Shaun P. Brennecke

Predictive Value of the sFlt-1:PlGF Ratio in Women with Suspected Preeclampsia

BACKGROUND The ratio of soluble fms-like tyrosine kinase 1 (sFlt-1) to placental growth factor (PlGF) is elevated in pregnant women before the clinical onset of preeclampsia, but its predictive value in women with suspected preeclampsia is unclear. METHODS We performed a prospective, multicenter, observational study to derive and validate a ratio of serum sFlt-1 to PlGF that would be predictive of the absence or presence of preeclampsia in the short term in women with singleton pregnancies in whom preeclampsia was suspected (24 weeks 0 days to 36 weeks 6 days of gestation). Primary objectives were to assess whether low sFlt-1:PlGF ratios (at or below a derived cutoff) predict the absence of preeclampsia within 1 week after the first visit and whether high ratios (above the cutoff) predict the presence of preeclampsia within 4 weeks. RESULTS In the development cohort (500 women), we identified an sFlt-1:PlGF ratio cutoff of 38 as having important predictive value. In a subsequent validation study among an additional 550 women, an sFlt-1:PlGF ratio of 38 or lower had a negative predictive value (i.e., no preeclampsia in the subsequent week) of 99.3% (95% confidence interval [CI], 97.9 to 99.9), with 80.0% sensitivity (95% CI, 51.9 to 95.7) and 78.3% specificity (95% CI, 74.6 to 81.7). The positive predictive value of an sFlt-1:PlGF ratio above 38 for a diagnosis of preeclampsia within 4 weeks was 36.7% (95% CI, 28.4 to 45.7), with 66.2% sensitivity (95% CI, 54.0 to 77.0) and 83.1% specificity (95% CI, 79.4 to 86.3). CONCLUSIONS An sFlt-1:PlGF ratio of 38 or lower can be used to predict the short-term absence of preeclampsia in women in whom the syndrome is suspected clinically. (Funded by Roche Diagnostics.).

Family history of pre‐eclampsia as a predictor for pre‐eclampsia in primigravidas

Objective: To assess the clinical utility of knowledge of a family history of pre‐eclampsia as a predictor for the development of pre‐eclampsia in primigravid women. Methods: 368 primigravid women were prospectively recruited from the outpatients department of an obstetric teaching hospital. Details of any family history of pre‐eclampsia were obtained from the women and their subsequent obstetric outcomes were observed. Results: Of 368 primigravid women, 34 (9.2%) developed pre‐eclampsia. Eighteen (4.9%) women of the total group stated that they had a mother (12), sister (five) or both (one) who had had pre‐eclampsia. Of these 18 women, five (27.8%) developed pre‐eclampsia. Of the women who had no family history, 29 (8.3%) developed pre‐eclampsia (relative risk, RR = 3.4; 95% CI, 1.5–7.6; P = 0.018). Four (22.2%) of the women with a positive family history developed severe pre‐eclampsia compared to 18 (5.1%) with a negative family history (RR = 4.3; 95% CI, 1.6‐11.5; P = 0.017). Conclusions: In a primigravida, a family history of pre‐eclampsia is associated with a fourfold increased risk of severe pre‐eclampsia. This clinical history identifies a group who warrant close clinical surveillance during pregnancy and who may be suitable for trials of prophylactic interventions.

GENETICS OF PRE-ECLAMPSIA

Pre-eclampsia is a serious complication of the second half of human pregnancy which occurs at frequencies of 1 % to 5% in most parts of the world. It is characterized clinically by high blood pressure, proteinuria, and generalized edema in association with a wide range of pathophysiological organ and system disturbances. Untreated, it can lead to the occurrence of epileptic-like grand-mal convulsions (eclampsia), which is a source of considerable maternal and fetal morbidity. The disease is unusual in having no known cause, although an immunological impairment is suspected. Here we review the evidence that the condition is genetic in origin. Several genetic models are discussed. Markedly raised incidences are seen in blood relatives (mothers, daughters, sisters, and granddaughters) but not in relatives by marriage (daughters-in-law, mothers-in-law). This suggests that the condition is caused by maternal genes. Other evidence, however, implicates the fetal genotype. The most decisive data supporting this s...

GAPDH, 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restriction.

