Normotensive Intrauterine Growth Restriction Research Articles

Placental expression of angiogenesis-related genes and their receptors in IUGR pregnancies: correlation with fetoplacental and maternal parameters

Objectives: Aberrations in placental vascular development compromising fetal supply of oxygen and essential nutrients can be a significant contributor to intrauterine growth restriction (IUGR). The development of placental vascular tree is under the influence of two families of growth factors, namely the vascular endothelial growth factor (VEGF) family and angiopoietin/TEK family. In this study, we have examined the expression of angiogenesis-related growth factors, mainly VEGF family and angiopoietin-TEK (endothelial-specific receptor tyrosine kinase) family genes in placentae from IUGR pregnancies uncomplicated by preeclampsia (PE) compared to normal pregnancies.Methods: Placentae from normotensive IUGR (n = 42) and appropriate for gestational age (AGA) pregnancies (n = 47) were collected and examined histologically. Clinical parameters were obtained from the medical records. Real-time quantitative PCR was performed to assess placental transcript abundance of VEGF, PGF, FLT1, ANGPT1, ANGPT2, and TEK normalized to a panel of reference genes. Associations of placental transcript abundance of the genes with maternal, placental, and neonatal parameters were tested.Results: Placental transcript abundance for VEGF (relative expression 10.81 versus 12.98, p < .001), PGF (12.14 versus 13.8, p < .001) and ANGPT2 (3.67 versus 9.55, p = .002) were significantly lower in IUGR placentae compared to AGA. The transcript level of VEGF showed significant negative correlation with birth weight (r = −0.419, p = .006), placental weight (r = −0.318, p = .040), placental length (r = −0.389, p = .011) and breadth (r = −0.308, p = .047) only in the IUGR group. Presence of histopathological features of hypoxia correlated with significantly higher transcript levels of PGF in IUGR placentae (12.6 versus 10.9, p = .046).Conclusion: The low levels of VEGF transcripts may be responsible for the impaired angiogenesis in IUGR placentae. The significance of higher relative expression of PGF in the presence of chronic hypoxia needs to be explored.

99. Concomitant intrauterine growth restriction alters the lipoprotein profile in preeclampsia

Preeclampsia (PE) is a heterogeneous disorder that can be subgrouped according to clinical and pathophysiological features. PE can be accompagnied by intrauterine growth restriction (IUGR) and both diseases share common features. Alterations in lipid metabolism have consistently been reported for both diseases. We investigated whether characteristic lipid changes can be related to either of both diseases. We hypothesized that in general IUGR displays a low cholesterol phenotype while pure PE is rather associated with high triglyceride concentrations. Serum lipid profile of patients with IUGR (n = 52), hypertensive IUGR (HIUGR, n = 28), and PE without IUGR (n = 56) were compared to a control group (CTRL, n = 167). Additionally, lipid components in serum of severe early-onset cases (<34th weeks of gestation) of normotensive IUGR, hypertensive IUGR (HIUGR), and PE without IUGR (each n = 10) were analyzed by extensive lipid subfractionation and compared to 30 control patients. Triglycerides are elevated in PE. However in IUGR LDL-cholesterol is reduced, irrespective of additional PE (IUGR 79 mg/dL; HIUGR 74 mg/dL vs. CTRL 133 mg/dL). The ApoB-profile shows a higher level of VLDL-ApoB in PE and a lower level of LDL-ApoB in IUGR, as expected. Interestingly, the HIUGR group shows features of both groups, high VLDL-ApoB level and a low ApoB-level in the LDL-subfractions. The presence of IUGR in PE alters lipid profiles. Study groups have to be selected carefully to avoid misinterpretation.

Serum Kisspeptin-10 Levels in Pregnant Women Complicated with Intrauterine Growth Restriction With or Without Preeclampsia

