Intrauterine Growth Restriction Placentas Research Articles

Differential expression of imprinted genes in normal and IUGR human placentas

Genomic imprinting refers to silencing of one parental allele in the zygotes of gametes depending upon the parent of origin. Loss of imprinting (LOI) is the gain of function from the silent allele that can have a maximum effect of doubling the gene dosage. LOI may play a significant role in the etiology of intrauterine growth restriction (IUGR). Using placental tissue from 10 normal and 7 IUGR pregnancies, we conducted a systematic survey of the expression of a panel of 74 “putatively” imprinted genes using quantitative RT-PCR. We found that 52/74 (~70%) of the genes were expressed in human placentas. Nine of the 52 (17%) expressed genes were significantly differentially expressed between normal and IUGR placentas; 5 were up-regulated (PHLDA2, ILK2, NNAT, CCDC86, PEG10) and 4 down-regulated (PLAGL1, DHCR24, ZNF331, CDKAL1). We also assessed LOI profile of 14 imprinted genes in 14 normal and 24 IUGR placentas using a functional and sensitive assay developed in our laboratory. Little LOI was observed in any placentas for 5 of the genes (PEG10, PHLDA2, MEG3, EPS15, CD44). With the 149 heterozygosities examined, 40 (26.8%) exhibited LOI > 3%. Some genes exhibited frequent LOI in placentas regardless of the disease status (IGF2, TP73, MEST, SLC22A18, PEG3), while others exhibited LOI only in IUGR placentas (PLAGL1, DLK1, H19, SNRPN). Importantly, there was no correlation between gene expression and LOI profile. Our study suggests that genomic imprinting may play a role in IUGR pathogenesis, but mechanisms other than LOI may contribute to dysregulation of imprinted genes.

Antioxidants activities and concentration of selenium, zinc and copper in preterm and IUGR human placentas

The aim of this study was to examine changes in activities of cytochrome c oxidase (CCO), glucose-6-phosphate dehydrogenase (G6PDH), Cu–Zn superoxide dismutase (Cu–Zn SOD), glutathione peroxidase (GSH-Px), glutathione (GSH) levels and copper (Cu), zinc (Zn) and selenium (Se) concentrations, and to assess the possible differences between preterm placentas, placentas from term pregnancies complicated by intrauterine growth restriction (IUGR) and full-term control placentas. The enzyme activities and the level of GSH decreased in IUGR and preterm placentas in comparison with the control group. CCO activity and GSH level in preterm placentas were markedly lower compared with the IUGR ( P<0.01; P<0.05) and control ( P<0.01; P<0.05) placentas, respectively. In IUGR placentas the level of Cu was reduced by 23% ( P<0.05) and Zn by 37%. In preterm placentas the level of Cu was reduced by 19% and Zn by 42%. Se level in IUGR and preterm placentas was higher ( P<0.05) by 28% and 32% than in control group, respectively. The strong relation was observed between birth weight and CCO activity, birth weight and Cu–Zn SOD activity, and a low level of Zn and Cu influenced the birth weight especially in IUGR cases. Moreover, the strong inverse correlation between Se level and birth weight, Se level and placental weight and Se level and CCO activity are new findings.

Amino Acid Transport Systems β and A in Fetal T Lymphocytes in Intrauterine Growth Restriction and With Tumor Necrosis Factor-α Treatment

Intrauterine growth restriction (IUGR) is associated with reduced activity of placental amino acid transport systems beta and A. Whether this phenotype is maintained in fetal cells outside the placenta is unknown. In IUGR, cord blood tumor necrosis factor (TNF)-alpha concentrations are raised, potentially influencing amino acid transport in fetal cells. We used fetal T lymphocytes as a model to study systems beta and A amino acid transporters in IUGR compared with normal pregnancy. We also studied the effect of TNF-alpha on amino acid transporter activity. In fetal lymphocytes from IUGR pregnancies, taurine transporter mRNA expression encoding system beta transporter was reduced, but there was no change in system beta activity. No significant differences were observed in system A mRNA expression (encoding SNAT1 and SNAT2) or system A activity between the two groups. After 24 or 48 h TNF-alpha treatment, fetal T lymphocytes from normal pregnancies showed no significant change in system A or system beta activity, although cell viability was compromised. This study represents the first characterization of amino acid transport in a fetal cell outside the placenta in IUGR. We conclude that the reduced amino acid transporter activity found in placenta in IUGR is not a feature of all fetal cells.

