Homeobox gene DLX4 expression is increased in idiopathic human fetal growth restriction

Idiopathic fetal growth restriction (FGR) is often associated with placental insufficiency. Previously, we isolated and characterized homeobox gene DLX4 from the placenta and provided evidence that DLX4 may regulate placental development. Here, we have investigated whether DLX4 expression levels were altered in idiopathic FGR. FGR-affected placentae were collected based on strict clinical criteria. DLX4 mRNA expression was analysed in placentae obtained from pregnancies complicated by idiopathic FGR and gestation-matched control pregnancies (n = 25 each). Initial RT-PCR results showed a qualitative increase in DLX4 mRNA in both FGR-affected placentae and gestation-matched controls. Real-time PCR showed a 3-fold increase in DLX4 mRNA levels in FGR-affected placentae compared with gestation-matched controls (P < 0.005). Western immunoblotting using a rabbit DLX4 polyclonal antibody revealed significantly increased levels of DLX4 protein in term FGR-affected placentae compared with term controls [5500.1 +/- 21.8 (n = 10) versus 3533.2 +/- 22.4 (n = 10); P < 0.001]. Qualitative immunohistochemical analyses of term placentae showed moderately increased immunoreactivity for DLX4 antigen in the FGR-affected placentae in syncytiotrophoblasts, residual cytotrophoblast cells and endothelial cells of the fetal capillaries compared with gestation-matched control term placentae. We conclude that the increased expression of homeobox gene DLX4 may be a contributing factor to the developmental abnormalities seen in the FGR-affected placentae.     

Activation of the novel prothrombinase, fg12, as a basis for the pregnancy complications spontaneous abortion and pre-eclampsia

PROBLEM: Impaired trophoblast invasion during the first trimester of pregnancy is linked to spontaneous abortion, and defective invasion in the second trimester to hypertension + proteinuria (pre-eclampsia). Hypertension developing during the third trimester of human pregnancy represents, in part, a corrective response in the mother to provide adequate placental perfusion for fetal growth when trophoblast has not to invaded and converted the myometrial porprtion of maternal spiral arteries into to low resistance-high capacitance conduits. Deportation of vesicles from hypoxemic trophoblast is thought to cause hypertension plus proteinuria, vascular damage and a systemic coagulopathy. Trophoblast invasion may be inhibited by local cytokines, such as TGF-betas but Thl-type cytokines associated with pre-eclapmsia and spontaneous abortions (e.g., IL-1, TNF-alpha, IFN-gamma) are not known to inhibit migration at in situ concentrations. Trophoblast invasion is also inhibited by the binding of surface integrins to fibronectin and fibrin, and fibrin production is stimulated by these Th1 cytokines via up-regulation of prothrombinases(s) such as fg12 which directly and via TNF-alpha-facilitated inflamation compromise trophoblast cell integrity. We, therefore, asked if fg12 expression and TNF-alpha are increased in first trimester human miscarriage and in third trimester pre-eclampsia. METHODS: fg12 mRNA was detected using in situ hybridization and fg12 protein by immunohistochemistry. TNF-alpha mRNA and protein were similarly tested. The techniques were validated using uterine sections from day 8.5 of CBA x DBA/2 pregnancies, and then were applied to sections of placentae from normal and pre-eclamptic pregnancies with and without intrauterine fetal growth restriction (IUGR). Fibrin was detectectd by immunohistochemistry. RESULTS: Expression of fg12 protein correlated with fg12 mRNA expression in mouse uteri and in placentae from normal human pregnancies. Increased expression of fg12 and TNF-alpha mRNA and protein, and increased fibrin deposition was detected in placental trophoblast. CONCLUSIONS: Activation of fg12 prothrombinase by Th1-type cytokines in pregnancy may lead to spontaneous abortion, or in ongoing pregnancy, to pre-eclampsia and/or IUGR.     

