Placenta

Title Page/Editorial Board

2010-03-01

Reagent specificity and scientific integrity: A necessary combination

D. Michael Nelson, Graham J. Burton — 2010-03-01

Integrity underpins the pursuit of science. As researchers, we depend on the reliability of many resources, reagents and techniques included, to perform experiments and test hypotheses. Appropriate controls are a key requirement for the interpretation of the resultant data, enabling us to make valid conclusions. The process of scientific discovery is done in good faith, and with rare exceptions the conclusions deduced are reliable.

Enhanced proapoptotic gene expression of XAF1, CASP8 and TNFSF10 in the bovine endometrium during early pregnancy is not correlated with augmented apoptosis

2010-01-21

Abstract: Bovine trophoblast cells release interferon-τ (IFNT), a type I IFN, as the pregnancy recognition signal. Since type I IFNs exert growth inhibitory and proapoptotic actions, the effect of the conceptus on components of the apoptosis pathways was determined in the bovine endometrium during the periimplantation period. Uteri of Simmental heifers were flushed post mortem at days 12, 15, and 18 of cycle or pregnancy for the recovery of conceptuses and the sampling of ipsilateral endometrial tissue at slaughter for quantitative RT-PCR, immunohistochemistry, caspase activity and TUNEL assays. Endometrium samples of pregnant animals revealed increased transcript levels for the proapoptotic genes XAF1 (day 15: 2.9-fold; day 18: 15.1-fold; p=0.005) and CASP8 (day 18: 2.4-fold; p=0.007). The mRNA expression increased significantly with the day of the cycle for the proapoptotic genes FASLG, TNFSF10, TNF and TNFSF1A (p=0.004, p=0.006, p=0.001 and p=0.007) and the antiapoptotic gene BIRC4 (p=0.03). We detected high amounts of FASLG transcripts in day 18 conceptuses (16-fold higher than day 18 endometria). This finding was validated at the protein level by immunohistochemistry. To further analyse the endometrial activation of the caspase cascade, the activities of initiator caspase 8 and effector caspases 3/7 were determined luminometrically. No difference between pregnant and cyclic animals was found for either caspase activity. Additionally, a TUNEL assay showed no increase of apoptotic cells in the pregnant endometrium. In conclusion, although the bovine conceptus induces the expression of proapoptotic genes, neither an activation of a caspase cascade nor an increase of apoptotic cells was noticed. These results suggest inhibitory mechanisms preventing endometrial cells from programmed cell death.

Differences in gene expression dependent on sampling site in placental tissue of fetuses with intrauterine growth restriction

2010-01-04

Abstract: Objective: The human placenta as part of the feto-placental unit may influence fetal endocrine systems and may therefore represent a very important link between intrauterine growth restriction (IUGR) and metabolic disorders in later life. We aimed to analyze the effect of sample origin on gene expression of placental factors potentially involved in fetal programming in IUGR versus appropriate for gestational age growth (AGA) to standardize sample collection procedure for a multicenter approach.Design: Placental gene expression of insulin-like growth factor-binding protein (IGFBP)-1, prolactin, corticotropin releasing hormone (CRH) and leptin was measured and compared between proximal, intermediate and peripheral region of the placenta in 22 IUGR (proven by anomalous placental Doppler velocimetry) and 19 AGA neonates.Results: Whereas no difference in gene expression was seen in the proximal portion, in the intermediate placental region mRNA expression of IGFBP-1 (p = 0.01), prolactin (p = 0.04), CRH (p = 0.01) and leptin (p = 0.04) was increased in IUGR samples compared to controls. At the placental periphery, gene expression of these placental transcripts showed a higher expression level in IUGR placentas without statistical significance, except for leptin (p = 0.03).Conclusion: Placental sampling site seems to be relevant for detecting differences in gene expression between IUGR and AGA neonates.