Expression Patterns of Two Serine Protease HtrA1 Forms in Human Placentas Complicated by Preeclampsia with and without Intrauterine Growth Restriction

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy-specific disorders that have in common abnormal placental implantation, a marked proliferation of villous cytotrophoblastic cells and focal necrosis of the syncytiotrophoblast. Several studies show an ischemic placenta with a high-resistance vasculature, which cannot deliver an adequate blood supply to the feto-placental unit. The cause of PE is a matter of debate, but recently studies in mice suggest that the primary feto-placental lesions are sufficient to initiate the disease. HtrA1, a member of the family of HtrA proteins, is a secreted multidomain protein with serine protease activity. It is expressed in first and third trimester of gestation. In specimens from the first trimester of gestation, immunostaining for HtrA1 is generally found in both layers of villous trophoblast, syncytiotrophoblast and cytotrophoblast. Cytoplasm of extravillous trophoblast and extracellular matrix of cell islands and cell columns are labeled for HtrA1. Specimens from third trimester of gestation show a more intense positivity for HtrA1 in the syncytiotrophoblast than in cytotrophoblast. The extravillous trophoblast and the decidual cells, is positive for HtrA1. The purpose of this study is to investigate the expression pattern of HtrA1 in placentas from PE without IUGR (maternal PE) and with IUGR (fetal PE) by quantitative western blotting and immunohistochemistry. By quantitative western blotting analysis we observed a significant upregulation of ∼30 kDa HtrA1 form in PE. Differently, we detected a significant total HtrA1 down-regulation in PE–IUGR. Moreover, immunostaining for HtrA1 was positive in the villous trophoblast, in the syncytial knots and irregularly in the fetal vessel walls in PE placentas while immunostaining for HtrA1was present particularly in the syncytial knots in PE–IUGR placentas. In conclusion, we suggest that the ∼30 kDa HtrA1 form can be correlated to maternal PE while that the significant down-regulation of total HtrA1 can be correlated to placental PE. These HtrA1 alterations could be considered as possible markers to discriminate placental PE from maternal PE.

Quantitative 3D Micro-CT Imaging of the Human Feto-placental Vasculature in Intrauterine Growth Restriction

Objective Placental vascular development matches fetal growth and development. Quantification of the feto-placental vasculature in placentas from pregnancies is complicated by intrauterine growth restriction (IUGR) revealed confounding results. Therefore, the feto-placental vascular volume in IUGR placentas was assessed by 3D micro-computed tomography (micro-CT). Methods and results Placental probes from IUGR ( n = 24) and healthy control placentas ( n = 40) were perfused in situ with Microfil or BaSO 4 and randomly chosen samples were scanned by micro-CT. Using 3D images, we quantitated the feto-placental vascular volume fraction (VVF). A subanalysis was performed at three different levels, reaching from the chorionic plate artery (level A), to intermediate arteries (level B) and capillary system (level C). Results were complemented by histology. The significance of differences in vascular volume measurements was tested with analysis of variance [ANOVA]. Results Microfil perfused placentas showed a total vascular volume fraction of 20.5 ± 0.9% in healthy controls. In contrast, the VVF decreased to 7.9 ± 0.9% ( p < 0.001) in IUGR placentas. Significant differences were found between Microfil and BaSO 4 perfused placentas in the vascular volume fraction using micro-CT and histology. Micro-CT demonstrated localized concentric luminal encroachments in the intermediate arteries in placentas complicated by IUGR. Conclusion Micro-CT imaging is feasible for quantitative analysis of the feto-placental vascular tree in healthy controls and pregnancies complicated by IUGR.