Abstract Background: Placental dysfunction is usually associated with a wide range of obstetric complications. Among the most important of these complications are preeclampsia (PE) and intrauterine growth restriction (IUGR), they are associated with a higher rate of perinatal mortality and morbidity. Kisspeptins are peptide products of the KISS-1 gene, which act through binding with the G-protein coupled receptor 54, Kisspeptin-10 (KP-10) produced by trophoblast cells, has an important role in controlling of migratory features and tro-phoblastic invasion. Studies that evaluate the role of KP-10 in pregnancy complications are sparse.Aim of the Work: To investigate serum KP-10 levels in correlation with some parameters in pregnancy complicated with IUGR with or without PE.Subjects and Methods: This study was performed on thirty pregnant woman equally divided into three groups, group I, ten uncomplicated pregnant women with normal blood pressure consider as control, group II, ten preeclamptic women com-plicated by IUGR and group III, ten normotensive pregnant women complicated by IUGR. The maternal serum was ana-lyzed for KP-10 levels and TNFa, while, the maternal urine was analyzed for protein detection. Also, gestational age, fetal birth weight, placental weight, BMI, and MAB were measured. Results: The present findings revealed significant decrease in maternal serum KP-10 levels in both of group II (p<0.05) and group III (p<0.01) rather than group I. Moreover, KP-10 was positively correlated with fetal birth weight and placental weight in group II and group III, while, negatively correlated with protienuria in group II and TNFa in group II and group III. However, no correlations were found with gestational age, BMI, and mean arterial blood pressure in all groups.Conclusion: These results propose that the decrease in KP-10 may be responsible for the pathogenesis that underlying the development of IUGR with preeclampsia and normotensive IUGR. So, its measurement might be helpful as a new biomar-ker in predicting poor placental dysfunction and adverse pregnancy outcome.

No evidence for association of MTHFR 677C>T and 1298A>C variants with placental DNA methylation

Background5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in one-carbon metabolism that ensures the availability of methyl groups for methylation reactions. Two single-nucleotide polymorphisms (SNPs) in the MTHFR gene, 677C>T and 1298A>C, result in a thermolabile enzyme with reduced function. These variants, in both the maternal and/or fetal genes, have been associated with pregnancy complications including miscarriage, neural tube defects (NTDs), and preeclampsia (PE), perhaps due to altered capacity for DNA methylation (DNAm). In this study, we assessed the association between MTHFR 677TT and 1298CC genotypes and risk of NTDs, PE, or normotensive intrauterine growth restriction (nIUGR). Additionally, we assessed whether these high-risk genotypes are associated with altered DNAm in the placenta.ResultsIn 303 placentas screened for this study, we observed no significant association between the occurrence of NTDs (N = 55), PE (early-onset: N = 28, late-onset: N = 20), or nIUGR (N = 21) and placental (fetal) MTHFR 677TT or 1298CC genotypes compared to healthy pregnancies (N = 179), though a trend of increased 677TT genotype in PE/IUGR together was observed (OR 2.53, p = 0.048). DNAm was profiled in 10 high-risk 677 (677TT + 1298AA), 10 high-risk 1298 (677CC + 1298CC), and 10 reference (677CC + 1298AA) genotype placentas. Linear modeling identified no significantly differentially methylated sites between high-risk 677 or 1298 and reference placentas at a false discovery rate < 0.05 and Δβ ≥ 0.05 using the Illumina Infinium HumanMethylation450 BeadChip. Using a differentially methylated region analysis or separating cytosine-guanine dinucleotides (CpGs) by CpG density to reduce multiple comparisons also did not identify differential methylation. Additionally, there was no consistent evidence for altered methylation of repetitive DNA between high-risk and reference placentas.ConclusionsWe conclude that large-scale, genome-wide disruption in DNAm does not occur in placentas with the high-risk MTHFR 677TT or 1298CC genotypes. Furthermore, there was no evidence for an association of the 1298CC genotype and only a tendency to higher 677TT in pregnancy complications of PE/IUGR. This may be due to small sample sizes or folate repletion in our Canadian population attenuating effects of the high-risk MTHFR variants. However, given our results and the conflicting results in the literature, investigations into alternative mechanisms that may explain the link between MTHFR variants and pregnancy complications, or in populations at risk of folate deficiencies, are warranted.

Acute-phase proteins in prediction of preeclampsia in patients with abnormal midtrimester uterine Doppler velocimetry.

Manifestation of preeclampsia is characterized by an inflammatory response and altered expression of acute-phase proteins. In this study, we examined the predictive value of serum amyloid A, progranulin, transthyretin, C-reactive protein and interleukin-6. Soluble endoglin was used as control. Maternal serum levels of the putative biomarkers were measured in 49 women with a midtrimester bilateral abnormal uterine artery Doppler velocimetry. Preeclampsia developed in 26.5%. 75.0% had an early-onset disease (<34+0weeks). Delivery <34+0weeks was indicated in 16.3%. 12.2% of patients developed a normotensive intrauterine growth restriction. All of the putative biomarkers were not predictive for preeclampsia. But serum levels of progranulin and also of soluble endoglin were increased in cases with development of a severe normotensive intrauterine growth restriction. Only soluble endoglin was predictive for the development of preeclampsia with an area under curve in the receiver operating curve analysis of 0.761 (P=0.006). Using a cut-off level of ≥9.14ng/mL, sensitivity, specificity, positive predictive value and negative predictive value were 53.9, 88.9, 63.6 and 84.2%, respectively. Inflammation is a late event during development of preeclampsia, and acute-phase proteins are not predictive for the disease in a high-risk population without clinical symptoms during the second trimester. Progranulin is a putative new biomarker for an early detection of intrauterine growth restriction in women without concomitant hypertensive disorders. Soluble endoglin improved predictive values for preeclampsia in patients with abnormal uterine Doppler.

