Trophoblast Giant Cells Research Articles

Early Expression of Pregnancy-Specific Glycoprotein 22 (PSG22) by Trophoblast Cells Modulates Angiogenesis in Mice1

Mouse and human pregnancy-specific glycoproteins (PSG) are known to exert immunomodulatory functions during pregnancy by inducing maternal leukocytes to secrete anti-inflammatory cytokines that promote a tolerogenic decidual microenvironment. Many such anti-inflammatory mediators also function as proangiogenic factors, which, along with the reported association of murine PSG with the uterine vasculature, suggest that PSG may contribute to the vascular adaptations necessary for successful implantation and placental development. We observed that PSG22 is strongly expressed around the embryonic crypt on Gestation Day 5.5, indicating that trophoblast giant cells are the main source of PSG22 during the early stages of pregnancy. PSG22 treatment up-regulated the secretion of transforming growth factor beta 1 and vascular endothelial growth factor A (VEGFA) in murine macrophages, uterine dendritic cells, and natural killer cells. A possible role of PSGs in uteroplacental angiogenesis is further supported by the finding that incubation of endothelial cells with PSG22 resulted in the formation of tubes in the presence and absence of VEGFA. We determined that PSG22, like human PSG1 and murine PSG17 and PSG23, binds to the heparan sulfate chains in syndecans. Therefore, our findings indicate that despite the independent evolution and expansion of human and rodent PSG, members in both families have conserved functions that include their ability to induce anti-inflammatory cytokines and proangiogenic factors as well as to induce the formation of capillary structures by endothelial cells. In summary, our results indicate that PSG22, the most abundant PSG expressed during mouse early pregnancy, is likely a major contributor to the establishment of a successful pregnancy.

Mouse Model of Abortion Induced by Brucella abortus Infection

The mechanisms of abortion induced by bacterial infection are largely unknown. We found that Brucella abortus, a causative agent of brucellosis and a facultative intracellular pathogen, caused abortion in pregnant mice. High rates of abortion are observed for bacterial infection on day 4.5 of gestation, but not for other days. Regardless of whether fetuses are aborted or not, the transmission of bacteria to the fetus and bacterial replication in the placenta are observed. There is a higher degree of bacterial colonization in the placenta than in other organs and many bacteria are detected in trophoblast giant cells in the placenta. The intracellular growth-defective virB4 mutant and attenuated vaccine strain S19 do not induce abortion. In the case of abortion, the induction of IFN-γ and RANTES production is observed at day 7.5 of gestation – the placental development period – for infection by the wild type strain but not by the virB4 mutant or S19. B. abortus-infected pregnant IFN-γ knockoutmice die within 15 days of infection, but nonpregnant IFN-γ knockout mice remain alive. The neutralization of IFN-γ or RANTES, in which production is induced by infection with B. abortus serves to prevent abortion. These results indicate that abortion induced by B. abortus infection is regulated by IFN-γ during the period of placental development, and the production and function of RANTES are correlated with IFN-γ.

Anti‐mouse renin expression in the rat kidney

The causes of toxemia of pregnancy are not fully understood. This project examines rat kidneys to determine if a mouse anti‐renin antibody will cross‐react rat renin‐producing cells in the rat. This will allow further study female reproductive tissues in the rat to examine for renin production. Renin is known as hormone produced by the juxtaglomerular cells of the afferent arteriole of the kidney in response low blood pressure. Also, renin is an immediate downstream target of Class I Hox gene regulation, important in embryonic development. In mice, renin is expressed in embryonic renal, adrenal and gonadal arteries, the adrenal glands, and tissue associated with embryonic pronephric and mesonephric kidneys. Using a renin/GFP reporter in transgenic mice, renin is expressed in placental giant trophoblast cells and uterine/ovarian artery cells of the tunica adventitia. In RCS rats, renin antibody stain that used anti‐rat renin and peroxidase labeled placental giant trophoblast cells and cells of the tunica adventitia of the uterine/ovarian artery. This project examines rat kidney tissue labeled by indirect immuno‐fluorescence with anti‐mouse renin antibody and fluorescent conjugate. Cells in the juxtaglomerular region of the kidney were renin positive. This demonstrates that anti‐mouse renin antibody can dependably label regions in the rat kidney that are known for renin production. This will allow for further studies to determine renin location in rat female reproductive tissues. Dr. Craig Jones of Roswell Park Cancer Institute generously donated mouse renin antibody and lab space for stain procedures. D'Youville College Faculty Council and Department of Math & Natural Sciences supported work.