Expression of insulin-like growth factor in the placenta of intrauterine growth-retarded human fetuses

Many cases of intrauterine growth retardation (IUGR) are the result of placental and fetal tissue insufficiency. Insulin-like growth factor-I (IGF-I) is known to play a role in placental and fetal growth. An immunocytochemical study was performed to localize IGF-I peptides in human placenta and umbilical cords of normal (n = 3) and IUGR (n = 3) fetuses. The peripartum fetal conditions were evaluated as well. Immunoreactive IGF-I was detected in the cytotrophoblast, syncytiotrophoblast, amnion, endothelial cells of fetal capillaries and in the decidua in both normal and IUGR placental tissue. A more robust immunostaining and increased numbers of positively stained cells were found in the decidua of IUGR placenta (p < 0.001). Intense immunostaining was also found in endothelial cells, smooth muscle cells and fibroblasts of the umbilical vein. IGF-I immunoreactivity was also present in stroma (Hofbauer cells and/or fibroblasts) of IUGR villi. Our results indicate that expression of IGF-I is high in specific sites in placenta and umbilical cords, which indicates a paracrine and/or endocrine function. The increased expression of IGF-I in placenta of IUGR fetuses indicates its involvement in restoring normal growth by means of a positive feed-back mechanism.     

Ontogeny of Hepatocyte Growth Factor (HGF) and Its Receptor (c-met) in Human Placenta : Reduced HGF Expression in Intrauterine Growth Restriction

Severe intrauterine growth restriction (IUGR) is characterized by abnormal placentation. Mouse gene knockout studies show that an absence of either hepatocyte growth factor (HGF) or its receptor, c-met, leads to intrauterine death secondary to severe IUGR with deficient placentation. In this study, immunocytochemistry localized HGF protein throughout placental villi across gestation, whereas c-met protein was localized only to the perivillous trophoblast and vascular endothelium. Within the IUGR placentae, a reduction in HGF immunostaining within the villous stroma was observed. HGF mRNA was strongly expressed in the perivascular tissue around the stem villous arteries throughout gestation, with weaker expression within the villous stroma and the terminal villi. c-met mRNA expression was limited to the perivillous trophoblast, particularly in the first trimester, with only a faint hybridization signal from the villous stroma. Placental mRNA expression was examined quantitatively using a ribonuclease protection assay: HGF and c-met mRNA expression increased from the first to the second trimester, reaching a zenith before decreasing again through the third trimester to term. HGF mRNA levels were significantly reduced in the IUGR placentae (P = 0.036), whereas c-met mRNA expression was within the normal range for gestation. These findings suggest that HGF derived from the perivascular tissue of stem villous arteries may play an important role in controlling normal villous development. Whereas reduced expression of HGF within IUGR placentae does not prove a causative link with abnormal villous development, the association lends support to this possibility.     

Hypoxia down-regulates placenta growth factor, whereas fetal growth restriction up-regulates placenta growth factor expression: molecular evidence for "placental hyperoxia" in intrauterine growth restriction