Comparative gene expression studies in the placenta may provide insights into molecular mechanisms of important genomic alterations in pregnancy disorders. Endogenous reference genes often referred to as housekeeping genes, are routinely used to normalise gene expression levels. For this reason, it is important that these genes be empirically evaluated for stability between placental samples including samples from complicated pregnancies. To address this issue, six candidate housekeeping genes including several commonly used ones (ACTB, GAPDH, 18S rRNA, TBP, SDHA and YWHAZ) were investigated for their expression stability in placentae obtained from pregnancies complicated by idiopathic FGR (n=25) and gestation-matched control pregnancies (n=25). Real-time PCR was performed using pre-validated gene expression assay kits. The geNorm program was used for gene stability measure (M) for the entire housekeeping genes in all control and FGR-affected placental samples. Results showed that GAPDH and 18S rRNA were most stable, with an average expression stability of M=0.441 and 0.443, respectively, followed by YWHAZ (M=0.472). SDHA, ACTB and TBP were the least stable housekeeping genes (M=0.495, 0.548 and 1.737, respectively). We recommend geometric averaging of two or more housekeeping genes to determine relative gene expression levels between FGR-affected and control placentae.

Inherited thrombophilia polymorphisms and pregnancy outcomes in Nulliparous women

OBJECTIVE: To estimate the association between five commonly inherited thrombophilia polymorphisms and adverse pregnancy outcomes in women who had no prior history of adverse pregnancy outcomes or personal or family history of venous thromboembolism. METHODS: Healthy nulliparous women (n=2,034) were recruited to this prospective cohort study before 22 weeks of gestation. Genotyping for factor V Leiden, prothrombin gene mutation, methylenetetrahydrofolate reductase enzyme (MTHFR) C677T, MTHFR A1298C, and thrombomodulin polymorphism was performed. Clinicians caring for women were blinded to the results of thrombophilia tests. The primary composite outcome was the development of severe preeclampsia, fetal growth restriction, placental abruption, stillbirth, or neonatal death. RESULTS: Complete molecular results and pregnancy outcome data were available in 1,707 women. These complications were experienced by 136 women (8.0%). Multivariable logistic regression demonstrated two statistically significant findings. Women who carried the prothrombin gene mutation had an odds ratio of 3.58 (95% confidence interval [CI] 1.20–10.61, P=.02) for the development of the composite primary outcome. Homozygous carriers of the MTHFR 1298 polymorphism had an odds ratio of 0.26 (95% CI 0.08–0.86, P=.03). None of the other polymorphisms studied showed a significant association with the development of the primary outcome in this cohort of women. CONCLUSION: Prothrombin gene mutation confers an increased risk for the development of adverse pregnancy outcomes in otherwise asymptomatic, nulliparous women, whereas homozygosity for MTHFR 1298 may protect against these complications. The majority of asymptomatic women who carry an inherited thrombophilia polymorphism have a successful pregnancy outcome. LEVEL OF EVIDENCE: II

The ABC transporter BCRP/ABCG2 is a placental survival factor, and its expression is reduced in idiopathic human fetal growth restriction

The efflux pump ATP binding cassette superfamily member G2 (ABCG2)/breast cancer resistance protein (BCRP) is highly expressed in human placenta. We have investigated the role of BCRP in the protection of the human placental trophoblasts from apoptosis and its expression in idiopathic fetal growth restriction, a condition associated with abnormal pla‐cental apoptosis. Inhibition of BCRP activity with the selective inhibitor Ko143 augmented cytokine (tumor necrosis factor‐α/interferon‐γ)‐induced apoptosis and phosphatidylserine externalization in primary tropho‐blast and trophoblast‐like BeWo cells. Silencing of BCRP expression in BeWo cells significantly increased their sensitivity to apoptotic injury in response to cytokines and exogenous C6 and C8 ceramides. BCRP silencing also increased intracellular ceramide levels after cytokine exposure but did not affect cellular protoporphyrin IX concentrations or sensitivity to activators of the intrinsic apoptotic pathway. BCRP expression in placentas from pregnancies complicated by idiopathic fetal growth restriction was decreased compared with controls, suggesting reduced transport of its substrates from the placenta. We conclude that BCRP may play a hitherto unrecognized survival role in the placenta, protecting the trophoblast against cytokine‐induced apoptosis and possibly other extrinsic activators via modulation of ceramide signaling. Decreased placental BCRP expression may result in reduced viability and hence functional deficit, contributing to the fetal growth restriction phenotype.—Evseenko, D. A., Murthi, P., Paxton, J. W., Reid, G., Emerald, B. S., Mohankumar, K. M., Lobie, P. E., Brennecke, S. P., Kalionis, B. Keelan, J. A. The ABC transporter BCRP/ ABCG2 is a placental survival factor, and its expression is reduced in idiopathic human fetal growth restriction. FASEB J. 21, 3592–3605 (2007)

