. The endotheliochorial placenta as a definitive form has an interhemal area consisting of maternal endothelium, interstitial lamina, trophoblast, individual or conjoint basal laminas, and fetal endothelium. We commonly think of such placentas as having hypertrophied maternal endothelium with abundant rough endoplasmic reticulum (rER), and as having hemophagous regions. Considering them as a whole, the trophoblast may be syncytial or cellular, fenestrated or nonfenestrated, and there may or may not be hemophagous regions. Variations also appear in the extent of hypertrophy of the maternal endothelium and in the abundance of rER in these cells. This combination of traits and a few other features produces many morphological variants. In addition to endotheliochorial as a definitive condition, a transitory endotheliochorial condition may appear in the course of forming a hemochorial placenta. In some emballonurid bats the early endotheliochorial placenta has two layers of trophoblast, but the definitive placenta lacks an outer syncytial trophoblast layer. In mollosid bats a well developed endotheliochorial placenta is present for a short time even after a definitive hemochorial placenta has developed in a different region. It is concluded that the endotheliochorial placenta is more widespread and diversified than originally thought, with the variant with cellular trophoblast in particular appearing in several species studied recently.     

Serological and immunohistochemical studies in pregnant women with systemic lupus erythematosus

The present report is a detailed, prospective analysis of 29 pregnancies in 19 patients with systemic lupus erythematosus (SLE). The clinical pregnant status was correlated with maternal serologic patterns. Poor obstetrical prognosis was associated with low or declining complement (C3 and C4) levels and rising antinuclear and anti-DNA antibody titers. Complement levels reflected clinical changes more accurately than either antinuclear factor. A smooth antepartum course and successful delivery occurred when complement values remained normal. Immunohistochemical studies showed granular or linear depositions of immunoglobulin and complement on syncytial trophoblast and stromal tissue in placenta with SLE. Slight deposits of IgG were observed on the stromal tissue in normal placenta. Immune complex deposition on the trophoblast correlated to both serologic evaluation and course of SLE pregnancy. Obstetrical prognosis can be evaluated by serial determinations of serologic status. These data suggest that declining levels of maternal complements in SLE may be partly due to immune complex deposition in the placenta, resulting in the increased placental dysfunction.     

Lateral placental growth occurs by trophoblast cell invasion of decidual veins

During human pregnancy, growth of the placenta is proportionally greater than the growth of the decidual surface, suggesting that trophoblast cells invade the decidua at the placenta's margin. We hypothesized that a method of lateral placental growth was trophoblast cell invasion of decidual veins. This was investigated in two in situ pregnancies and in tissues from 100 women undergoing elective termination at 8-12 weeks of gestation. Decidua was compared to normal secretory endometrium. Histological sections were stained by immunohistochemistry to identify trophoblast cell and vascular antigens, as well as vascular cell adhesion molecule (VCAM), integrin subunits beta(1)and beta(4), and oncofetal fibronectin. Dilated veins were observed in all decidua but not in the secretory endometrium. Decidual and myometrial veins contained villi, trophoblast cell islands and syncytial elements. Decidual endothelial cells expressed vascular cell adhesion molecule (VCAM). Villous trophoblast cells were integrin subunits beta(4)positive and beta(1)negative. Trophoblast cell islands in the placenta and within decidual veins were integrin subunits beta(1)positive and beta(4)negative. Trophoblast cell islands and villi attached to veins, and mononuclear cells, invaded decidual stroma. Oncofetal fibronectin was present at sites of trophoblast invasion.These findings suggest that a method of lateral placental growth is trophoblast cell invasion of veins.     