Early placental development occurs in an environment of relative hypoxia. Hypoxia promotes angiogenesis and up-regulates vascular endothelial growth factor (VEGF) expression while it down-regulates placenta growth factor (PIGF) that possess 53% homology with VEGF. Morphological studies show poor placental vascular development and an increase in the mitotic index of cytotrophoblasts in intrauterine growth restriction (IUGR). We hypothesized that the reported relatively high oxygen level in the intervillous space in contact with IUGR placental villi will limit angiogenesis by changes in VEGF and PIGF expression and function. Western immunoblot analysis demonstrates a diametric expression of PIGF and VEGF proteins throughout pregnancy with PIGF levels increasing and VEGF levels decreasing, consistent with placental oxygenation. In IUGR placentae, the ratio of PIGF/GAPDH mRNA was increased by 2.3-fold (p < 0.03) and PIGF protein levels were also increased, (p < 0.05) as compared with gestationally-matched normal placentae. PIGF mRNA and protein were localized to the trophoblast bilayer and villous mesenchyme of the human placenta throughout gestation. In vitro studies demonstrated that increasing oxygen tension (hyperoxia) up-regulated PIGF protein in term placental villous explants, whereas hypoxic culture of a term trophoblast choriocarcinoma cell line (BeWo) down-regulated PIGF mRNA and protein and VEGFR-1 (Flt-1) autophosphorylation. The addition of PIGF-1 to a spontaneously transformed first trimester cytotrophoblast cell line stimulated DNA synthesis while PIGF-2 had little effect. VEGF and PIGF exert their biological actions by means of a common receptor VEGFR-1. In the first trimester trophoblast cells, PIGF-1 increased the association of phosphorylated extracellular signal-related kinase (ERK) with VEGFR-1 immunoprecipitates while both PIGF-1 and PIGF-2 also potentiated endogenous VEGF mediated association of phosphorylated extracellular related kinase (ERK) with VEGFR-2 (KDR). More importantly, the addition of PIGF-1 had little effect while PIGF-2 inhibited cell growth in cultured endothelial cells derived from human umbilical vein. Nitric oxide (NO) is reported to promote angiogenesis and PIGF-2 inhibited the basal release of NO from the first trimester trophoblast. The tissue expression and functional studies support the hypothesis of "placental hyperoxia" in early-onset IUGR because hypoxia down-regulates trophoblast PIGF levels, PIGF expression is increased in IUGR, and PIGF-2 inhibits endothelial cell growth. Taken together, these changes provide a cellular explanation for the observed poor angiogenesis in the pathogenesis of IUGR and show that the two PIGF isoforms may modulate trophoblast and endothelial cell function differently, possibly through potentiation of VEGF mediated activation of VEGF-2.     

11Beta-hydroxysteroid dehydrogenase type 2 in human pregnancy and reduced expression in intrauterine growth restriction

The type 2 isoform of 11beta-hydroxysteroid dehydrogenase (11beta- HSD2), which inactivates cortisol (F) to cortisone (E), has been suggested to play a role in the ontogeny of the fetal pituitary-adrenal axis and also protect the developing fetus from the deleterious effects of circulating maternal glucocorticoids. The abundance of 11beta-HSD2 in the placenta and other fetal tissues was inferred from the F/E ratio in 17 term deliveries in both umbilical arterial (1.73 +/- 0.24, mean +/- SE) and umbilical venous blood (1.16 +/- 0.14) compared with adult peripheral venous blood (7.76 +/- 0.57, n = 70). Using sensitive assays for 11beta-HSD2 and an in-house human 11beta-HSD2 antibody, the expression and activity of this enzyme in fresh frozen human placenta increased progressively from first (8-12 weeks, n = 16) and second (13- 20 weeks, n = 9) to third trimester (term) pregnancies (39-40 weeks, n = 50). Placental 11beta-HSD2 activity was significantly reduced in deliveries complicated by intrauterine growth restriction (IUGR) [25-36 weeks, n = 12, activity 380 pmol/mg/h median (225-671; 95% confidence interval)], compared with the term deliveries [888 (725-1362)] and with appropriately grown pre-term deliveries [27-36 weeks, n = 14, activity 810 (585-1269)], P < 0.05. In human pregnancy placental 11beta-HSD2 activity increases markedly in the third trimester of pregnancy at a time when maternal circulating levels of glucocorticoid are rising. The finding of attenuated placental 11beta-HSD2 activity in IUGR suggests that glucocorticoids may, in part, contribute to impaired fetal growth and that this is closely controlled in normal gestation through placental 11beta-HSD2 expression.      

The role of insulin-like growth factor binding protein-1 phosphoisoforms in pregnancies with impaired placental function identified by doppler ultrasound.