Expression pattern of tumor necrosis factor alpha in placentae of idiopathic fetal growth restriction

Tumor Necrosis Factor-Alpha (TNF-α) is one of the proinflammatory cytokines that provokes a variety of biological effects on the placenta. The increased placental exposure to TNF-α have induced impaired fetal development in experimental animals, but no data are available on the expression and localization of TNF-α in human placenta of idiopathic fetal growth restriction (FGR). The aim of this study was to characterize the immunohistochemical expression and localization of TNF-α in idiopathic FGR placentae in comparison with those of appropriate for gestational age (AGA) fetuses. 75 human placentae were collected between April, 2010 and March, 2011; 50 placentae were collected from pregnancies associated with idiopathic FGR and 25 placentae from AGA pregnancies. Histological and Immunohistochemical methodologies were employed in formalin fixed paraffin-embedded sections from the placentae of all subjects. Area percent of TNF-α immunostaining was evaluated using image analysis technique. In both AGA and idiopathic FGR placentae, cytoplasmic TNF-α was localized in the decidual and chorionic trophoblasts and in the endothelium of decidual and chorionic vessels. Trophoblast giant cells (TGC) in the decidua and chorionic villi of AGA specimens show deficient or negative TNF-α immunoexpression while those of idiopathic FGR show positive immunostaining. The mean area percent of TNF-α staining was greater in idiopathic FGR placentae (5.93 ± 0.69) compared to AGA ones (3.28 ± 0.41) (p = 0.001). Enhanced placental expression and specific cellular localization and of TNF-α are expected to contribute to impaired fetal development in idiopathic FGR and the TGCs are proposed to be an obvious source of this cytokine in such cases.

Delivery of AP-2α siRNA into cultured bovine trophoblast cells by electroporation repressed key placenta-specific gene expression

Binucleate trophoblast giant cells (BNC) characteristically appear early in gestation in the bovine placenta. They secret pivotal hormones and cytokines for feto-maternal communication, for example, expression of placental lactogens (CSH1), prolactin-related protein 1 (PRP1) and pregnancy-associated glycoprotein 1 (PAG1) are necessary for pregnancy establishment in bovine. These genes transcription are regulated in a temporal and spatial manner, however, molecular mechanisms by which these gene transcriptions are regulated in this manner have not been firmly elucidated. In this study, a cell culture model for bovine trophoblast cells was initially established, small interfering RNA duplexes against Activator Protein-2α (TFAP2A) was transfected into the cells by electroporation, and transcripts of CSH1, PRP1 and PAG1 were measured by qPCR. The results showed that trophoblast giant cells were confluent for 90% after cultured for 10days, and BNC constituted of a population of more than 45% of the total cells. Using a fluorescein-labeled non-silencing siRNA duplex, an electroporation protocol yielding routinely >93% positive cells could be established, and siRNA duplex transfection demonstrated an efficient knockdown of cellular AP-2α mRNA level by 72.30±3.28% in electroporated cells. Finally, CSH1, PRP1 and PAG1 genes expression were effectively down-regulated by 65.45±6.38% (P<0.01), 40.73±11.72% (P<0.01) and 11.59±1.88% (P<0.05), respectively. It was therefore suggested that electroporating siRNA into bovine trophoblast cells could be an efficient method to manipulate BNC function and to study the regulation mechanism of specific gene transcription without the use of chemical transfection reagents. It was suggested that AP-2α could be at least involved in the regulation of expression CSH1 and PRP1 transcripts.