Effect of Maternal Tobacco Smoke Exposure on the Placental Transcriptome

2010-01-21

Abstract: Smoking in pregnancy increases a woman's risk of preterm delivery resulting in serious neonatal health problems and chronic lifelong disabilities for the children (e.g., mental retardation, learning problems). To study the effects of tobacco smoke on the placental transcriptome, we performed gene expression profiling on placentas from women exposed to tobacco smoke in pregnancy (N = 12) and from those without significant exposure (N = 64).Gene expression profiles were determined by Illumina HumanRef-8 v2 Expression BeadChips with 18,216 gene probes. Microarray data were normalized by quantile method and filtered for a detection P-value <0.01. Differential gene expression was determined by moderated t-statistic. A linear model was fitted for each gene given a series of arrays using lmFit function. Multiple testing correction was performed using the Benjamini and Hochberg method.Abundant levels of transcripts were found for genes encoding placental hormones (CSH1, CSHL1), pregnancy-specific proteins (PSG3, PSG4, PAPPA), and hemoglobins (HBB, HBG, HBA). Comparative analysis of smokers vs nonsmokers revealed the differential expression of 241 genes (P < 0.05). In smoker cohort, we detected high up-regulation of xenobiotic genes (CYP1A1, CYP1B1, CYB5A, COX412), collagen genes (e.g., COL6A3, COL1A1, COL1A2), coagulation genes (F5, F13A1) as well as thrombosis-related genes (CD36, ADAMTS9, GAS6).In smokers, we identified deregulated genes that show tissue non-specific induction and may be considered as general biomarkers of tobacco smoke exposure. Further, we also found genes specifically deregulated in the exposed placentas. Functional annotation analysis suggested processes and pathways affected by tobacco smoke exposure that may represent molecular mechanisms of smoke-induced placental abnormalities.

Validation of Placental Vascular Sonobiopsy for Obtaining Representative Placental Vascular Indices by Three-Dimensional Power Doppler Ultrasonography

M.G. Tuuli, M. Houser, L. Odibo, K. Huster, G.A. Macones, A.O. Odibo — 2010-01-11

Abstract: Objective: Placental vascular sonobiopsy has been proposed for obtaining a representative sample of the placental vascular tree when evaluation of the whole placenta is not feasible. We tested the hypothesis that placental vascular indices from sonobiopsy correlate well with those from the entire placenta.Methods: Three-dimensional power Doppler ultrasound examinations were performed in 120 singleton pregnancies at 11–14 weeks' gestation. The VOCAL™ program was used to calculate placental vascularization index (VI), flow index (FI) and vascularization flow index (VFI) from stored images of each placenta by whole placenta evaluation and placenta vascular sonobiopsy. The mean of each index from four spherical sonobiopsies were compared to those from evaluation of the entire placenta for their degree of correlation and agreement.Results: The mean VI and VFI from the two techniques were similar (13.9 [95% CI 12.3–15.8] versus 14.3 [95% CI 12.1–17.0], p = 0.62 and 6.1 [95% CI 5.2–7.1] versus 6.1 [95% CI 5.0–7.4], p = 0.93, respectively) and significantly correlated (Pearson's r = 0.70 [95% CI 0.60–0.78, p < 0.001] and r = 0.69 [95% CI 0.58–0.77, p < 0.001], respectively). The mean FI from the two techniques were significantly different (44.5 [95% CI 42.9–46.1] versus 41.3 [95% CI 39.6–43.0], p = 0.001), but correlated (r = 0.59 [95% CI 0.46–0.70, p < 0.001]).Conclusion: Our findings suggest that placenta vascular indices from sonobiopsy have a good correlation with those from evaluation of the entire placenta. Sonobiopsy may be a valid alternative for evaluation of the placental vascular tree when visualization of the entire placenta is not feasible. Measurements of VI and VFI appear to be more reliable than FI in sonobiopsy specimen.