Circulating and Placental Endoglin Concentrations in Pregnancies Complicated by Intrauterine Growth Restriction and Preeclampsia

Inadequate trophoblast invasion and spiral artery remodeling leading to poor placental perfusion and hypoxia are believed to underlie preeclampsia (PE) and intrauterine growth restriction (IUGR). Recent studies implicate increased circulating endoglin as a contributor to the pathogenesis of PE. The objective of this study was to determine whether placental and circulating endoglin concentrations are altered in pregnancies complicated by intrauterine growth restricted (IUGR) infants and to address the role of hypoxia on the regulation of placental endoglin. We analyzed 10 placentas each from normal pregnant (NP), PE, and IUGR subjects. Endoglin levels were 2.5-fold higher in preeclamptic placentas compared to NP (15.4 ± 2.6 versus 5.7 ± 1.0, p < 0.01). In contrast, endoglin levels were similar in NP and IUGR placentas (5.7 ± 1.0 vs 5.9 ± 1.1, p = NS). Placentas from pregnancies with both PE and IUGR exhibited endoglin levels comparable to the PE group and significantly different from normotensive pregnancies with and without IUGR pregnancies (mean 14.9 ± 4.0, n = 9, p = 0.013). Soluble endoglin concentrations in maternal plasma were comparable in NP and IUGR, but higher in women with PE ( n = 10 per group, p < 0.05). Despite a 2-fold increase in hypoxia inducible factor, HIF-1α, we did not observe endoglin upregulation in NP, PE, or IUGR placental villous explants exposed to hypoxia (2% oxygen). In contrast to PE, placental or circulating endoglin is not increased in normotensive women delivering small, asymmetrically grown (IUGR) infants at term. The placentas of women with IUGR appear to be fundamentally different from PE women with respect to endoglin, despite the proposed common pathology of deficient trophoblast invasion/spiral artery remodeling and poor placental perfusion.

Placental Stress, Protein Synthesis Inhibition, and Growth Restriction.

Intrauterine growth restriction (IUGR) affects 4-7% of births and poses a major clinical challenge. It is associated with an increased risk of stillbirth and neonatal morbidity and mortality, and also a predisposition to type II diabetes, obesity and cardiovascular disease in adult life. There are many potential causes, including genetic disorders, infection, and maternal undernutrition, but the majority of cases remain unexplained. Nonetheless, these cases are frequently associated with deficient physiological conversion of the maternal spiral arteries supplying the placenta, suggesting malperfusion may contribute to the aetiology. We hypothesise that the retention of smooth muscle within the walls of the arteries leads to spontaneous vasoconstriction, greater intermittent perfusion of the placenta and ischaemia-reperfusion-type stress. One of the potential consequences of malperfusion is endoplasmic reticulum (ER) stress, which can lead to protein synthesis inhibition through activation of the unfolded protein response. We tested for this by examining placentas from 6 cases of IUGR alone, 6 cases of IUGR complicated by early-onset preeclampsia (PE+IUGR) and 6 normal placentas. All placentas were collected with informed patient consent, were non-laboured and delivered by caesarean section. We observed multiple blocks to protein synthesis in the pathological placentas. Firstly, increased phosphorylation of eukaryotic initiation factor 2α (eIF2α) indicated suppression of translation initiation in the IUGR placentas compared to controls, and was further increased in PE+IUGR placentas. Consequently the levels of many proteins were reduced. In particular, AKT was reduced at the protein, but not mRNA, level and cyclin D1 protein was also lowered. These changes could be reproduced in trophoblast-like cell lines by induction of ER stress, and were associated with reduced proliferation. Phosphorylation of eIF2α blocks translation initiation by acting as a competitive inhibitor of the guanine-nucleotide-exchange factor, eIF2B. Activity of eIF2B can also be regulated through phosphorylation by kinases such as GSK-3β. We observed increased activity of GSK-3β and levels of P-eIF2B in the pathological placentas, representing a second block to protein synthesis. These blocks to translation initiation are sufficient to account for the smaller placental phenotype in IUGR pregnancies. Our findings are consistent with ER stress, which is one of at least four pathways that lead to phosphorylation of eIF2α. Indeed, we observed dilatation of the ER cisternae in the syncytiotrophoblast in the pathological placentas. Molecular evidence of ER stress was seen in IUGR placentas, but was more severe in PE+IUGR placentas where it was associated with activation of the pro-apoptotic protein CHOP. This may account for the deportation of placental apoptotic debris into the maternal circulation, which has been proposed as a causal factor in preeclampsia. To conclude, we provide the first evidence that ER stress may play a key role in limiting placental growth in complicated pregnancies. The severity of that stress may influence the balance between proliferation and apoptosis, and so determine the clinical outcome of the pregnancy. Supported by the Wellcome Trust (069027/Z/02/Z).