Aberrations of early trophoblast differentiation predispose to pregnancy failure: lessons from the anti-phospholipid syndrome

OBJECTIVES: The epithelium of the human placenta comprises an inner cytotrophoblast (CT) which proliferates and fuses with the outer differentiated syncytiotrophoblast (ST). Turnover has been studied focussing on second and third trimester placentas but with a paucity of data describing the normal first trimester trophoblast. The aim of this study was to compare the nuclear CT:ST ratio in normal and pathological pregnancy and thus establish the relationship between cytotrophoblast and syncytiotrophoblast nuclear number during early gestation. METHODS: Archival first trimester material from placentas from healthy pregnancy and recurrent miscarriage (anti-phospholipid syndrome) was stained with H&E, cytokeratin-7 and Mib-1. The area of trophoblast as a fraction of total villous area was calculated and the number of sectioned cytotrophoblast and syncytiotrophoblast nuclei as well as the number of proliferating cytotrophoblast was evaluated. RESULTS: Normal features of trophoblast development during the first trimester (rise in trophoblast area, increase in number of syncytiotrophoblast nuclei, increase in number of proliferating cytotrophoblast, decrease in the nuclear CT:ST ratio) are absent/reversed in tissues from recurrent miscarriage (decreasing trophoblast area, constant number of syncytiotrophoblast nuclei, decreasing number of proliferating trophoblast, constant nuclear CT:ST ratio). CONCLUSIONS: Proliferation of cytotrophoblast in early gestation provides a pool of trophoblast stem cells critical for ongoing placental development. Premature cytotrophoblast differentiation in favour of syncytial fusion results in deficiencies of cytotrophoblast and rarification of villous trophoblast. Abnormal trophoblast differentiation in early gestation may be due to a premature onset of maternal perfusion of the placenta and may be a likely antecedent for conditions associated with failure of placentation such as recurrent miscarriage.     

The localisation of copper/zinc superoxide dismutase in human normal term placenta by an immunofluorescence technique.

The antioxidant enzyme copper and zinc containing superoxide dismutase (Cu/Zn SOD) was localised in normal human term placenta to the syncytial trophoblast, using an immunofluorescence technique. Possible physiological roles of Cu/Zn SOD in the human placenta are discussed.     

Expression of a cytokeratin 18 neo-epitope is a specific marker for trophoblast apoptosis in human placenta

In epithelial cells the caspase-mediated cleavage of cytokeratin 18 during apoptosis leads to the formation of a specific neo-epitope, recognized by the antibody M30. To test whether this antibody can be used as a specific marker for apoptotic trophoblast, we have stained serial sections of villi and junctional zone of first and third trimester human placenta with antibodies against cytokeratins 7 and 18, and against active caspase 3, with M30 and with the TUNEL reaction. Comparison of M30 immunoreactivities with TUNEL positivity and immunoreactivities for cytokeratins 7 and 18 clearly demonstrates that M30 specifically labels late apoptotic trophoblast cells. This finding is supported by the fact that in trophoblast, M30 immunoreactivities largely overlap with those for active caspase 3. As compared to the TUNEL test, the M30 immune reaction appears to be a highly reproducible marker for apoptotic trophoblast. This antibody stains a larger number of cells within the apoptosis cascade as compared to the TUNEL reaction, since cytokeratin 18 cleavage starts earlier than cleavage of DNA and since endonuclease activation can be bypassed in some trophoblast cells. The data suggest that M30 is superior to the TUNEL reaction as a marker for the detection of trophoblast apoptosis since it is easier to handle, more specific for apoptosis and less prone to artifacts.     

Light and electron microscopic observations on a human placenta 2 weeks after fetal death

Morphologic evidence for the viability of the placenta 2 weeks after fetal death at the twenty-seventh week of gestation was obtained using light and electron microscopy. The syncytial trophoblast appeared normal for this stage of gestation. There was an abundance of cytotrophoblastic elements, which showed many intermediate stages in cytoplasmic complexity from simple cells with little endoplasmic reticulum and an abundance of ribosomal and glycogen granules to cells with the cytoplasmic characteristics of syncytial trophoblast. The fetal mesenchyme was devascularized and showed a marked de position of collagen and basement membrane-like material. Follicles in the ovary showed evidence of luteinizing influence. The single corpus luteum appeared nonfunctional as judged by light and electron microscopic criteria.     