Contractile activity, membrane potential, and cytoplasmic calcium in human uterine smooth muscle in the third trimester of pregnancy and during labor

OBJECTIVE This study was undertaken to investigate in human tissue samples the mechanisms underlying spontaneous and prostaglandin F(2)(alpha)-induced contractions during the final trimester of pregnancy and labor. STUDY DESIGN Membrane potential and cytoplasmic calcium were recorded simultaneously with contraction in uterine strips obtained from the lower segment during cesarean delivery. RESULTS Between week 28 of gestation and term there was a progressive increase in the frequency of spontaneous contractions and a decrease in the negative potential of the membrane. The response to prostaglandin F(2alpha) was biphasic. The initial excitatory component remained stable toward term. A later inhibitory component, which was underpinned by increased activity of the sodium-potassium adenosine triphosphatase pump, decreased at the time of labor. CONCLUSIONS There is a gradual increase in excitability in uterine muscle throughout the third trimester of human pregnancy. The initial component of the prostaglandin response is a large contraction that is kept brief by a subsequent inhibitory component of the response, which ensures that full relaxation occurs between contractions.

Implementation of the sFlt-1/PlGF ratio for prediction and diagnosis of pre-eclampsia in singleton pregnancy: implications for clinical practice

Pre-eclampsia (PE) is a leading cause of maternal and fetal/neonatal morbidity and mortality worldwide. Clinical diagnosis and definition of PE is commonly based on the measurement of non-specific signs and symptoms, principally hypertension and proteinuria1–3. However, due to the recognition that measurement of proteinuria is prone to inaccuracies and the fact that PE complications often occur before proteinuria becomes significant, most recent guidelines also support the diagnosis of PE on the basis of hypertension and signs of maternal organ dysfunction other than proteinuria3–5. Furthermore, the clinical presentation and course of PE is variable, ranging from severe and rapidly progressing early-onset PE, necessitating preterm delivery, to late-onset PE at term. There may be associated intrauterine growth restriction (IUGR), further increasing neonatal morbidity and mortality. These features suggest that the classical standards for the diagnosis of PE are not sufficient to encompass the complexity of the syndrome. Undoubtedly, proper management of pregnant women at high risk for PE necessitates early and reliable detection and intensified monitoring, with referral to specialized perinatal care centers, to reduce substantially maternal, fetal and neonatal morbidity6,7. In the decade since Maynard et al.8 reported that excessive placental production of soluble fms-like tyrosine kinase receptor-1 (sFlt-1), an antagonist of vascular endothelial growth factor and placental growth factor (PlGF), contributes to the pathogenesis of PE, extensive research has been published demonstrating the usefulness of angiogenic markers in both diagnosis and the subsequent prediction and management of PE and placenta-related disorders. Various reports have demonstrated that disturbances in angiogenic and antiangiogenic factors are implicated in the pathogenesis of PE and have possible relevance in the diagnosis and prognosis of the disease. Increased serum levels of sFlt-1 and decreased levels of PlGF, thereby resulting in an increased sFlt-1/PlGF ratio, can be detected in the second half of pregnancy in women diagnosed to have not only PE but also IUGR or stillbirth, i.e. placenta-related disorders. These alterations are more pronounced in early-onset rather than late-onset disease and are associated with severity of the clinical disorder. Moreover, the disturbances in angiogenic factors are reported to be detectable prior to the onset of clinical symptoms (disease), thereby allowing discrimination of women with normal pregnancies from those at high risk for developing pregnancy complications, primarily PE9–30. Plasma concentrations of angiogenic/antiangiogenic factors are of prognostic value in obstetric triage: similar to the progressively worsening clinical course observed in women with early-onset PE, changes in the angiogenic profile leading to a more antiangiogenic state can be found. Current definitions of PE are poor in predicting PE-related adverse outcomes. A diagnosis of PE based on blood pressure and proteinuria has a positive predictive value of approximately 30% for predicting PE-related adverse outcomes31. Estimation of the sFlt-1/PlGF ratio allows identification of women at high risk for imminent delivery and adverse maternal and neonatal outcome23,30,32–35. Moreover, it has also been shown that the time-dependent slope of the sFlt-1/PlGF ratio between repeated measurements is predictive for pregnancy outcome and the risk of developing PE, and repeated measurements have been suggested36. However, the ‘optimal’ time interval for a follow-up test remains unclear. Finally, high values are closely related to the need to deliver immediately22,23,37. Additionally, in normal and complicated pregnancies, angiogenic factors are correlated with Doppler ultrasound parameters, mainly uterine artery (UtA) indices38–42. Combining the sFlt-1/PlGF ratio with UtA Doppler ultrasound, at the time of diagnosis of early-onset PE, has prognostic value mainly for perinatal complications, being limited for the prediction of maternal complications37,43. The additional measurement of the sFlt-1/PlGF ratio has been shown to improve the sensitivity and specificity of Doppler measurement in predicting PE44–48, supporting its implementation in screening algorithms. Whereas studies on the predictive efficacy of the sFlt-1/PlGF ratio in the first trimester have yielded contradictory results49, reports on the use of this marker as an aid in prediction from the mid trimester onwards have led to its suggested use as a screening tool, especially for identifying all women developing PE and requiring delivery within the subsequent 4 weeks50–52. This short review of the literature highlights that measurement of the sFlt-1/PlGF ratio has the potential to become an additional tool in the management of PE, particularly as automated tests that allow rapid and easy measurement of these markers are now widely available. Nevertheless, although these markers were incorporated recently into the German guidelines53, no formal recommendation regarding how to use sFlt-1, PlGF or the sFlt-1/PlGF ratio has been established in any official protocol. The purpose of this paper is to answer questions that are frequently asked around the use of the sFlt-1/PlGF ratio in the diagnosis and prediction of PE and regarding the implications for clinical practice, in particular, ‘When?’ and ‘In which women?’ should the sFlt-1/PlGF ratio be measured and, ‘What should be done with the results?’, and to provide guidance to educate physicians on the use of the sFlt-1/PlGF ratio in clinical practice. To achieve this, international experts in the use of angiogenic markers have strived to develop a consensus statement on the clinical use of the sFlt-1/PlGF ratio and the consequential management in pregnant women with suspected PE or at a high risk of developing PE.