This study was performed to investigate the hypothesis that insulin-like growth factor binding protein-1 (IGFBP-1) is involved in the pathogenesis of trophoblast invasion and impaired placentation in human pregnancy. The role of total and non-phosphorylated IGFBP-1 in women with fetal growth restriction and in high risk pregnancies identified by uterine artery Doppler ultrasound screening was examined. This was a prospective study of women booked for antenatal care having second trimester anomaly scans and Doppler screening between 22-26 weeks gestation. Women were divided into three groups and compared: normal uterine artery Doppler and normal fetal growth (control group, n = 10); abnormal Doppler and normal fetal growth [bilateral uterine artery notches (BN; n = 16); abnormal Doppler and intrauterine growth restriction (IUGR; n = 8)]. Maternal serum was collected, stored and assayed simultaneously for total and non-phosphorylated IGFBP-1. There was elevated total and non-phosphorylated IGFBP-1 (mean 44.99 +/- 12.19 and 29.61 +/- 10.38 microg/l respectively) in the IUGR group compared with controls (mean 17.96 +/- 3.24 and 12.18 +/- 1.55 microg/l, P < 0.05). This finding suggests that the various IGFBP-1 isoforms, the degree of phosphorylation and the ratios of these different forms locally may be important during trophoblast invasion and may be implicated in clinical manifestations of impaired placentation later in the second trimester.     

Bcl—2蛋白在正常、妊高征及胎儿宫内发育迟缓胎盘中的表达

目的 通过检测Bcl-2蛋白在正常各期胎盘、妊高征胎盘和胎儿宫内发育迟缓胎盘中的表达，探讨Bcl-2蛋白在胎盘的发育过程中的功能及妊高征和胎儿宫内发育迟缓的病理机制。方法 取正常的8－10周、20－22周、足月胎盘以及妊高征和胎儿宫内发育迟结的胎盘组织固定，石蜡包坦，用ABC法抗Bcl-2免疫组织化学染色，光镜观察。结果 正常早孕绒毛合体细胞滋养怪、细胞滋养层细胞核、绒毛吵轴细胞阳性，细胞滋养层的胞质、绒毛中轴基质阴性。正常中期和足月胎盘滋养层细胞、血管内皮细胞阳性。与正常足月胎盘相比，妊高征、台儿宫内发育迟缓胎盘几科不着色。结论 Bcl-2蛋白在正常各期胎盘中均有表达，而在妊高征和胎儿宫内发育迟缓胎盘几乎不表达，Bcl-2蛋白表达异常与妊高征与胎儿宫内发育迟缓的发病机制有关。     

Endothelial nitric oxide synthase in placental villous tissue from normal, pre-eclamptic and intrauterine growth restricted pregnancies

Nitric oxide (NO) regulates blood flow in the human placenta. As increased resistance to blood flow is seen in the fetal-placental vasculature in pregnancies complicated by pre-eclampsia and/or intrauterine growth restriction (IUGR), we examined expression of endothelial nitric oxide synthase (eNOS) in these placentas. Placental villous tissue sections were obtained from normotensive control (n = 5), IUGR alone (n = 5) or pre-eclamptic (with or without IUGR (n = 9) patients, immunostained for eNOS and scored for localization, type (punctate or diffuse) and intensity of eNOS staining in syncytiotrophoblast and placental vessels. The significance of differences was calculated using the Mann-Whitney U-test. No differences in intensity or type of immunostaining in syncytiotrophoblast were seen. Placentas from patients with pre- eclampsia with or without IUGR had a significantly more basal distribution of eNOS in syncytiotrophoblast. eNOS immunostaining was absent in terminal villous capillary and faint in stem villous vessel endothelium of normal placentas, but was intense in the endothelium of both of these types of vessels in the IUGR and pre-eclampsia groups, with significantly greater staining seen in stem vessels of patients with IUGR alone. This increased eNOS expression and hence increased NO production in the fetal-placental vasculature may be an adaptive response to the increased resistance and poor perfusion in these pathological pregnancies.      