Spatial and temporal expression of folate-related transporters and metabolic enzymes during mouse placental development

It is well understood that maternal folate deficiency can cause abnormal fetal development. However, the extent to which placental development and function are also dependent upon folate uptake and metabolism remains unclear. To understand which trophoblast cell types may be affected by folate deficiency or abnormal folate metabolism, we completed a comprehensive spatial and temporal protein expression analysis of folate receptor (Folr), folate transporters (proton-coupled folate receptor [Slc46a1 or PCFT] and reduced folate carrier-1 [Rfc1]) and folate metabolic enzymes (5,10-methylenetetrahydrofolate reductase [Mthfr] and methionine synthase [Mtr]) in histological sections of mouse placentas from early development (E8.5) until term (E18.5). We observed that the highest level of protein expression was during early development (E8.5-E10.5), prior to the formation of the three main layers of the mature placenta suggesting that folate uptake and metabolism may be required for placental development, itself. As expected, the labyrinth trophoblast cells, which are responsible for nutrient transport, expressed these proteins throughout pregnancy, including robust expression in the sinusoidal trophoblast giant cells that line the maternal blood spaces. Other trophoblast giant cell (TGC) subtypes (parietal-TGCs and canal-TGCs), whose function does not include nutrient transport, expressed folate transporters and enzymes from E8.5 onwards. Remarkably, these proteins were also detected in glycogen trophoblast cells from E12.5–E18.5 suggesting a new role in folate uptake and metabolism for these cells. Together, these data provide evidence that folate may be necessary for normal placental development and function, and perturbations in its availability or metabolism may lead to secondary effects on fetal development.

Angiopoietin-like gene expression in the mouse uterus during implantation and in response to steroids

The purpose of this work was to determine if and where Angiopoietin-like genes are expressed in the mouse uterus during the implantation period of pregnancy and to determine if uterine expression of such genes is controlled by estrogen or progesterone. We found that all six known murine angiopoietin-like genes were expressed in the mouse uterus during implantation. The expression of four genes was controlled by either estrogen or progesterone. Only the levels of angiopoietin-like 4 (Angptl4) mRNA dramatically increased in implantation segments of the uterus during decidualization and was conceptus-independent. Due to this increased expression and the fact that angiopoietin-like 4 protein plays a role in lipid metabolism and angiogenesis in other tissues, only the expression of Angptl4 was further examined in the uterus and developing placenta. Angptl4 mRNA was localized to subpopulations of the endometrial stromal fibroblast and endothelial cell populations during decidualization. It was also localized to the ectoplacental cone, trophoblast giant cells and parietal endoderm of the conceptus at this time. By mid-pregnancy, Angptl4 mRNA was localized mainly to the mesometrial lymphoid aggregate region plus mesometrial endothelial cells of the uterus, as well as in various cell types of the conceptus. Additional work showed that Angptl4 expression increases in mouse endometrial stromal cells as they undergo decidualization in vitro. As in other cell types, the expression of Angptl4 in endometrial stromal cells was increased in response to an agonist of the peroxisome proliferator activated receptors. Taken together, the results of this work support the hypothesis that locally expressed Angptl4 might play a role in local uterine/placental lipid metabolism and vascular changes during implantation and thus provide a basis for future research.