Quantitative 3D micro-CT Imaging of the Human Fetoplacental Vasculature in Intrauterine Growth Restriction and Diabetes Mellitus

Ziele: The quantitative changes in the fetoplacental vascular volume in placentas from pregnancies complicated by intrauterine growth restriction (IUGR) and diabetes mellitus (DB) are not well defined. The purpose of this study was to increase understanding of the substantial variation in the fetoplacental vascular volume in IUGR and DB assessed by 3D micro-computed tomography (micro-CT). Methode: Samples from IUGR (n=32), diabetic (n=24), and healthy control placentas (n=40) were infused in situ with Microfil or BaSO4 and scanned by micro-CT. Using those 3D images, we quantitated the complete fetoplacental vascular volume fraction (VVF). A subanalysis was performed at three different levels, reaching from the corion plate artery (level A), to intermediate arteries (level B) and capillary system (level C). Results were complemented by histology. Ergebnis: Microfil perfused placentas showed a total vascular volume fraction of 20.5±0.9% in healthy controls. In contrast, the VVF decreased to 16.7±0.9 (p<0.001) and 7.9±0.9 (p<0.001) in DB and IUGR placentas, respectively. Heterogeneity in the decrease in the vascular volume fraction in diabetic placentas was found to be present in intermediate arteries (Level B) and the capillary system (Level C) but not in the corion plate artery (Level A)

Release of inositol phosphoglycan P-type by the human placenta following insulin stimulus: A multiple comparison between preeclampsia, intrauterine growth restriction, and gestational hypertension

Objective. Abnormal metabolism of inositol phosphoglycan P-type (P-IPG) has been described in insulin-resistant states. Recently, a definite link between P-IPG and preeclampsia has been reported. P-IPG release after insulin stimulus has been described in the placental tissue of healthy women and a complete absence of P-IPG release has been found in preeclamptic samples, associated with disturbed insulin signaling. This study was undertaken to assess the release of this mediator in intrauterine growth restriction (IUGR) and hypertensive disorders other than preeclampsia.Methods. Seven women with IUGR, seven with gestational hypertension, 11 with preeclampsia, and 12 controls were recruited for this study. Fresh placental membranes were prepared and incubated with human recombinant insulin. Bioactivity of P-IPG released after insulin stimulus was assessed using a specific bioassay. A multiple comparison between groups was carried out. The study population provided a statistical power of 0.94.Results. P-IPG release was highest and lowest from healthy and preeclamptic samples, respectively (p < 0.01). Specimens from patients with IUGR and gestational hypertension released less P-IPG than did controls (p < 0.05).Conclusions. Abnormal release of P-IPG from placentas of IUGR and gestational hypertensive mothers seems to confirm an association between these disorders of human pregnancy and insulin resistance.

453: Placental arginase II is increased with preeclampsia but not with intrauterine growth restriction at term