Altered Platelet Function in Intrauterine Growth Restriction: A Cause or a Consequence of Uteroplacental Disease?

Objective A limited number of platelet function studies in intrauterine growth restriction (IUGR) have yielded conflicting results. We sought to evaluate platelet reactivity in IUGR using a novel platelet aggregation assay. Study Design Pregnancies with IUGR were recruited from 24 weeks' gestation (estimated fetal weight < 10th centile) and had platelet function testing performed after diagnosis. A modification of light transmission aggregometry created dose-response curves of platelet reactivity in response to multiple agonists at differing concentrations. Findings were compared with healthy third trimester controls. IUGR cases with a subsequent normal birth weight were analyzed separately. Results In this study, 33 pregnancies retained their IUGR diagnosis at birth, demonstrating significantly reduced platelet reactivity in response to all agonists (arachidonic acid, adenosine diphosphate, collagen, thrombin receptor-activating peptide, and epinephrine) when compared with 36 healthy pregnancy controls (p < 0.0001). Similar results were obtained for cases demonstrating an increasing in utero growth trajectory. When IUGR preceded preeclampsia or gestational hypertension, platelet function was significantly reduced compared with normotensive IUGR. Conclusion Using this comprehensive platelet assay, we have demonstrated a functional impairment of platelets in IUGR. This may reflect platelet-derived placental growth factor release. Further evaluation of platelet function may aid in the development of future platelet-targeted therapies for uteroplacental disease.

Abstract 235: Chronic Endothelin Type A Receptor Blockade Abolishes Age-dependent Hypertension In Female Intrauterine Growth Restricted Rat Offspring.

Birth weight is inversely associated with blood pressure. Placental insufficiency in the rat induces low birth weight indicative of intrauterine growth restriction (IUGR) associated with sex-dependent hypertension in IUGR offspring in young adulthood. However, placental insufficiency programs the development of age-dependent hypertension in the female IUGR rat by 12 months of age. Blood pressure increases with age and the prevalence of hypertension is significantly increased in low birth weight women at 60 relative to 50 years of age. Yet, the exact mechanisms that mediate age-dependent hypertension following a developmental insult are unknown. We previously noted that endothelin mediated via its endothelin type A receptor (ET A R) plays a significant role in blood pressure regulation in hypertensive male IUGR but not in normotensive female IUGR rats in young adulthood. Thus, we hypothesized that endothelin mediated via its ET A R would play a contributory role in the development of age-dependent hypertension in the female IUGR rat. Mean arterial pressure (MAP) was measured via arterial catheter in conscious female control and female IUGR offspring at approximately 12 and 18 months of age in animals that received vehicle or an ET A R antagonist (ABT-627, 5 mg/kg BW, Abbott) in the drinking water for 4 days prior to measure of MAP. MAP was significantly increased in IUGR (135±2 mmHg; P &lt;0.05,) relative to control (121±2 mmHg) at 12 months of age (N=9 per group). MAP was remained significantly elevated in IUGR (149±5 mmHg, P &lt;0.05) relative to control (135±5 mmHg) at 18 months of age (N=5 per group) despite age-dependent increases in MAP in IUGR and control rats. Chronic ET A R blockade significantly decreased MAP in treated IUGR (113±2 mmHg; P &lt;0.05 vs. untreated IUGR, N=6) but blockade of the ET A R had no impact on blood pressure in treated control (134±9 mmHg; N=5) at 18 months of age. Thus, these findings indicate that age augments the importance of endothelin via its ET A R in the etiology of hypertension programmed by IUGR but does not contribute to the age-dependent increase in blood pressure in the female control. Furthermore, this study highlights the need for sex- and age-specific considerations in the management of hypertension following a developmental insult.