Galectin fingerprinting detects differences in expression profiles between bovine endometrium and placentomes as well as early and late gestational stages

The concept of a network within the family of adhesion/growth regulatory galectins implies distinct spatio-temporal expression profiles. To test this assumption immunohistochemically for bovine placenta, placentomal (P) and interplacentomal tissues (IP) were collected at a slaughterhouse from the three stages of pregnancy (early gestation = day 30–130; mid gestation = day 130–220; late gestation = day 220–275). The specimens were snap-frozen or fixed in Bouin’s solution, then embedded in paraffin. Gene expression for galectins-1, -3, -4 and -9 in P and IP of late gestational stages was monitored by RT-PCR. Galectin-type-specific antibodies were used for immunohistochemical localization. In IP, galectin-1 was present in stroma cells and early gestational trophoblast giant cells (TGC), whereas galectin-3 was confined to uterine epithelial cells. In contrast, both galectins were found in epithelia of P tissue. Uterine epithelial cells and blood vessel walls were positive for galectin-4, while galectin-9 was detected predominantly in uterine epithelial cells and late gestational TGC. Our study thus reveals individual profiles among the galectins tested, an indication for specific functions exerted by each protein in the bovine endometrium and placenta.

6-Mercaptopurine-induced histopathological changes and xanthine oxidase expression in rat placenta

The placenta secures the embryo and fetus to the endometrium and releases a variety of steroid and peptide hormones that convert the physiology of a female to that of a pregnant female. Chemical-induced alteration or deviation of placental function in the maternal and extraembryonic tissue can ultimately lead to pregnancy loss, congenital malformation and fetal death. The 6-mercaptopurine (6-MP), an anti-leukemic drug, is known to produce undesired effects on some organs, then the placenta/embryo toxicity of 6-MP was investigated in pregnant rats given 60 mg/kg with two intraperitoneal injections on gestation days (GD) 11 and 12. The rats were sacrificed and their placentas were collected on GD13 or 15. On GD15 small and limb-defected embryos were found in the 6-MP-treated rats. Placental weights were significantly reduced on GD15, as well as a reduced number of cells was detected in the labyrinth zone with both the labyrinth and basal zones having thinned. Cleaved caspase-3-positive cells increased in number in the labyrinth zone, while in the basal zone, glycogen cells reduced with cytolysis. The number of spongiotrophoblasts and trophoblastic giant cells also increased by 6-MP treatment. The 6-MP-treatment resulted in the increased xanthine oxidase (Xdh) expression in the placenta, which gene is related to the ischemic condition of tissues. These data suggest that apoptosis of the labyrinth zone cells may lead to decreased materno-fetal exchange. Moreover, subsequent ischemia in the placental tissue may occur and induce Xdh expression.

The effects of Carthamus tinctorius L. on placental histomorphology and survival of the neonates in mice.

Carthamus tinctorius L. (Safflower) is a member of the asteraceae family which is used as a food additive but also has medicinal applications. This study investigated its effects on placental histomorphology and survival of mice neonates. Eighty Balb/C pregnant mice were randomly distributed into one control and three experimental groups (n=20). The control group received only distilled water, whereas experimental groups were administered intraperitoneally C. tinctorius extract at doses of 0.7, 1.4, and 2.8 mg/kg during the organogenesis period (6(th) to 16(th) day of gestation). On the 17(th) day of gestation, half of the animals were euthanized; their fetuses and placentas were removed and histomorphological study was performed. In the rest of the animals after parturition, the number of neonates was counted. Survival rates were periodically calculated for neonates within days 5, 15, 25, and 42 after birth. The results were evaluated by one-way ANOVA. The results showed that treatment with 1.4 and 2.8 mg/kg C. tinctorius extract caused reduction in the trophoblastic giant cells ratio and increasing in the proportion of labyrinthine interhemal membrane (LIM). Moreover, the size of the labyrinthine zone per whole placenta, weight, diameter, and thickness of the placenta in the mice administered with 1.4 and 2.8 mg/kg C. tinctorius extract became lower than those of controls (p<0.05). In addition, in the treated mice with 1.4 and 2.8 mg/kg C. tinctorius extract, the number of neonate was drastically decreased on days 5, 15, 25, and 42 after birth. It is concluded that treatment with C. tinctorius extract in doses of 1.4 and 2.8 mg/kg induces toxic changes in the placental structure so caution should be paid to popular consumption of this plant both as an alternative medicine and as a food additive.