Decreased Placental Methylation at the H19/IGF2 Imprinting Control Region is Associated with Normotensive Intrauterine Growth Restriction but not Preeclampsia

D.K. Bourque, L. Avila, M. Peñaherrera, P. von Dadelszen, W.P. Robinson — 2010-01-11

Abstract: Many genes exhibiting genomic imprinting, parent-of-origin differences in gene expression, are involved in regulating placental and fetal growth. The goal of the present study was to assess whether abnormal regulation of imprinted genes is associated with intrauterine growth restriction (IUGR) and/or preeclampsia (PET).Methods: Genomic DNA was extracted from at least two whole villi samples from control (N=22), IUGR (N=13), PET (N=17), and PET+IUGR (N=21) placentas. Methylation was assessed using the Illumina GoldenGate Methylation Cancer Panel I array and Pyrosequencing and MS-SNuPE assays.Results: The 11p15.5 ICR1 (associated with H19 and IGF2) methylation showed considerable intra-placental variability. Nonetheless, average methylation at this site was significantly decreased in normotensive IUGR placentas (p<0.001), but not in any other group. Methylation at ICR2 (KvDMR1; associated with CDKN1C and other maternally expressed 11p15.5 genes) was not significantly altered in any group and no significant changes in expression levels were observed in the genes controlled by this region. There were no significant methylation changes observed in any candidate imprinted gene evaluated by the Illumina array. LINE-1 methylation, a marker of whole genome methylation, was also similar in all groups.Conclusions: Reduced methylation of ICR1 is associated with normotensive IUGR but not IUGR associated with preeclampsia, suggesting a different etiology of IUGR in this group. A reduction in placental IGF2 could be an adaptive response to restrict fetal growth in the presence of abnormal placentation or a response to poor fetal growth itself.

Mesenchymal stem cells in human placental chorionic villi reside in a vascular Niche

2010-01-08

Abstract: The chorionic villi of human term placentae are a rich source of mesenchymal stem cells (PMSCs). The stem cell “niche” within the chorionic villi regulates how PMSCs participate in placental tissue generation, maintenance and repair, but the anatomic location of the niche has not been defined. A number of cell surface markers for phenotypic characterisation of mesenchymal stem cells (MSCs) were employed to identify the stem cell niche within the chorionic villi of first trimester and term human placenta. This included antibodies to pericyte cell surface markers STRO-1 and 3G5, which have been used to identify mesenchymal stem cells in other tissues, but have not been studied in placental tissues. PMSCs were isolated from term human placentae and shown to have stem cell properties by their ability to grow on untreated plastic culture ware, capacity for forming clones (i.e. clonogenicity) and their capability to differentiate into adipocytes, chondrocytes and osteocytes. Western analysis confirmed that STRO-1 and 3G5 are present in placental protein extracts and in PMSCs. Immunocytochemistry revealed PMSCs were positive for MSC cell surface markers (STRO-1, 3G5, CD105, CD106, CD146, CD49a, α-SMA) and negative for haematopoietic stem cell markers (CD117, CD34) and endothelial markers (CD34, vWF). Immunohistochemistry with antibodies to MSC cell surface markers on first trimester and term tissues revealed a vascular niche for PMSCs. Dual-label immunofluorescence analysis was used to compare STRO-1 antibody staining with that of endothelial cell marker vWF and found no significant overlap in staining. This indicated that some PMSCs have a pericyte-like phenotype. We propose that the vascular niche harbours a pool of PMSCs that can give rise to committed progenitors for tissue maintenance and repair, and that PMSCs contribute to vessel maturation and stabilization.

Decidual NK cell-derived conditioned medium enhances capillary tube and network organization in an extravillous cytotrophoblast cell line