Placenta creta and placenta praevia creta

The placental bed in placenta creta and placenta praevia creta was studied from pregnancy or immediate postpartum hysterectomy specimens. In all cases placental villi were seen in direct contact with myometrium, the sine qua non of placenta creta, but was focal in some cases. There was no apparent diminution of decidua parietalis or, in cases of focal accreta, of adjacent basalis. In all cases the extravillous trophoblast was mainly uninuclear or binuclear, in contrast to the placental bed syncytial giant cells seen in late normal placentation. There was an apparent proliferation of interstitial trophoblast at the junction of placenta with myometrium, but the density of interstitial trophoblast deeper in the myometrium was lower than it is in normal placentation. An unusual uteroplacental vasculature was seen in which physiological changes were present in large arteries of the radial/arcuate system deep in the myometrium, while there were also spiral arteries more superficially without physiological changes. These findings suggest that in placenta creta there is defective interaction between maternal tissues, particularly decidua, and migratory trophoblast in the early stages of placentation resulting in undue adherence of the placenta or penetration into the uterus coupled with the development of an abnormal uteroplacental circulation.     

Human trophoblast contains an intracellular protein reactive with an antibody against CD133--a novel marker for trophoblast

CD133 is a protein expressed on the cell membrane of a subfraction of haematopoietic stem and progenitor cells, as well as on some epithelial cells. Previously available antibodies against CD133 recognized only the glycosylated protein, localized to membrane protrusions or microvilli. Due to this, immature intracellular stages of the CD133 protein could not be visualized using these antibodies. We describe reactivity of a commercially available antibody against CD133, called AC133-2, with an intracellular protein in trophoblast. Both villous and extravillous cytotrophoblast, as well as syncytiotrophoblast were stained by AC133-2 in cryostat sections of first trimester and term placenta. Villous stroma was not stained. AC133-2 reactivity was seen in methanol-fixed primary trophoblast cells and trophoblast-derived cell lines, and was coexpressed with cytokeratin-7. CD133 messenger RNA was present in trophoblast and trophoblast-derived cell lines, but also in cells not displaying any reactivity with CD133 antibodies. AC133-2 recognized a 55-60 kDa protein on Western blots of cell extracts including trophoblast. The exact nature of this protein is not yet understood. However, AC133-2 is applicable as a positive marker for the characterization of all subtypes of trophoblast and for trophoblast cell lines.     

Development of the haemophagous region and labyrinth of the placenta of the tenrec, Echinops telfairi

Our purpose was to determine how the central haemophagous region and cellular haemomonochorial labyrinth of the tenrec placenta are formed. The haemophagous region is preceded by a region of invasion of the endometrium by trophoblast comprising a cytotrophoblast layer covered by syncytial trophoblast and contiguous with numerous masses of multinucleate trophoblast. The trophoblast intrudes into the endometrium, eliminating the stroma, although small vessels and clumps of glandular epithelium persist. This extensive central region is connected to the forming disk by a ring of chorioallantois covered by a single layer of columnar trophoblast. Later the multinucleate masses and syncytial trophoblast degenerate. The unilaminar cytotrophoblast remains, is elaborated into folds, and phagocytoses glandular secretion, cell debris and erythrocytes. As the central area is transforming, fetal capillaries move into the cytotrophoblast pads surrounding the central zone. Prior to this, the cytotrophoblast has formed a multilayered structure and interrupted maternal vessels to create an anastomotic network of blood spaces lined by cytotrophoblast. The invasion of fetal capillaries transforms this preplacental pad into a cellular haemomonochorial labyrinth with the uninvaded portion forming an underlying spongy zone. Thus interaction of the trophoblast with the endometrium is substantially different in the central zone compared to the area of the preplacental pad.     

Trophoblast deportation part II: a review of the maternal consequences of trophoblast deportation

The deportation of trophoblast debris from the placenta was first documented over 100 years ago, and today we know that the deported material ranges from multinucleated syncytial knots/sprouts to trophoblast-derived nanoparticles. However little is known about the effect of trophoblast debris on maternal physiology since it is difficult to investigate these effects in vivo in women. Animal models have been reported but they have provided relatively little information. Most of our current knowledge regarding the effects of trophoblast debris on maternal systems is provided by studies using trophoblast debris obtained from in vitro models of the human placenta. Herein we review the animal models and the in vitro studies, which, between them, suggest that deported trophoblast material may play a role in tolerising the maternal immune system during normal pregnancy, and conversely that in pathological pregnancies aberrant maternal immune and/or endothelial/vascular responses may result from a change in either the quantity or quality of deported trophoblast debris.     