Utility of Diagnosing Various Histological Patterns of Diffuse Chronic Hypoxic Placental Injury

To examine the clinicopathologic correlations of three histological patterns of diffuse chronic hypoxic placental injury (preuterine [PR], uterine [UH], and postuterine [PU]), a retrospective statistical analysis of a large 14-year placental database was performed. Of 5097 placentas between 20 and 43 weeks of gestation examined consecutively, 4413 did not feature histological chronic placental hypoxia, while 684 did. In the latter, maternal hypertensive disorders, diabetes mellitus, abnormal cardiotocography and Dopplers, cesarean sections, inductions of labor, and fetal growth restriction, as well as other placental hypoxic lesions and decidual arteriolopathy, were statistically significantly more common than in the remaining placental material. Two hundred eighty-nine PR cases featured the most advanced gestational age and meconium staining; 237 UH cases featured severe preeclampsia, decidual arteriolopathy, villous infarction, membrane laminar necrosis, microscopic chorionic pseudocysts, excessive extravillous trophoblasts, and maternal floor multinucleate trophoblastic giant cells; and 158 PU cases featured the lowest placental weight and the highest prevalence of abnormal Dopplers, umbilical cord compromise, fetal growth restriction, cesarean section rate, and complicated 3rd stage of labor. The specificity of chronic hypoxic patterns of placental injury was much higher than the sensitivity, with the highest specificity for an excessive amount of extravillous trophoblasts. Diagnosing various hypoxic patterns of placental injury by histology may help to clarify the etiopathogenesis of a significant proportion of complications of pregnancy and abnormal fetal or neonatal outcomes. The patterns should help to retrospectively diagnose placental hypoxia, even in clinically unsuspected cases.

Variation in Macrophage Migration Inhibitory Factor [MIF] immunoreactivity during bovine gestation

Macrophage Migration Inhibitory Factor (MIF) is a proinflammatory cytokine involved in several aspects of the immune response. MIF appears to play important roles in materno-fetal immuno-tolerance during placental establishment, modulation and growth as studied in epitheliochorial porcine and hemochorial human and mouse placentae. Here we studied the bovine placenta being multiplex, villous and synepitheliochorial with a low degree of invasion, to see if MIF could be involved. Placental tissues sampled from 12 cows at 9 stages of gestation (days 18–250), and endometrial tissues from two non-pregnant animals were processed for immunohistochemistry. Bovine MIF was detected by Western blot using anti-human MIF monoclonal antibodies. An immunoreactive band of approximately 12kDa confirmed similarities between bovine and human MIFs. Compared to the non-pregnant stage with very faint staining, the caruncular epithelium during pregnancy showed stronger staining for MIF. The intercaruncular epithelium in non-pregnant endometrium showed some reaction apically with increasing intensity at uterine gland openings; in contrast, at day 18 of gestation this staining was markedly increased. During gestation both caruncular and trophoblast epithelium of the placentomes were positive with different intensity in relation to the gestational stage. In the uterine glands, some strongly stained cells were present. The mature binucleated trophoblast giant cells were negative throughout pregnancy. During reestablishment of vascularisation, the vasculature in the caruncular area showed MIF reactivity. While supporting involvement of MIF in different placental types, the spatio-temporal variation in the bovine placenta suggests a regulatory role for MIF mainly in the interhemal barrier and during vascular development.

Checkpoint Kinase 1 Prevents Cell Cycle Exit Linked to Terminal Cell Differentiation

Trophoblast stem (TS) cells proliferate in the presence of fibroblast growth factor 4, but in its absence, they differentiate into polyploid trophoblast giant (TG) cells that remain viable but nonproliferative. Differentiation is coincident with expression of the cyclin-dependent kinase (CDK)-specific inhibitors p21 and p57, of which p57 is essential for switching from mitotic cell cycles to endocycles. Here, we show that, in the absence of induced DNA damage, checkpoint kinase-1 (CHK1), an enzyme essential for preventing mitosis in response to DNA damage, functions as a mitogen-dependent protein kinase that prevents premature differentiation of TS cells into TG cells by suppressing expression of p21 and p57, but not p27, the CDK inhibitor that regulates mitotic cell cycles. CHK1 phosphorylates p21 and p57 proteins at specific sites, thereby targeting them for degradation by the 26S proteasome. TG cells lack CHK1, and restoring CHK1 activity in TG cells suppresses expression of p57 and restores mitosis. Thus, CHK1 is part of a "G2 restriction point" that prevents premature cell cycle exit in cells programmed for terminal differentiation, a role that CHK2 cannot play.