INTRAUTERINE GROWTH RESTRICTION AT TERM ARUNDHATHI JEYABALAN, AUGUSTINE RAJAKUMAR, STACY MCGONIGAL, NINA MARKOVIC, CARL HUBEL, SIDNEY MORRIS, University of Pittsburgh, Obstetrics, Gynecology and Reproductive Sciences, Pittsburgh, Pennsylvania, University of Pittsburgh, Epidemiology, Pittsburgh, Pennsylvania, University of Pittsburgh, Obstetrics, Gynecology and Reproductive Sciences, Pennsylvania, University of Pittsburgh, Molecular Genetics and Biochemistry, Pittsburgh, Pennsylvania OBJECTIVE: Inadequate trophoblast invasion and spiral artery remodeling leading to poor placental perfusion are believed to underlie preeclampsia (PE) and intrauterine growth restriction (IUGR). Arginase competes for L-arginine, a substrate for both arginase(s) and nitric oxide synthase(s), thereby reducing nitric oxide bioavailability. This may limit nitric oxide-dependent vasodilation (contributing to reduced placental perfusion) and increase peroxynitrite (contributing to oxidative stress in the placenta). The objective of this study was to test the hypothesis that placental arginase II is increased in pregnancies complicated by PE or IUGR. STUDY DESIGN: We utilized 10 placentas each from normal pregnant (NP), PE, and IUGR subjects. Diagnosis of PE was based on the Working Group Report (2000) and hyperuricemia of 1 standard deviation above normal levels for gestational age. IUGR (without PE) was defined as infant birth weight less than the 10th percentile and asymmetrical fetal growth profile in normotensive women. Arginase II protein expression was measured by Western analysis. Densitometric ratios for matched pairs of PE/NP and IUGR/NP are presented as mean SE and analyzed by two-sided sign test. RESULTS: Baseline demographic characteristics were similar in all groups. Arginase II protein was detectable in all placental samples. Placental arginase II expression was 50% higher in women with PE compared to controls (PE/NP ratio of 1.51 0.25, p 0.02). In contrast, arginase II levels were similar in NP and IUGR placentas (ratio 1.06 0.24, p 0.75). CONCLUSION: Placental arginase II protein mass is higher with preeclampsia compared to uncomplicated pregnancies, but not with normotensive women delivering small, asymmetrically grown infants. This suggests that placentas from women with IUGR at term may be fundamentally different from PE women with respect to arginase II. The increased placental arginase II may reduce nitric oxide bioavailability leading to deficient spiral artery remodeling, poor placental perfusion, and increased oxidative stress in preeclampsia.

Placental Expression of Soluble fms-Like Tyrosine Kinase 1 is Increased in Singletons and Twin Pregnancies with Intrauterine Growth Restriction

Intrauterine growth restriction (IUGR) is characterized by decreased placental perfusion. Low oxygen has been shown to increase soluble fms-like tyrosine kinase 1 (sFlt-1) expression in the human placenta. The objective of this study was to examine sFlt-1 expression in different types of IUGR pregnancies, including early-onset severe cases characterized by abnormal umbilical and uterine artery Doppler and discordant IUGR twins in which the normal cotwin represents the optimal control because both placentas share the same uterine environment. Placentas from four subgroups were collected: early severe IUGR with umbilical artery absent end diastolic flow (n = 19), small for gestational age with normal uterine and umbilical artery Doppler (n = 11), severely growth-restricted dichorionic and monochorionic twins with abnormal umbilical artery Doppler (n = 9), preeclamptic twins (n = 3), and age-matched normal singletons (n = 19) and twin controls (n = 8). Expression of sFlt-1 mRNA and protein was significantly increased in IUGR placentas compared with small for gestational age and normal control placentas. sFlt-1 expression levels were also significantly greater in the small IUGR twin placentas from discordant twin pregnancies compared with the normal cotwin. In preeclamptic twins, sFlt-1 expression was increased in only one of the two placentas. Our results demonstrate that sFlt-1 expression is increased in severe IUGR placentas with abnormal umbilical artery Doppler of singletons and also in discordant IUGR twins. Reduced placental perfusion may contribute to the increased expression of sFlt-1 in IUGR pregnancies. Our data are compatible with differential sFlt-1 expression in placentas from discordant twins.

A hierarchical analysis of transcriptome alterations in intrauterine growth restriction (IUGR) reveals common pathophysiological pathways in mammals