Prediction of preeclampsia and induced delivery at

BackgroundWomen with bilateral abnormal uterine artery Doppler velocimetry (UtADV) are at increased risk for an adverse pregnancy outcome. This study aimed to determine if additional assessment of midtrimester angiogenic factors improves the predictive accuracy of Doppler results for various outcome parameters.MethodsWomen with a bilateral abnormal UtADV, which was defined as a postsystolic incision and/or an increased pulsatility index greater than the 95th centile, and a singleton pregnancy were prospectively recruited between 19 + 0 and 26 + 6 weeks of gestation. Maternal serum levels of placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFLT-1) were measured with a fully automated immunoassay and their ratio was calculated.ResultsAngiogenic factors could predict the development of preeclampsia (PE), as well as induced delivery at <34 weeks of gestation, but failed to predict the development of normotensive intrauterine growth restriction. Twelve (24.0%) of the 50 recruited women developed PE. Nine of these patients had early-onset disease (<34 + 0 weeks). Six (12.0%) patients were delivered at <34 + 0 weeks. The most useful test results in the prediction of PE and induced delivery at <34 + 0 weeks were observed using the sFLT-1/PlGF >95th centile ratio with a sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of 66.7%, 89.5%, 66.7%, and 89.5% for PE, and 85.7%, 86.1%, 50.1%, and 97.4% for induced delivery, respectively. Positive and negative likelihood ratios were 6.33 (95% CI 2.31–17.38) and 0.37 (95% CI 0.17–0.84) for PE, and 6.14 (95% CI 2.76–13.69) and 0.17 (0.03–1.02) for induced delivery, respectively. Corresponding odds ratios were 17.0 (95% CI 3.5–83.0) and 37.0 (95% CI 3.8–363.9), respectively.ConclusionsMeasurement of angiogenic factors improves the specificity of an abnormal UtADV for prediction of PE. Compared with prediction of PE an abnormal sFLT-1/PlGF ratio revealed higher sensitivity for prediction of induced delivery at <34 + 0 weeks. The NPV of 97% will help to reassure most patients with an abnormal UtADV and a normal sFLT-1/PlGF ratio.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2393-14-292) contains supplementary material, which is available to authorized users.

Widespread DNA hypomethylation at gene enhancer and low CpG density regions in placentas associated with early-onset preeclampsia

s / Placenta 34 (2013) A1–A99 A86 P2.88. WIDESPREAD DNA HYPOMETHYLATION AT GENE ENHANCER AND LOW CPG DENSITY REGIONS IN PLACENTAS ASSOCIATEDWITH EARLY-ONSET PREECLAMPSIA John Blair , Ryan Yuen , Brendan Lim , Deborah McFadden , Peter von Dadelszen , Wendy Robinson 1 University of British Columbia, Vancouver, BC, Canada; University of Toronto, Toronto, ON, Canada Preeclampsia is a serious complication of pregnancy that can affect both maternal and fetal outcomes. Early-onset preeclampsia (EOPET) is a severe form of preeclampsia that is associated with altered physiological characteristics and gene expression in the placenta. DNA methylation is a relatively stable epigenetic modification to DNA that can reflect gene expression, and can provide insight into the mechanisms underlying such expression changes. Objectives: We sought to profile DNA methylation changes in EOPET to better characterize the pathology underlying this condition and the relationship to clinical features. Methods: DNA methylation was profiled in placentas from 20 EOPET and 20-gestational age matched control pregnancies using the Infinium HumanMethylation450 Beadchip. This analysis was followed up using the HT-12v4 Expression BeadChip microarray, bisulfite pyrosequencing and bioinformatic analysis. Results: We identified 38,840 CpG sites with significant (FDR 12.5% methylation difference compared to the gestational age-matched normotensive controls. Significant sites were enriched at the enhancers and low CpG density regions of the associated genes and the majority (74.5%) of these sites were hypomethylated in EOPET. No stratification of the EOPET DNA methylation profile was apparent based on presence/absence of intrauterine growth restriction (IUGR) or severe proteinuria. CpG sites from four genes relevant to preeclampsia (INHBA, BHLHE40, SLC2A1, and ADAM12) were followed up using bisulfite pyrosequencing in a larger cohort, and they showed different extent of changes in related pregnancy disorders (late-onset preeclampsia (LOPET) and normotensive IUGR (nIUGR)). Genome-wide expression in a subset of samples showed that some of the gene expression changes were negatively correlated with DNA methylation changes, particularly for genes that are responsible for angiogenesis (such as EPAS1 and FLT1). Conclusion: Based on DNA methylation profiling, we conclude that there are widespread DNA methylation alterations in EOPET that may be associated with changes in placental function. This property may provide a useful tool for early screening of such placentas. http://dx.doi.org/10.1016/j.placenta.2013.06.254 P2.89. MORPHOLOGY OF THE MOUSE MESOMETRIAL DECIDUA DURING EARLY PREGNANCY Alex Kors Vidsiunas , Sergio Ferreira de Oliveira 2 Nove de Julho University, Sao Paulo-SP, Brazil; University of Sao Paulo, Sao

Widespread DNA hypomethylation at gene enhancer regions in placentas associated with early-onset pre-eclampsia.