Chorionic Disk Extravillous Trophoblasts in Placental Diagnosis

To study clinical and placental associations of increased amount of chorionic disk extravillous trophoblast (IAEVT), the frequency of selected clinical and placental parameters of 189 consecutive cases with IAEVT, defined as more than 5 cell islands and/or placental septa per placental section, were compared with those for all remaining 1,006 placentas examined during the same period. IAEVT was statistically significantly associated with preeclampsia, decidual arteriolopathy, placental infarction, and several chronic placental hypoxic lesions (uterine hypoxic pattern of hypoxic placental injury, microscopic chorionic pseudocysts, massive perivillous fibrin deposition, and trophoblastic multinucleated giant cells in decidua) and absence of meconium staining and umbilical cord abnormalities. The amount of chorionic disk extravillous trophoblast is increased in association with clinical conditions and placental lesions associated with chronic hypoxia of uterine origin, ie, placental malperfusion. Counting placental septa and cell islands is a valuable surrogate test of chronic placental hypoxia.

The expression of Fas/FasL and apoptosis in yak placentomes

To clarify the status and distribution of Fas and Fas-Ligand (FasL) in yak's placentomes, immunohistochemistry (IHC) was carried out to analyze the expression and location of Fas and FasL in paraffin embedded sections. The area of positive stained sites was selected and measured using image analyses software (Image Pro-Plus 6.0). So the positive index (PI) was calculated to estimate the intensity of protein expression according to the percentage of positive area in corresponding compartment of the placentomes. In cotyledonary villi, Fas mainly presented on the villous trophoblast cells in early pregnancy. The positive index reached a maximum of 20.7 ± 8.8 at the third month of pregnancy. Then Fas was declined rapidly along with the progress of gestation and the value was 2.8 ± 1.3 after the 7th month of pregnancy. However, in caruncular crypts, Fas was mainly localized to isolated cells or clustered cells of the uterine stroma underlying the caruncular epithelium. The intensity was lower and the positive index was changed between 4.7 ± 0.9 and 8.5 ± 1.6 throughout gestation. For FasL, it gave a distinct immunostained distribution. In cotyledonary villi, FasL was localized dominantly and strongly in the cytoplasm of binuclear, mononuclear and trinuclear trophoblast giant cells (TGC). The positive index of FasL maintained a moderate level all through the gestation. In caruncular crypts, the expression of FasL was weak and the positive index was declined. Only in the first two months, maternal uterine epithelial cells intensely expressed FasL and the index reached to the maximum of 19.8 ± 5.2. The result of subcellular localization of Fas ligand using immunoelectron microscopy technology indicated that FasL was subcellular located in some intracellular vesicles of TGC. This means the vesicles of trophoblast giant cells itself can express FasL. By the TUNEL method, apoptosis was detected in yak placentomes. The amount of apoptotic cells was rare. The fetal chorionic trophoblast cells and caruncular crypt epithelium cells demonstrated higher percentage of apoptosis in middle pregnancy, which suggested that apoptosis plays an important role in placental cellular regeneration. In addition, the apoptosis of maternal caruncular stromal cells provides a local mechanism for maternal immunotolerance to the fetus and this mechanism was mediated by Fas–FasL pathway.