Intra-uterine growth restriction (IUGR) is a frequent disease, affecting up to 10% of human pregnancies and responsible for increased perinatal morbidity and mortality. Moreover, low birth weight is an important cause of the metabolic syndrome in the adult. Protein depletion during the gestation of rat females has been widely used as a model for human IUGR. By transcriptome analysis of control and protein-deprived rat placentas, we were able to identify 2543 transcripts modified more than 2.5 fold (1347 induced and 1196 repressed). Automatic functional classification enabled us to identify clusters of induced genes affecting chromosome structure, transcription, intracellular transport, protein modifications and apoptosis. In particular, we suggest the existence of a complex balance regulating apoptosis. Among repressed genes, we noted several groups of genes involved in immunity, signalling and degradation of noxious chemicals. These observations suggest that IUGR placentas have a decreased resistance to external aggression. The promoters of the most induced and most repressed genes were contrasted for their composition in putative transcription factor binding sites. There was an over-representation of Zn finger (ZNF) proteins and Pdx1 (pancreatic and duodenal homeobox protein 1) putative binding sites. Consistently, Pdx1 and a high proportion of ZNF genes were induced at the transcriptional level. A similar analysis of ZNF promoters showed an increased presence of putative binding sites for the Tata box binding protein (Tbp). Consistently again, we showed that the Tbp and TBP-associated factors (Tafs) were up-regulated in IUGR placentas. Also, samples of human IUGR and control placentas showed that human orthologous ZNFs and PDX1 were transcriptionally induced, especially in non-vascular IUGR. Immunohistochemistry revealed increased expression of PDX1 in IUGR human placentas. In conclusion, our approach permitted the proposition of hypotheses on a hierarchy of gene inductions/repressions leading to massive transcriptional alterations in the IUGR placenta, in humans and in rodents.

Placental HIF-1α, HIF-2α, membrane and soluble VEGF receptor-1 proteins are not increased in normotensive pregnancies complicated by late-onset intrauterine growth restriction

Inadequate trophoblast invasion and spiral artery remodeling leading to poor placental perfusion are believed to underlie the pregnancy pathologies preeclampsia (PE) and intrauterine growth restriction (IUGR). The main objective of this study was to investigate hypoxia-inducible transcription factor-alpha (HIF-alpha) and downstream genes (VEGF receptor-1) Flt-1 and soluble fms-like tyrosine kinase 1 (sFlt-1) proteins in IUGR placentas. Placentas from normal pregnant (NP; n = 18), PE (n = 18), and IUGR (n = 10) patients were investigated. Normotensive patients with IUGR delivered babies at >or= 37 wk of gestation with birth weights of <10% and asymmetrical growth. HIF-1 alpha, -2 alpha, Flt-1, and sFlt-1 protein, and mRNA were assessed by Western and Northern blot analyses, respectively. The results are expressed as ratios of the densitometric values for each pair of pathologic and normal placentas, a ratio of 1.0 indicating no difference. Comparable to our earlier studies, the PE/NP ratios for HIF-1 alpha, -2 alpha, and Flt proteins were significantly increased by 50-100% (all P < 0.01 vs. 1.0). Unexpectedly, the IUGR/NP ratios for HIF-1 alpha and -2 alpha proteins were 1.03 +/- 0.07 and 0.96 +/- 0.16, respectively, and for Flt and sFlt were 1.14 +/- 0.15 and 0.95 +/- 0.12, respectively (all P = not significant vs. 1.0). Northern blot analysis revealed comparable levels of HIF-alpha mRNA in abnormal and normal placentas. In contrast to PE, HIF-alpha proteins and regulated genes are not increased in placentas from normotensive pregnant women delivering small, asymmetrically grown babies >or= 37 wk of gestation. The absence of an increase in HIF-alpha protein is not due to insufficient HIF-alpha mRNA for protein synthesis. Thus, the placentas from women with PE and late IUGR are fundamentally different at the molecular level.

Expression of Estrogen Receptor-α, Estrogen Receptor-β and Placental Endothelial and Inducible NO Synthase in Intrauterine Growth-Restricted and Normal Placentas

Nitric oxide seems to play important roles in the physiology of placental blood circulation, although their expression in pathological placentas and their role is still unclear. Therefore, the aim of this study was to investigate the expression of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta) and the endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) in intrauterine growth-restricted (IUGR) placentas, hemolysis, elevated liver enzymes, low platelets (HELLP) placentas and in normal healthy control placentas. Slides of paraffin-embedded placental tissue were obtained after delivery from patients diagnosed with IUGR, HELLP and normal term placentas and analyzed for eNOS and iNOS, as well as ERalpha/beta expression. Intensity of immunohistochemical reaction was analyzed using a semi-quantitative score, and statistical analysis was performed. In addition, Western blot experiments were performed for comparison of staining intensities obtained by immunohistochemistry and Western blot. Expression of eNOS and iNOS is significantly reduced in trophoblast cells of placentas with HELLP. However, ERbeta expression in HELLP placentas demonstrated a significantly elevated expression intensity compared to normal controls. ERalpha expression was not significantly different in all three pathologies investigated. We speculate that the increased ERbeta expression in both syncytiotrophoblast and extravillous trophoblast cells represents accelerated proliferation of placental tissue or can be seen as a compensatory effect in HELLP placentas.