Intra-uterine growth restriction downregulates the hepatic toll like receptor-4 expression and function

Maternal starvation is a significant cause of intrauterine growth restriction (IUGR) in the world and increases the risk of infection in the neonate. We examined the effect of maternal starvation on Toll like receptor (TLR)4 expression in hepatic, splenic and intestinal tissues obtained from the adult IUGR offspring of prenatal calorie restricted rats. The hepatic TLR4 protein concentration was undetectable in the IUGR rats that had restricted milk intake during the suckling period (SM/SP; n = 4. p < 0.05) as compared to the normal growth controls (CM/CP; n = 4), and access to ad lib milk intake during the sucking period partially corrected the hepatic TLR4 expression (SM/CP; n = 4). IUGR had no effect on the splenic (n = 4) or intestinal (n = 4) TLR4 mRNA levels. In the liver, IUGR led to a 20% increase in baseline tumor necrosis factor (TNF)-alpha mRNA expression (p < 0.03) and a 70% increase in interleukin-1beta (IL-1beta) mRNA expression (p < 0.008) as compared to the control rats (CM/CP; n = 7). LPS-induced hepatic TNF-alpha release was significantly higher in SM/SP as compared to CM/CP. We propose that IUGR dysregulates TLR4 expression and function in the offspring, which may help explain the increased risk of Gram-negative sepsis and inflammatory diseases in this population.     

Homeobox gene ESX1L expression is decreased in human pre-term idiopathic fetal growth restriction

Fetal growth restriction (FGR) is a clinically significant pregnancy disorder in which the fetus fails to achieve its full growth potential in utero. This study involved idiopathic FGR, which is frequently associated with placental dysfunction. Here, we investigated mRNA levels of the human placental homeobox gene ESX1L in pre-term and term idiopathic FGR pregnancies compared with gestation-matched controls. Real-time PCR quantitation showed ESX1L levels in control placentae decreased between pre-term and term [0.7 +/- 0.20 (27-35 weeks, n = 13) versus 0.2 +/- 0.06 (36-41 weeks, n = 12), t-test, P < 0.005]. ESX1L levels in FGR-affected placentae were significantly lower than in gestation-matched controls, and there was no significant change between pre-term FGR and term FGR [0.32 +/- 0.04 (27-36 weeks, n = 11) versus 0.31 +/- 0.02 (36-41 weeks, n = 14), t-test, P = 0.82]. Multiple linear regression analysis revealed a rapid decline in ESX1L expression in control placentae [0.075-fold of the calibrator for each week of gestation (95% CI = -0.105 to -0.045, P < 0.0005)]. In FGR-affected placentae, ESX1L levels were lower than in gestation-matched controls, and the decline in ESX1L levels with gestation was not significant [0.001-fold of the calibrator for each week of gestation (95% CI = -0.030 to 0.010, P < 0.3]. The linear relationship between ESX1L mRNA levels in FGR-affected placentae and gestation-matched controls during gestation was significantly different (likelihood ratio test for interaction, P = 0.0005). Our findings were consistent with a potential role for the ESX1L gene within the growth control mechanism of the fetus, through its effect on placental function.     

Down-regulation of placental transport of amino acids precedes the development of intrauterine growth restriction in rats fed a low protein diet

Intrauterine growth restriction (IUGR) represents an important risk factor for perinatal complications and for adult disease. IUGR is associated with a down-regulation of placental amino acid transporters; however, whether these changes are primary events directly contributing to IUGR or a secondary consequence is unknown. We investigated the time course of changes in placental and fetal growth, placental nutrient transport in vivo and the expression of placental nutrient transporters in pregnant rats subjected to protein malnutrition, a model for IUGR. Pregnant rats were given either a low protein (LP) diet ( n = 64) or an isocaloric control diet ( n = 66) throughout pregnancy. Maternal insulin, leptin and IGF-I levels decreased, whereas maternal amino acid concentrations increased moderately in response to the LP diet. Fetal and placental weights in the LP group were unaltered compared to control diet at gestational day (GD) 15, 18 and 19 but significantly reduced at GD 21. Placental system A transport activity was reduced at GD 19 and 21 in response to a low protein diet. Placental protein expression of SNAT2 was decreased at GD 21. In conclusion, placental amino acid transport is down-regulated prior to the development of IUGR, suggesting that these placental transport changes are a cause, rather than a consequence, of IUGR. Reduced maternal levels of insulin, leptin and IGF-1 may link maternal protein malnutrition to reduced fetal growth by down-regulation of key placental amino acid transporters.     