Protective Role of Heme Oxygenase-1 in Listeria monocytogenes-Induced Abortion

It is well-known fact that various pathogens, including bacteria, virus, and protozoa, induce abortion in humans and animals. However the mechanisms of infectious abortion are little known. In this study, we demonstrated that Listeria monocytogenes infection in trophoblast giant cells decreased heme oxygenase (HO)-1 and B-cell lymphoma-extra large (Bcl-XL) expression, and that their overexpression inhibited cell death induced by the infection. Furthermore, HO-1 and Bcl-XL expression levels were also decreased by L. monocytogenes in pregnant mice. Treatment with cobalt protoporphyrin, which is known to induce HO-1, inhibited infectious abortion. Taken together, our study indicates that L. monocytogenes infection decreases HO-1 and Bcl-XL expression and induces cell death in placenta, leading to infectious abortion.

Lineage-specific function of the noncoding Tsix RNA for Xist repression and Xi reactivation in mice.

The noncoding Tsix RNA is an antisense repressor of Xist and regulates X inactivation in mice. Tsix is essential for preventing the inactivation of the maternally inherited X chromosome in extraembryonic lineages where imprinted X-chromosome inactivation (XCI) occurs. Here we establish an inducible Tsix expression system for investigating Tsix function in development. We show that Tsix has a clear functional window in extraembryonic development. Within this window, Tsix can repress Xist, which is accompanied by DNA methylation of the Xist promoter. As a consequence of Xist repression, reactivation of the inactive X chromosome (Xi) is widely observed. In the parietal endoderm, Tsix represses Xist and causes reactivation of an Xi-linked GFP transgene throughout development, whereas Tsix progressively loses its Xist-repressing function from embryonic day 9.5 (E9.5) onward in trophoblast giant cells and spongiotrophoblast, suggesting that Tsix function depends on a lineage-specific environment. Our data also demonstrate that the maintenance of imprinted XCI requires Xist expression in specific extraembryonic tissues throughout development. This finding shows that reversible XCI is not exclusive to pluripotent cells, and that in some lineages cell differentiation is not accompanied by a stabilization of the Xi.

Ablation of Tpbpa-positive trophoblast precursors leads to defects in maternal spiral artery remodeling in the mouse placenta

The placenta is composed of multiple trophoblast cell types that have diverse endocrine, vascular and nutrient transport functions. We have developed a transgenic system to investigate the developmental and functional roles of specific cell types using conditional expression of a cytotoxin to induce cell ablation in transgenic mice. The Tpbpa gene is expressed in ectoplacental cone cells starting between embryonic days (E) 7.5 and 8.5, and later in the spongiotrophoblast layer of the mature placenta. Tpbpa-positive cells are progenitors of many trophoblast subtypes including three subtypes of trophoblast giant cells (TGCs) and glycogen trophoblast cells. We used a Cre recombinase transgene driven by the Tpbpa promoter to irreversibly activate a diphtheria toxin A (DTA) transgene. Cre/DTA double transgenic placentas showed dramatic reduction of Tpbpa-positive spongiotrophoblast cells by E10.5 and conceptuses died by ~E11.5. The number of cells associated with maternal blood spaces, spiral artery TGCs (SpA-TGCs) and canal TGCs, and glycogen trophoblast cells were reduced. The loss of these specific trophoblast subtypes, especially SpA-TGCs, was correlated with a decrease in maternal spiral artery diameters, indicating a critical role of these cells in modulating the maternal vasculature. In contrast, parietal TGCs were not significantly reduced by progenitor cell ablation, suggesting that there is compensatory growth of this population and indeed a population of Ascl2 (Mash2)-positive/Tpbpa-negative cells was increased in the spongiotrophoblast layer in the Cre/DTA double transgenics. Our work demonstrates that the Tpbpa-positive lineage is essential for placental function and particularly critical for maternal vasculature remodeling.