Pre-eclampsia is a serious complication of pregnancy that can affect both maternal and fetal outcomes. Early-onset pre-eclampsia (EOPET) is a severe form of pre-eclampsia that is associated with altered physiological characteristics and gene expression in the placenta. DNA methylation is a relatively stable epigenetic modification to DNA that can reflect gene expression, and can provide insight into the mechanisms underlying such expression changes. This case–control study focused on DNA methylation and gene expression of whole chorionic villi samples from 20 EOPET placentas and 20 gestational age-matched controls from pre-term births. DNA methylation was also assessed in placentas affected by late-onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR). The Illumina HumanMethylation450 BeadChip was used to assess DNA methylation at >480 000 cytosine-guanine dinucleotide (CpG) sites. The Illumina HT-12v4 Expression BeadChip was used to assess gene expression of >45 000 transcripts in a subset of cases and controls. DNA methylation analysis by pyrosequencing was used to follow-up the initial findings in four genes with a larger cohort of cases and controls, including nIUGR and LOPET placentas. Bioinformatic analysis was used to identify overrepresentation of gene ontology categories and transcription factor binding motifs. We identified 38 840 CpG sites with significant (false discovery rate <0.01) DNA methylation alterations in EOPET, of which 282 had >12.5% methylation difference compared with the controls. Significant sites were enriched at the enhancers and low CpG density regions of the associated genes and the majority (74.5%) of these sites were hypomethylated in EOPET. EOPET, but not associated clinical features, such as intrauterine growth restriction (IUGR), presented a distinct DNA methylation profile. CpG sites from four genes relevant to pre-eclampsia (INHBA, BHLHE40, SLC2A1 and ADAM12) showed different extent of changes in LOPET and nIUGR. Genome-wide expression in a subset of samples showed that some of the gene expression changes were negatively correlated with DNA methylation changes, particularly for genes that are responsible for angiogenesis (such as EPAS1 and FLT1). Results could be confounded by altered cell populations in abnormal placentas. Larger sample sizes are needed to fully address the possibility of sub-profiles of methylation within the EOPET cohort. Based on DNA methylation profiling, we conclude that there are widespread DNA methylation alterations in EOPET that may be associated with changes in placental function. This property may provide a useful tool for early screening of such placentas. This study identifies DNA methylation changes at many loci previously reported to have altered gene expression in EOPET placentas, as well as in novel biologically relevant genes we confirmed to be differentially expressed. These results may be useful for DNA- methylation-based non-invasive prenatal diagnosis of at-risk pregnancies.

Early onset pre-eclampsia is associated with altered DNA methylation of cortisol-signalling and steroidogenic genes in the placenta.

Placental cortisol is inactivated in normotensive pregnancies, but is frequently present in pre-eclampsia associated placentae. Since glucocorticoids are strongly associated with the programming of long-term health, we assessed DNA methylation of genes involved in cortisol signalling and bioavailability, and hormonal signalling in the placenta of normotensive and hypertensive pregnancies. Candidate genes/CpG sites were selected through analysis of Illumina Infinium HumanMethylation450 BeadChip array data on control (n = 19) and early onset pre-eclampsia (EOPET; n = 19) placental samples. DNA methylation was further quantified by bisulfite pyrosequencing in a larger cohort of control (n = 111) cases, in addition to EOPET (n = 19), late onset pre-eclampsia (LOPET; n = 18) and normotensive intrauterine growth restriction (nIUGR; n = 13) cases. DNA methylation (percentage points) was increased at CpG sites within genes encoding the glucocorticoid receptor (NR3C1 exon 1D promoter; +8.46%; P<0.01) and corticotropin releasing hormone (CRH) binding protein (CRHBP intron 3; +9.14%; P<0.05), and decreased within CRH (5′ UTR; −4.30%; P = 0.11) in EOPET-associated placentae, but not in LOPET nor nIUGR cases, compared to controls. Differential DNA methylation was not observed among groups at the 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) gene promoter. Significant hypomethylation was observed in pre-eclampsia but not nIUGR placentae for steroidogenic genes, including CYP11A1 (exon1; EOPET; −9.66%; P<0.00001, and LOPET; −5.77%; P<0.001), 3β-hydroxy-delta-5-steroid dehydrogenase type 1 (HSD3B1 exon 2; EOPET; −12.49%; P<0.00001, and LOPET; −6.88%; P<0.001), TEA domain family member 3 (TEAD3 intron 1; EOPET; −12.56%; P<0.00001) and CYP19 (placental-specific exon 1.1 promoter; EOPET; −10.62%, P<0.0001). These data represent dysregulation of the placental epigenome in pre-eclampsia related to genes involved in maintaining the hormonal environment during pregnancy and highlights particular susceptibility in the early onset syndrome.