Maternal taurine supplementation in the late pregnant rat stimulates postnatal growth and induces obesity and insulin resistance in adult offspring

An adequate supply of taurine during fetal life is important for normal beta-cell development and insulin action. An altered availability of taurine may programme glucose metabolism in utero and result in type 2 diabetes in adult age. We examined whether maternal taurine supplementation in late pregnant rats affects postnatal growth, adult body composition, insulin sensitivity and endogenous insulin secretion in intrauterine growth restricted (IUGR) and normal offspring. Uterine artery ligation or sham operations were performed on gestational day (GD) 19. Taurine supplementation was given to half of the dams from GD 18 until term, resulting in four groups of offspring: sham ( n = 22), sham/taurine ( n = 22), IUGR ( n = 22) and IUGR/taurine ( n = 24). The offspring were studied at 12 weeks of age. In offspring with normal birth weight, fetal taurine supplementation markedly stimulated postnatal growth. In sham/taurine females, fat depots, plasma free fatty acid and leptin concentrations were increased, and insulin sensitivity was reduced. Insulin sensitivity was unaltered in IUGR and IUGR/taurine offspring. However, whereas IUGR offspring showed little catch-up growth, 50% of IUGR/taurine animals displayed complete catch-up at 12 weeks of age, and these animals had increased fat depots and reduced insulin sensitivity. In conclusion, taurine supplementation in late gestation resulted in accelerated postnatal growth, which was associated with adult obesity and insulin resistance in both IUGR and normal offspring. This effect was particularly evident in females. These data suggest that fetal taurine availability is an important determinant for postnatal growth, insulin sensitivity and fat accumulation.     

Altered cell kinetics in cultured placental villous explants in pregnancies complicated by pre-eclampsia and intrauterine growth restriction

Placentae in pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are characterized by morphological variations, apoptosis, and syncytial shedding, features that are linked to inappropriate oxygen and inflammatory cytokines. Cell turnover within the placenta is dynamic. In this study, these cellular events have been investigated longitudinally using a placental explant model. Intrinsic variations between normal (n = 14), PE (n = 16) and IUGR pregnancies (n = 11), and their responses to oxygen (3% and 17%) and exogenous tumour necrosis alpha (TNFalpha), were recorded. Placental explants were assessed for apoptotic morphology, immunolocalization of MIB-1 (a proliferation marker), lactate dehydrogenase (a necrosis marker), and human chorionic gonadotrophin (hCG, a marker of cytotrophoblast differentiation). Explants under TNFalpha and 17% O2 revealed progressive degeneration of syncytiotrophoblast (ST) followed by restoration of hCG, localized to newly differentiated cytotrophoblasts. This differentiation was significantly enhanced in PE and IUGR. Responses to 3% O2 were similar between groups: a sharp decline in hCG and failure to recover thereafter. Exaggerated cell death was recorded in PE and IUGR explants exposed to TNFalpha and 3% O2. All significant changes in apoptosis were confined to ST and stromal compartments. Enhanced cell death was predominantly apoptotic in PE and necrotic in IUGR. 3% O2 promoted cell proliferation in normal placentae but this response was not reciprocated in PE and IUGR. Elevated hCG in PE and IUGR explants may represent a placental predisposition to differentiation in vivo. In addition, the increased susceptibility of villous components to cell death, in the absence of stimulated proliferation, may provide a powerful mechanism for aberrant or adaptive placental cell turnover in utero.     