Monocarboxylate transporter 8 expression in the human placenta: the effects of severe intrauterine growth restriction

Thyroid hormones (THs) are essential for normal fetal development, with even mild perturbation in maternal thyroid status in early pregnancy being associated with neurodevelopmental delay in children. Transplacental transfer of maternal THs is critical, with increasing evidence suggesting a role for 3,3',5-tri-iodothyronine (T3) in development and function of the placenta itself, as well as in development of the central nervous and other organ systems. Intrauterine growth restriction (IUGR) is associated with fetal hypothyroxinaemia, a factor that may contribute to neurodevelopmental delay. The recent description of monocarboxylate transporter 8 (MCT8) as a powerful and specific TH membrane transporter, and the association of MCT8 mutations with profound neurodevelopmental delay, led us to explore MCT8 expression in placenta. We describe the expression of MCT8 in normal human placenta throughout gestation, and in normal third-trimester placenta compared with that associated with IUGR using quantitative reverse transcriptase PCR. MCT8 mRNA was detected in placenta from early first trimester, with a significant increase with advancing gestation (P=0.007). In the early third trimester, MCT8 mRNA was increased in IUGR placenta compared with normal samples matched for gestational age (P<0.05), but there was no difference between IUGR and normal placenta in the late third trimester. Western immunoblotting findings in IUGR and normal placentae were in accord with mRNA data. MCT8 immunostaining was demonstrated in villous cytotrophoblast and syncytiotrophoblast as well as extravillous trophoblast cells from the first trimester onwards with increasingly widespread immunoreactivity seen with advancing gestation. In conclusion, expression of MCT8 in placenta from early gestation is compatible with an important role in TH transport during fetal development and a specific role in placental development. Altered expression in placenta associated with IUGR may reflect a compensatory mechanism attempting to increase T3 uptake by trophoblast cells.

Expression of Inhibin/activin Subunits alpha (-α), beta A (-β A) and beta B (-β B) in Placental Tissue of Normal and Intrauterine Growth Restricted (IUGR) Pregnancies

During human pregnancy the placenta produces a variety of proteins like steroid hormones and their receptors that are responsible for the establishment and ongoing of the feto-placental unit. Inhibins are dimeric glycoproteins, composed of an alpha-subunit and one of two possible beta-subunits (beta (A) or beta (B)). Aims of the present study were the determination of the frequency and tissue distribution patterns of the inhibin/activin subunits in human placental tissue of normal pregnancies and pregnancies complicated with fetal growth restriction (IUGR). Slides of paraffin embedded placental tissue were obtained after delivery from patients diagnosed with IUGR (n = 6) and normal term placentas (n = 8). Tissue samples were fixed and incubated with monoclonal antibodies inhibin/activin-subunits -alpha, -beta (A), -beta (B). Intensity of immunohistochemical reaction on the slides was analysed using a semi-quantitative score and statistical analysis was performed (P<0.05). A significant lower expression of the inhibin-alpha subunit in IUGR extravillous trophoblast compared to normal pregnancies was observed, while the inhibin-alpha immunostaining was significantly upregulated in syncytiotrophoblast. Additionally, a significant down-regulation of inhibin-beta (B) subunit in extravillous trophoblast cells in IUGR syncytiotrophoblast cells was demonstrated. A co-localisation of inhibin-alpha and the beta-subunits was also observed, suggesting a production and secretion of intact inhibin A and inhibin B. Although the precise role of these inhibin/activin subunits in human placenta and IUGR pregnancies is still unclear, they could be involved in autocrine/paracrine signalling, contributing to several aspects like angiogenesis and tissue remodelling.