Hypomethylation of the LEP gene in placenta and elevated maternal leptin concentration in early onset pre-eclampsia

In pre-eclampsia, placental leptin is up-regulated and leptin is elevated in maternal plasma. To investigate potential epigenetic regulation of the leptin (LEP) gene in normal and complicated pregnancy, DNA methylation was assessed at multiple reported regulatory regions in placentae from control pregnancies (n=111), and those complicated by early onset pre-eclampsia (EOPET; arising <34weeks; n=19), late onset pre-eclampsia (LOPET; arising ⩾34weeks; n=18) and normotensive intrauterine growth restriction (nIUGR; n=13). The LEP promoter was hypomethylated in EOPET, but not LOPET or nIUGR placentae, particularly at CpG sites downstream of the transcription start site (−10.1%; P<0.0001). Maternal plasma leptin was elevated in EOPET and LOPET (P<0.05), but not nIUGR, compared with controls. EOPET cases showed a trend towards biallelic LEP expression rather than skewed allelic expression observed in control placentae, suggesting that loss of normal monoallelic expression at the LEP locus is associated with hypomethylation, leading to increased overall LEP expression.

Carboxyhemoglobin levels in umbilical cord blood of women with pre-eclampsia and intrauterine growth restriction

Pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are associated with abnormal placentation. Heme oxygenase (HO) and carbon monoxide (CO) are involved in normal placental development and function and vasomotor control in the placenta. The objective of our study was to measure CO levels, as assessed by carboxyhemoglobin (COHb) levels in the umbilical cord arterial blood of women with PE, normotensive IUGR (<10th percentile for birth weight), and normotensive pregnancies with appropriate-for-gestational age (AGA) infants. We prospectively analyzed COHb levels in the umbilical arterial blood of women with PE, normotensive IUGR, and normotensive AGA pregnancies. Exclusion criteria included cigarette smoke exposure, hemolytic disorders, a positive direct anti-globulin test, chronic hypertension, fever, and any significant medical illness. COHb levels were measured using the ABL 725 blood gas analyzer. There were 41 women in the normotensive AGA group, 42 in the PE group, and 36 in the normotensive IUGR group. Maternal age, mode of delivery, gravidity, parity, and gender of the infants were similar in the three groups. Gestational age and birth weight were significantly higher in the normotensive AGA group compared with the other two groups. COHb levels were significantly lower in the PE group compared with the normotensive AGA group (0.38±0.06% vs. 0.77±0.11%, P<0.05). COHb levels, although lower in the normotensive IUGR group compared with the normotensive AGA group, did not reach statistical significance. Our data suggests the HO-CO system may have a role in the pathogenesis of PE. We also, for the first time, provide information on umbilical arterial COHb levels in normotensive IUGR pregnancies.

OS065. Placental growth factor as a diagnostic and prognostic test forplacental complications of pregnancy

Placental growth factor (PlGF) levels in maternal circulation are altered in pre-eclampsia and intrauterine growth restriction (IUGR) (Benton et al.) and may have utility in identifying cases associated with placental dysfunction and stratifying pregnancy survival. We sought to determine if a positive PlGF test measured on the Triage PlGF rapid assay (Alere, San Diego) agrees with the clinical diagnosis and predicts preterm delivery. EDTA-blood was collected from women admitted to the AMC Obstetrics Department after 20 weeks of gestation with informed consent and according to the protocol approved by the AMC Medical Ethical Board (10/127). Plasma free PlGF levels from women diagnosed with early-onset pre-eclampsia (n=28), normotensive IUGR (N=6) and pregnancy complications excluding pre-eclampsia and IUGR (n=18) were quantified using the Triage PlGF immunoassay. Samples were collected before GA 34+6 and analyzed in batch assay. Results were interpreted against GA-dependent cutoffs set at the 5th centile for gestational age in normal pregnancy (Knudsen et al). PlGF levels below the cutoffs were assigned "positive" according to the product insert. The proportion of subjects with a positive PlGF test result was calculated for each group, together with the proportion of subjects requiring preterm delivery. Twenty-eight women developed early-onset pre-eclampsia and, of these, 27/28 (96.4%) had a positive PlGF test. The woman with negative PlGF test presented to clinic at GA 34+3 with hypertension and suspected pre-eclampsia, but delivered at GA 39+4. Six women developed normotensive IUGR, of which 4 had a positive PlGF test, and in each the PlGF level was below the limit of detection of the test. The 2 women with a negative PlGF test had twin pregnancies. Eighteen women developed pregnancy complications excluding pre-eclampsia and IUGR. Six had at least one serial sample with a positive PlGF test. Of these, one woman had partial placental abruption, 3 had PPROM, one had spontaneous labour at GA 33+3, and one had bleeding after elective embryo reduction and neonatal death during delivery at 23+3. The proportion of women with a positive PlGF test, where the date of delivery is known, requiring preterm delivery was 34/37 (91.9%). These preliminary data suggest that a positive PlGF test by Triage may identify placentally-mediated pregnancy complications and that a very low level of PlGF identifies women at increased risk for preterm delivery.