Y. Hu, G. Eastabrook, R. Tan, C.D. MacCalman, J.P. Dutz, P. von Dadelszen — 2010-01-18

Abstract: Extravillous cytotrophoblast (EVT) migration, invasion and endovascular differentiation are regulated by a variety of growth factors, cytokines and adhesion molecules. Decidual natural killer cells (dNK) and their secreted cytokines probably modulate these processes. In this study, we used dNK-derived conditioned medium (dNK-CM) to investigate whether or not (i) dNK-CM was able to enhance capillary tube and network formation of an EVT cell line, HTR8/SVneo, on Matrigel, (ii) PI3K/AKT pathway and p38 MAPK pathway activation were involved, and (iii) HTR8/SVneo surface ICAM-1 played a role in the process of HTR8/SVneo endovascular differentiation. The results demonstrated that HTR8/SVneo constitutively form ‘vascular’ tubes and networks after culture on Matrigel. dNK-CM enhanced and maintained tube and network formation, acquiring an endothelium-like angiogenic morphology followed by increased VEGF-C production. HTR8/SVneo cell expression level of VE-cadherin, PECAM-1, VCAM-1 and αvβ3 was unaltered by dNK-CM, whereas ICAM-1 expression level was increased. Anti-human ICAM-1 blocking antibody inhibited HTR8/SVneo migration and partially reversed dNK-CM-mediated enhancement of HTR8/SVneo tube and network formation. PI3K/AKT and p38 MAPK pathways were activated in dNK-CM-mediated enhancement of HTR8/SVneo tube and network formation. The PI3K/AKT and p38 MAPK pathway inhibitors (LY294002 and SB202190, respectively) decreased dNK-CM-stimulated ICAM-1 induction, HTR8/SVneo migration, and reversed tube and network formation. The results suggest that dNK cell-secreted growth factors and cytokines participate in the regulation of HTR8/SVneo endothelium-like tube formation. Adhesion molecules, particularly ICAM-1, expressed on EVT may participate in the process. To our knowledge, this is the first report of a role for ICAM-1 in EVT angiogenesis, as previously reported for endothelial cells.

Peroxisome proliferator-activated receptors are altered in pathologies of the human placenta: Gestational diabetes mellitus, intrauterine growth restriction and preeclampsia

2010-01-04

Abstract: Background: Common complications of pregnancy arise in part from dysfunctional placental development, and include gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR) and preeclampsia (PE). Peroxisome proliferator-activated receptors (PPARs), and their partner retinoid X receptor a (RXRα), mediate trophoblast differentiation and thus may offer insight into the pathophysiology of these diseases.Methods: Human placentae were obtained from women at term with GDM and were compared to uncomplicated term placentae. Placentae from women who delivered preterm with IUGR, PE or co-existing PE and IUGR were compared to matched controls. Quantitative RT-PCR and Western blotting were used to examine mRNA and protein expression of PPARα, PPARδ, PPARγ and RXRα. DNA binding activity of PPAR isoforms were measured in nuclear protein extracts.Results: GDM was associated with significantly lower placental PPARγ mRNA and protein, PPARα protein and RXRα protein expression, while PPAR DNA binding activity remained unchanged. Placentae from women with PE did not demonstrate any changes in mRNA or protein expression or PPAR DNA binding activity, while IUGR/PE placenta showed significant increases in PPARα protein, PPARγ mRNA and protein and RXRα mRNA and protein expression. Significantly elevated protein expression of PPARα and RXRα were associated with IUGR placentae. IUGR and IUGR/PE placentae had significantly higher PPARγ DNA binding activity compared to controls.Conclusions: The data presented herein suggest that PPARs may be involved in the pathophysiology of GDM, PE and IUGR.

Fatty acids alter glycerolipid metabolism and induce lipid droplet formation, syncytialisation and cytokine production in human trophoblasts with minimal glucose effect or interaction

2010-01-20

Abstract: The diabetic pregnancy is characterized by maternal hyperglycaemia and dyslipidaemia, such that placental trophoblast cells are exposed to both. The objective was to determine the effects of hyperglycaemia, elevated non-esterified fatty acids (NEFA) and their interactions on trophoblast cell metabolism and function.Trophoblasts were isolated from normal term human placentas and established in culture for 16h prior to experiments. Glucose utilisation, fatty acid oxidation and fatty acid esterification were determined using radiolabelled metabolic tracer methodology at various glucose and NEFA concentrations. Trophoblast lipid droplet formation including adipophilin mRNA expression, viability, apoptosis, syncytialisation, secretion of hormones and pro-inflammatory cytokines were also assessed.Glucose utilisation via glycolysis was near maximal at the low physiological glucose concentration of 4mM; whereas NEFA esterification into triacylglycerol and diacylglycerol increased linearly with increasing NEFA concentrations without evidence of plateau. Culture of trophoblasts in 0.25mM NEFA for 24h upregulated fatty acid esterification processes, inhibited fatty acid oxidation, inhibited glycerol release (a marker of lipolysis) and promoted adipophilin and lipid droplet formation, all consistent with upregulation of fatty acid storage and buffering capacity. NEFA also promoted trophoblast syncytialisation and TNFα, IL-1β, IL-6 and IL-10 production without effects on cell viability, apoptosis or hormone secretion. Hyperglycaemia caused intracellular glycogen accumulation and reduced lipid droplet formation, but had no other effects on trophoblast metabolism or function.NEFA have effects on trophoblast metabolism and function, mostly independent of glucose, that may have protective as well as pathophysiological roles in pregnancies complicated by diabetes and/or obesity.