The Placenta in Pre-eclampsia and Intrauterine Growth Restriction: Studies on Exchange Surface Areas, Diffusion Distances and Villous Membrane Diffusive Conductances

We test the null hypothesis that the morphometric diffusive conductance of the placental villous membrane does not alter in pregnancies complicated by intrauterine growth restriction (IUGR) or pre-eclampsia (PE). Placentas were collected from cases of normotensive IUGR, pure PE, PE + IUGR and from control pregnancies. Microscopical fields on formalin-fixed, trichrome-stained histological sections were randomly sampled for location and orientation. Using stereological methods, the exchange surface areas of peripheral (terminal and intermediate) villi and their fetal capillaries and the arithmetic and harmonic mean thicknesses of the villous membrane (maternal aspect of trophoblast to luminal aspect of vascular endothelium) were estimated. An index of the variability in thickness of this membrane, and an estimate of its oxygen diffusive conductance, was derived secondarily. Group comparisons were drawn using two-way analysis of variance to identify main effects (of PE or IUGR) and interaction effects (between PE and IUGR). PE did not have significant effects on placental morphology and there were no significant effects of PE or IUGR on membrane thickness or its variability. In contrast, IUGR (with or without PE) was associated with reduced surface areas and this was the principal factor leading to a smaller membrane diffusive conductance in these placentas. When account was taken of fetal mass, specific conductance showed no effects of PE or IUGR despite the mass-specific conductance in pure IUGR placentas appearing to be smaller than that in controls. The decline in total conductances is indicative of perturbations operating at the levels of villous trophoblast and fetal vasculature and these may contribute to fetal hypoxic stress.

Placental cell turnover in health and disease

Pre-eclampsia (PE) and intra-uterine growth restriction (IUGR) cause significant maternal and perinatal morbidity and mortality. Placental dysfunction is central to the development of both conditions. Although the pathophysiology of these conditions is unknown, there is common placental pathology with an increase in apoptotic cell death seen within the trophoblast. In addition, in pre-eclampsia, apoptotic fragments of syncytiotrophoblast have been detected in the maternal circulation. Both hypoxia and reactive oxygen species have been proposed as potential mediators of the insults to the placenta in pre-eclampsia and IUGR resulting in apoptosis. Cell proliferation and apoptosis are tightly regulated by oncoproteins. The increased apoptosis observed within trophoblast is associated with an alteration in oncoprotein expression within placental tissue. Further investigation of these oncoproteins capable of detecting or responding to cell damage may improve understanding of the pathophysiology of pre-eclampsia and IUGR.

Unbalanced Placental Expression of Imprinted Genes in Human Intrauterine Growth Restriction

Imprinted genes control fetal and placental growth in mice and in rare human syndromes, but the role of these genes in sporadic intrauterine growth restriction (IUGR) is less well-studied. We measured the ratio of mRNA from a maternally expressed imprinted gene, PHLDA2, to that from a paternally expressed imprinted gene, MEST, by Northern blotting in 38 IUGR-associated placentae and 75 non-IUGR placentae and found an increase in the PHLDA2/MEST mRNA ratio in IUGR (p=0.0001). Altered expression of PHLDA2 and MEST was not accompanied by changes in DNA methylation within their imprinting centers, and immunohistochemistry showed PHLDA2 protein appropriately restricted to villous and intermediate cytotrophoblast in the IUGR placentae. We next did a genome-wide survey of mRNA expression in 14 IUGR placentae with maternal vascular under-perfusion compared to 15 non-IUGR placentae using Affymetrix U133A microarrays. In this series six imprinted genes were differentially expressed by ANOVA with a Benjamini-Hochberg false discovery rate of 0.05, with increased expression of PHLDA2 and decreased expression of MEST, MEG3, GATM, GNAS and PLAGL1 in IUGR placentae. At lower significance, we found IGF2 mRNA decreased and CDKN1C mRNA increased in the IUGR cases. We confirmed the significant reduction in MEG3 non-translated RNA in IUGR placentae by Northern blotting. In addition to imprinted genes, the microarray data highlighted non-imprinted genes acting in endocrine signaling (LEP, CRH, HPGD, INHBA), tissue growth (IGF1), immune modulation (INDO, PSG-family genes), oxidative metabolism (GLRX), vascular function (AGTR1, DSCR1) and metabolite transport (SLC-family solute carriers) as differentially expressed in IUGR vs. non-IUGR placentae.

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

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