Sensitivity and specificity of finding of multinucleate trophoblastic giant cells in decidua in placentas from high-risk pregnancies

This is a retrospective analysis of sensitivity and specificity of clustered placental basal plate multinucleate trophoblastic giant cells for various clinical conditions and placental lesions associated with fetal and placental hypoxia. Selected clinical and placental parameters of 375 consecutive cases of placentas with clusters of multinucleate trophoblastic giant cell (at least 3 cells with at least 3 nuclei) in the decidua (study group) were compared with all remaining 2674 placentas concurrently studied (control group) in 20-week-or-more high-risk pregnancies. Multinucleate trophoblastic giant cell was found in 12.3% of placentas. The study group had statistically significantly more cases of preeclampsia, abnormal Dopplers, induction of labor, and cesarean sections, with its placentas lighter and with more common other hypoxic lesions than in the control-group placentas. The multinucleate trophoblastic giant cell prevalence negatively correlated with gestational age (R = -0.56), peaking at the turn of the second and the third trimesters of pregnancy and declining afterward, and most strongly correlated with the excessive amount of extravillous trophoblasts in the chorionic disc (R = +0.33). The sensitivity of multinucleate trophoblastic giant cells was, on average, 3 times lower than the specificity, the latter averaging greater than 90%. In conclusion, finding of multinucleate trophoblastic giant cells is not exclusively limited to uteroplacental malperfusion of preeclampsia but is also seen in other types of high-risk pregnancy and in association with other placental hypoxic lesions and patterns. Multinucleate trophoblastic giant cells most likely reflect a premature fusion of extravillous trophoblasts because of several factors, likely including also hypoxia. Being highly specific, finding the multinucleate trophoblastic giant cells is unlikely to give a false-positive result and therefore has high value in retrospectively explaining the perinatal morbidity and mortality.

Endopolyploid and proliferating trophoblast cells express different patterns of intracellular cytokeratin and glycogen localization in the rat placenta

The presence of keratin intermediate filaments is a characteristic of trophoblast differentiation. Meantime, their intracellular localization in the functionally different subtypes of placental trophoblast is poorly investigated in rodent, whereas their placentae are being broadly investigated in recent years as a model of the feto-maternal interaction. The purpose was to study the intracellular distribution of cytokeratin filaments in correlation with glycogen deposits, both being important constituents of the trophoblast cells in rat placenta. Different rat trophoblast cell populations exhibited different patterns of cytokeratin immunolocalization. The most intensive immunostaining was observed in the highly endopolyploid SGTCs (secondary giant trophoblast cells) at the border with decidua basalis. The most prominent cytokeratin-positive threads were found at the periphery of cytoplasm and in the extensive system of cytoplasmic sprouts by which the SGTC connect each other. Similar cytokeratin intensity and distribution was detected in the TSC (trabecular spongiotrophoblast cells) of the junctional zone of placenta that line the lacunae with the maternal blood. Clusters of highly proliferative pre-glycogen as well as glycogen cells showed some weaker cytokeratin signals mostly in the perinuclear and peripheral zones of cytoplasm. At the 11.5th to the 13.5th day of gestation, the interstitial and endovascular invasive endopolyploid TGTCs (tertiary giant trophoblast cells) prove the intensive cytokeratin staining throughout the cytoplasm and its sprouts. Meantime, the TGTCs were glycogen negative. By contrast, glycogen was heavily accumulated in the glycogen cells that belong both to the junctional zone of placenta and the cuff of the central arterial channel underlying the monolayer of endovascularly invading TGTCs. Thus, the TGTCs that are first to penetrate into the depth of the uterine wall do not contain glycogen but are accompanied by the glycogen-rich cells. The SGTC also contained the prominent deposits of glycogen at the periphery of cytoplasm and in the cytoplasmic sprouts. At the 16th day of gestation, an extensive interstitial invasion of the cytokeratin-positive glycogen trophoblast cells from the junctional zone was observed. The patterns of cytokeratin and glycogen intracellular localization are specific for each subtype of the rat trophoblast; that is, most probably, accounted for by the functional diversity of different trophoblast populations, i.e. patterns of invasion/phagocytosis and their involvement in a barrier at the feto-maternal interface.