Expression of tumor associated antigens CA 15-3 and CA 19-9 in trophoblast of the normal human placenta

Mucin 1 (MUC1) is abundantly expressed by various organs, including human placenta and endometrium. Since glycan modifications of MUC1 are potentially relevant for physiological as well as pathological processes, this study was aimed at establishing an expression profile of two MUC1 glycoepitopes, CA 15-3 and CA 19-9, in trophoblast throughout pregnancy. Immunohistochemical analysis of normal placenta demonstrated that trophoblast cells express both mucin antigens throughout gestation with a distinct staining pattern. The staining of villous trophoblast was non-uniform for both antigens, and stronger for CA 15-3. Only a proportion of extravillous trophoblast of the cell column, in decidual stroma or lining blood vessels was also stained. Whether the studied MUC 1 glycoforms can be linked to trophoblast cells invasion remains to be established.     

Distensible transtrophoblastic channels in the rat placenta

Paracellular pathways in the haemotrichorial placenta of the rat were studied by electron microscopy using lanthanum hydroxide as an electron dense marker. Near term placentae were dually perfused in situ, adding lanthanum to the fetal perfusate. In some placentae outflow pressure on the fetal side was elevated (between 10 and 25 cm H(2)O) to promote filtration of fluid in a fetomaternal direction. Under normal pressure conditions lanthanum particles lined the subendothelial spaces and tubular structures in the inner, syncytial layer of trophoblast. Further penetration of lanthanum into the tubules was blocked by coarse lanthanum aggregates. Elevated fetal hydrostatic pressure resulted in a fluid shift across the placenta (filtration rate 50+/-16 per cent of fetal arterial inflow rate), distending the tubules in the inner trophoblast layer. Lanthanum particles gradually appeared in tubular structures in the middle (syncytial) layer and in the lateral intercellular spaces in the outer (cellular) layer. Finally lanthanum reached the maternal surface of the trophoblast. These pressure effects were only partially reversible. When the fetal pressure was returned to control values, some distension of the tubules persisted and the entire length of the paracellular pathways remained accessible to lanthanum. It is concluded that the placental barrier in the rat contains pressure dependent paracellular pathways connecting the maternal and fetal extracellular compartments.     

Phagocytic properties of cultured murine trophoblast

The phagocytic potential of cultured trophoblast from early (ectoplacental cone (EPC); day 7.5 post coitum) and mid-term (placenta; day 12 to 14 post coitum) pregnancy in the mouse has been examined using a variety of test particles and culture conditions. In suspension, small numbers (less than 1 per cent) of large placental trophoblast cells showed limited phagocytic uptake of Staphylococcus aureus but not of opsonized sheep red blood cells (RBCs). In contrast, trophoblast phagocytosis was never seen in monolayer placental cell culture. Placental macrophages consistently exhibited phagocytic uptake of both opsonized sheep RBCs and S. aureus under these conditions. In monolayer culture, EPC trophoblast phagocytosed S. aureus, but there was only limited uptake of RBCs (mouse or sheep) or spermatozoa. When cultured in a three-dimensional matrix (blood and plasma clots), however, EPC trophoblast demonstrated extensive phagocytosis of both RBCs and sperm. These results are discussed with reference to the use of in vitro systems for examining developmental processes, and a possible role for trophoblast phagocytosis in early gestation is proposed.     

The immunohistochemical localization of pregnancy-specific beta 1-glycoprotein in postimplantation rat trophoblast

Pregnancy-specific beta 1-glycoprotein (SP1) was identified by peroxidase-antiperoxidase (PAP) immunohistochemistry in the placenta of inbred strains of rat between 10 and 21 days of gestation. SP1 was located predominantly in basal zone trophoblast and in intravascular trophoblast of decidual vessels, but it was absent from labyrinthine trophoblast. No perivascular cells were identified by SP1 staining, but occasional clusters of interstitial trophoblast stained for SP1. The results suggest that basal and labyrinthine trophoblast are functionally different.