Genes or placenta as modulator of fetal growth: evidence from the insulin-like growth factor axis in twins with discordant growth

To determine whether fetal growth is regulated by placental and/or fetal factors, we measured maternal and fetal concentrations of insulin-like growth factor-I (IGF-I), IGF-II and insulin-like growth factor binding protein-1 (IGFBP-1) (total and non-phosphorylated) in dichorionic (DC) and monochorionic (MC) twins with (DC, n = 13; MC, n = 12) or without (DC, n = 13; MC, n = 12) discordant birth weight. In the discordant MC pregnancy, growth-restricted (IUGR) twins had lower IGF-II concentrations (P < 0.001) but similar IGF-I concentrations compared to the appropriate for gestational age(AGA) co-twin. The differences in IGF-II concentrations showed a positive association with percentage birth weight discordance (r = 0.60; P < 0.05) in MC twins. In contrast, IUGR DC twins had lower IGF-I concentrations (P < 0.05) but similar IGF-II concentrations compared to the AGA co-twins. There was a positive correlation between IGF-I concentrations and birth weight (r = 0.47; P < 0.05) in DC twins. Total IGFBP-1 concentrations were higher in both MC and DC IUGR twins (P < 0.05) compared to AGA twins. A negative association was found between total IGFBP-1 concentrations and birthweight of both MC (r = 0.47; P < 0.05) and DC (r = 0.58; P < 0.01) twins. No such differences in IGF concentrations were found between concordant MC and DC twin pairs. The maternal IGF concentrations were comparable between the MC and DC groups. These data suggest that growth discordances of twins exposed to the same maternal environment may be due to variations in either IGF-I or IGF-II/IGFBP-1, depending upon the functioning of the placenta.     

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.     

Homeobox gene Distal-Less 3 is a regulator of villous cytotrophoblast differentiation and its expression is increased in human idiopathic foetal growth restriction

Human idiopathic foetal growth restriction (FGR) is frequently associated with placental insufficiency. In our previous studies, we have reported the isolation and characterisation of the homeobox gene Distal-less 3 (DLX3) in the human placenta. In this study, we have investigated the level of DLX3 expression in idiopathic FGR-affected placentae and determined its functional role in villous trophoblast differentiation. FGR-affected placentae (n?=?25) were collected based on well-defined clinical criteria and matched for gestation with control uncomplicated pregnancies (n?=?25). Real-time polymerase chain reaction and immunoblotting showed increased DLX3 mRNA and protein expression in FGR-affected placentae compared with gestation-matched controls. Qualitative immunohistochemistry revealed DLX3 localisation in the syncytiotrophoblast, cytotrophoblasts and endothelial cells surrounding the foetal capillaries in both FGR-affected and control placentae. Down-regulation of DLX3 in primary villous trophoblast cells and a trophoblast-derived cell line showed decreased expression of differentiation markers, 3βHSD, βhCG and syncytin. Therefore, we conclude that increased DLX3 expression in FGR may contribute to trophoblast dysfunction observed in FGR.     

Impaired cell fusion and differentiation in placentae from patients with intrauterine growth restriction correlate with reduced levels of HERV envelope genes

One leading cause of perinatal morbidity and mortality is intrauterine growth restriction (IUGR). Several causes for IUGR have been proposed involving cytotrophoblast dysfunction. Envelope genes of the human endogenous retrovirus (HERV)-W (Syncytin-1), -FRD (Syncytin-2), and -P(b) have fusogenic properties, whereas envelope genes of HERV-R, -V1, and -V2 have putative placental functions. All six HERV envelope genes and three known cellular receptors were analyzed for expression in human control and IUGR placentae (n = 38) and in cultured cytotrophoblasts from control and IUGR (n = 8) placentae. All envelope genes demonstrated downregulation in IUGR compared to control placentae tissues, which were confirmed with cultured cytotrophoblasts. Examination of the Syncytin-1 and Syncytin-2 receptors ASCT-1/-2 and MFSD2 showed that MFSD2 was significantly expressed lower in IUGR than in control placentae and cytotrophoblasts. A reduction of Syncytin-1 protein expression was confirmed for IUGR placentae with immunoblotting and paraffin tissue sections. Embedded placental IUGR tissues showed an overall disorganized syncytiotrophoblast layer with fewer nuclei. Cytotrophoblasts from IUGR placentae demonstrated a lower cell fusion index and nuclei per syncytiotrophoblast in vitro. Fusogenic and non-fusogenic envelope genes are dysregulated in IUGR placentae and may contribute to the etiology of growth restriction in utero.