The neurocognitive outcome of IUGR children born to mothers with and without preeclampsia

Objective: To examine long-term behavioral and neurodevelopmental outcome of children born growth restricted and exposed to hypertension in utero at 9 years of age. Methods: Somatic growth and neurocognitive outcomes were evaluated at age 9–10 years of age in 42 children born with intra uterine growth restriction (IUGR) after pregnancies complicated by hypertensive disorder (17 with maternal preeclampsia and 25 after gestational hypertension). This study group was compared to a control group of 78 IUGR children born after normotensive pregnancy. Results: Only weight was found to be significantly lower in the hypertensive-IUGR group, versus the normotensive IUGR children. No significant differences were found in any of the neurocognitive parameters including IQ, school achievements, and neurodevelopmental score at age 9–10 years. Conclusion: IUGR is a well known risk factor for later cognitive difficulties but maternal hypertensive disorder does not seem to add significantly to this risk.

T13.5 Placental growth factor as a diagnostic for early onset pre-eclampsia and normotensive intrauterine growth restriction of placental origin

T13.6 – Table 2. Comparison table Normal Chronic hypertension Superimposed hypertension Pre-eclampsia Gestational hypertension Normal – P = 0.377 P = 0.031 P< 0.0001 P = 0.002 Chronic hypertension P = 0.377 – P = 0.107 P = 0.003 P = 0.021 Superimposed hypertension P = 0.031 P = 0.107 – P = 0.218 P = 0.673 Pre-eclampsia P< 0.0001 P = 0.003 P = 0.218 – P = 0.201 Gestational hypertension P = 0.002 P = 0.021 P = 0.673 P = 0.201 – sensitivity and specificity increased to 100% and 92%, respectively. Combined, a positive PlGF test was able to diagnosis early onset pre-eclampsia or nIUGR with 94% sensitivity and 93% specificity. Conclusion: This evidence suggests that PlGF can be used to confirm diagnosis of early onset pre-eclampsia and identify nIUGR of placental origin from mimics. Funding: Biosite, CIHR, MSFHR, CFRI T13.6 Urinary placental growth factor differentiates the hypertensive disorders of pregnancy Neil Campbell1, Robert Ogle1, Annemarie Hennessy2, Charlene Thornton1. 1Royal Prince Alfred Hospital, Sydney, Australia; 2University of Western Sydney and Heart Research Institute, Australia Objective: The objective of this study is to establish whether urinary placental growth factor (uPlGF) is capable of discriminating between the different types of hypertensive disease in pregnancy in the third trimester. Design: This is a prospective descriptive case control study. Setting: Royal Prince Alfred Hospital, Urban tertiary referral hospital in Sydney, Australia. The Canterbury Hospital, district general hospital, Sydney, Australia Patients: Women in the third trimester of pregnancy who were currently inpatients on the antenatal ward or patients attending the antenatal outpatients department. Interventions: Patients in the third trimester of pregnancy were selected from the above settings, reviewed for eligibility and demographic data extracted. Patients were then asked to provide a urine specimen some of which was analysed for protein and creatinine and the rest stored and then later analysed for urine PlGF level. These levels after correction for creatinine concentration were then used in the final analysis. Measurements & main results: We have identified 36 women in the hypertensive group (preeclampsia (n=15), Gestational hypertension (n=13), Chronic hypertension (n=8), Preeclampsia superimposed on chronic hypertension (n=5)) and 47 women in the normal control group. There is a statistically significant difference in PlGF levels between the preeclamptic group and the normotensive controls (P<0.0001) and also between the gestational hypertensive group and the normotensive group (P=0.002) Distributions of the preeclamptic group and gestational hypertensive group taken as a whole when compared to the chronic hypertensive group and preeclampsia superimposed on chronic hypertension group also taken as a whole showed a significant difference as well (P=0.007). Table 1. Group characteristics

Subsequent Pregnancy Outcome after First Pregnancy with Normotensive Early-Onset Intrauterine Growth Restriction at