Genome-Wide Association Scan Identifies a Risk Locus for Preeclampsia on 2q14, Near the Inhibin, Beta B Gene

Elucidating the genetic architecture of preeclampsia is a major goal in obstetric medicine. We have performed a genome-wide association study (GWAS) for preeclampsia in unrelated Australian individuals of Caucasian ancestry using the Illumina OmniExpress-12 BeadChip to successfully genotype 648,175 SNPs in 538 preeclampsia cases and 540 normal pregnancy controls. Two SNP associations (rs7579169, p = 3.58×10−7, OR = 1.57; rs12711941, p = 4.26×10−7, OR = 1.56) satisfied our genome-wide significance threshold (modified Bonferroni p<5.11×10−7). These SNPs reside in an intergenic region less than 15 kb downstream from the 3′ terminus of the Inhibin, beta B (INHBB) gene on 2q14.2. They are in linkage disequilibrium (LD) with each other (r2 = 0.92), but not (r2<0.80) with any other genotyped SNP ±250 kb. DNA re-sequencing in and around the INHBB structural gene identified an additional 25 variants. Of the 21 variants that we successfully genotyped back in the case-control cohort the most significant association observed was for a third intergenic SNP (rs7576192, p = 1.48×10−7, OR = 1.59) in strong LD with the two significant GWAS SNPs (r2>0.92). We attempted to provide evidence of a putative regulatory role for these SNPs using bioinformatic analyses and found that they all reside within regions of low sequence conservation and/or low complexity, suggesting functional importance is low. We also explored the mRNA expression in decidua of genes ±500 kb of INHBB and found a nominally significant correlation between a transcript encoded by the EPB41L5 gene, ∼250 kb centromeric to INHBB, and preeclampsia (p = 0.03). We were unable to replicate the associations shown by the significant GWAS SNPs in case-control cohorts from Norway and Finland, leading us to conclude that it is more likely that these SNPs are in LD with as yet unidentified causal variant(s).

HLA‐G deletion polymorphism and pre‐eclampsia/eclampsia

Objective To investigate a HLA‐G deletion polymorphism in pre‐eclamptic pedigrees and the general population.