Estrogen-regulated Expression and Distribution of Id-1 in the Mouse Uterus

S.-H. Hong, H.-Y. Nah, C.-H. Kim — 2009-12-23

Abstract: Id genes are involved in proliferation and differentiation in various cell types. However, it remains to be fully investigated how Id genes are regulated in highly dynamic uterine tissue. Using cDNA microarray, we previously observed that Id-1 was upregulated in uteri from ovariectomized (OVX) mice exposed to 17β-estradiol (E2). Here, we further examined the expression pattern of Id-1 and its regulation of by E2 and progesterone (P4) in the mouse uterus. Increased Id-1 transcripts in OVX mice uterus exposed to E2 were efficiently reduced by pretreatment with ICI 182,780 (an antagonist for nuclear estrogen receptor), suggesting that E2 induced expression of Id-1 is mediated via classical estrogen receptor (ER). In contrast, P4 treatment did not enhance or antagonize the action of E2 on Id-1 expression. Laser capture microdissection revealed that Id-1 is exclusively expressed in luminal epithelial cells. Notably, Id-1 was significantly upregulated at implantation sites on day 4.5 of pregnancy due to strong expression of Id-1 in blastocyst stage embryos. The present results show that the expression of Id-1 in the mouse uterus is tightly regulated by E2 via classical ER genomic pathway, not P4 suggesting an important role in uterine physiological events such as the estrous cycle and implantation process.

Comparison of Phospholipid Molecular Species between Terminal and Stem Villi of Human Term Placenta by Imaging Mass Spectrometry

Y. Kobayashi, T. Hayasaka, M. Setou, H. Itoh, N. Kanayama — 2010-01-29

Abstract: Placental villi play pivotal roles in the feto-maternal transportation and phospholipids constitute major part of villous membrane. However, the functional contributions as well as pathological roles of placental phospholipids are yet to be fully clarified, because tissue distribution of phospholipids in the placental villi has not been identified. Recently, we have been developing and optimizing an imaging system based on a matrix-assisted laser desorption ionization (MALDI)-based mass spectrometer, which provides clear two-dimensional molecular identification with highly sensitive mass spectrometry from mixtures of ions generated on tissue surfaces. In the present study, we applied this technology to the molecular identification of phospholipids in the human term placenta and found that sphingomyelin (d18:1/16:0) and phosphatidylcholine (16:0/20:4) were distributed differently between stem and terminal villi. This methodology detected a distinct tissue distribution of phosphatidylcholine (16:0/20:4) of terminal villi, coupling with arachidonic acid (AA), which might be a clue leading to the future investigation of the possible involvement the synthetic cascade of eicosanoids in the physiology as well as pathological development of terminal villi, such as fetal growth restriction and/or fetal hypoxia, since terminal villi plays the central roles for nutrient and oxygen supply from maternal to fetal circulation.

Investigation of an antibody reported to identify leukocyte immunoglobulin-like receptors (LILRB2) on placental vascular smooth muscle

Joan S. Hunt, Claudia Veronica Zaga Clavellina — 2009-11-16

In 2008, our group published data concerning expression of two inhibitory HLA-G receptors in human term placentas, leukocyte immunoglobulin-like receptor B1 (LILRB1, also known as ILT-2) and LILRB2 (also known as ILT-4). The main tool for identifying these receptors was immunohistology using two monoclonal antibodies (mAb) supplied to our laboratory by Amgen, Inc., Thousand Oaks, CA. Cells identified as positive were placental stromal cells (LILRB1+/LILRB2+) and vascular smooth muscle (SM) cells (LILRB1neg/LILRB2+).

IFPA Pages

2010-03-01