Objective. The aim of this study was to evaluate maternal and fetal outcome of the subsequent pregnancy of primiparous women with a history of early-onset intrauterine growth restriction (IUGR), prompting delivery before 34 weeks of gestation, without concomitant maternal hypertensive disease. Design. Retrospective cohort study. Setting. Tertiary center in the Netherlands. Population. Women with a normotensive first pregnancy complicated by early-onset severe IUGR, prompting delivery before 34 weeks of gestation. Methods. Reproductive follow-up data were recorded for 22 women with a normotensive first pregnancy complicated by early-onset severe IUGR before 34 weeks, referred to the University Medical Centre Utrecht, the Netherlands, between 1993 and 2005. Main Outcome Measures. Main outcome measures were recurrent IUGR, perinatal mortality, preterm delivery, preeclampsia (PE), pregnancy-induced hypertension, and other major obstetric complications, for example placental abruption. Results. Mean gestational age at delivery was 29.4 weeks for the index pregnancy compared to 36.4 weeks for the next pregnancy. IUGR recurred in six pregnancies (27.3%). Four subsequent pregnancies were complicated by hypertensive disorders. Perinatal mortality was 72.7% in the index pregnancy, compared to 13.6% in the second pregnancy. Overall, 11 women (54.5%) had an uneventful pregnancy. Conclusion. Women with first pregnancy early-onset IUGR, without concomitant maternal hypertensive disease, frequently develop severe perinatal complications in their subsequent pregnancy.

An association between cytomegalovirus infection and pre‐eclampsia: a case–control study and data synthesis

Pre-eclampsia shares several similarities with atherosclerotic heart disease. We explored whether, like atherosclerosis, there is a potential link between cytomegalovirus (CMV) infection and pre-eclampsia. CMV IgG, IgM and IgA antibodies were determined by enzyme-linked immunosorbent assays in serums from pre-eclampsia (n = 78), normotensive intrauterine growth restriction (nIUGR) (n = 30) and normal pregnancy controls (n = 109). Data were analyzed by chi-squared, Kruskal-Wallis ANOVA and Mann-Whitney U-tests. Further, we conducted a comprehensive review of published studies on the relation between CMV infection and pre-eclampsia. Risk ratios (RRs) and 95% confidence interval (CI), according to CMV infection status, were calculated using Review Manager. Women with pre-eclampsia had increased CMV IgG seropositivity compared with nIUGR (p < 0.01) and normal pregnancy controls (p < 0.01). In addition, CMV IgG antibody level was higher in pre-eclampsia than normal pregnancy controls (p < 0.001). No difference was observed in CMV IgM or IgA among study groups. Data synthesis revealed that women with CMV infection were at higher risk in the development of pre-eclampsia, compared with women without CMV infection. Combined results for six studies yielded a RR of 1.5 (95% CI 1.2-1.9). CMV infection seems to affect the occurrence of pre-eclampsia. Evaluation of the relation between CMV infection and pre-eclampsia may provide mechanistic insights into pre-eclampsia-related inflammation.

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

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.

Toll‐like receptors 2 and 4 and the cryopyrin inflammasome in normal pregnancy and pre‐eclampsia

Pre-eclampsia involves a maternal inflammatory response that differs from both normal pregnancy and normotensive intrauterine growth restriction (IUGR). Our objective was to examine neutrophil Toll-like receptor (TLR), cryopyrin, nuclear factor-kappaB (NF-kappaB) subunit and interleukin-1beta (IL-1beta), and inflammatory cytokine profiles in women with pre-eclampsia or normotensive IUGR, as well as in normal pregnancy and non-pregnancy controls. A case-control study was performed. We examined the messenger RNA (mRNA) and protein expressions of TLR4 and TLR2, mRNA levels of cryopyrin, IL-1beta, NF-kappaB subunits p50 and p65, as well as maternal serum inflammatory cytokine profiles (IL-2, IL-6, tumour necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma] and IL-10) in women with and without pre-eclampsia using real-time reverse transcription polymerase chain reactions, flow cytometry and multiplex immunoassays. A single tertiary maternity hospital in Vancouver, Canada. Women with early-onset pre-eclampsia (<34 weeks of gestation, n = 25), women with late-onset pre-eclampsia (>or=34(+0) weeks of gestation, n = 25), women with normotensive IUGR (n = 25), women with normal pregnancy (n = 75) and non-pregnancy (n = 25) controls. Women with pre-eclampsia (as a single combined group of early- and late-onset, and particularly in women with early-onset pre-eclampsia) had increased TLR2 and TLR4 mRNA and protein expressions elevated cryopyrin, NF-kappaB subunit, and IL-1beta mRNA expression, and TNF-alpha:IL-10 and IL-6:IL-10 ratios compared with other groups. These data suggest that TLRs and cryopyrin may modulate the innate immune response of the maternal syndrome of pre-eclampsia, and might also trigger the differential inflammatory response existing between early onset pre-eclampsia and normotensive IUGR.