Ulrik Dolberg Anderson

Review: Biochemical markers to predict preeclampsia

Worldwide the prevalence of preeclampsia (PE) ranges from 3 to 8% of pregnancies. 8.5 million cases are reported yearly, but this is probably an underestimate due to the lack of proper diagnosis. PE is the most common cause of fetal and maternal death and yet no specific treatment is available. Reliable biochemical markers for prediction and diagnosis of PE would have a great impact on maternal health and several have been suggested. This review describes PE biochemical markers in general and first trimester PE biochemical markers specifically. The main categories described are angiogenic/anti-angiogenic factors, placental proteins, free fetal hemoglobin (HbF), kidney markers, ultrasound and maternal risk factors. The specific biochemical markers discussed are: PAPP-A, s-Flt-1/PlGF, s-Endoglin, PP13, cystatin-C, HbF, and α₁-microglobulin (A1M). PAPP-A and HbF both show potential as predictive biochemical markers in the first trimester with 70% sensitivity at 95% specificity. However, PAPP-A is not PE-specific and needs to be combined with Doppler ultrasound to obtain the same sensitivity as HbF/A1M. Soluble Flt -1 and PlGF are promising biochemical markers that together show high sensitivity from the mid-second trimester. PlGF is somewhat useful from the end of the first trimester. Screening pregnant women with biochemical markers for PE can reduce unnecessary suffering and health care costs by early detection of mothers at increased risk for PE, thus avoiding unnecessary hospitalization of pregnant women with suspect or mild PE and enabling monitoring of the progression of the disease thereby optimizing time for delivery and hopefully reducing the number of premature births.

Fetal hemoglobin and α1-microglobulin as first- and early second-trimester predictive biomarkers for preeclampsia

OBJECTIVE The aim of this study was to evaluate fetal hemoglobin (HbF) and α(1)-microglobulin (A1M) in maternal serum as first-trimester biomarkers for preeclampsia (PE). STUDY DESIGN The design was a case-control study. We included 96 patients in the first trimester of pregnancy (60 with PE and 36 controls). Venous serum samples were analyzed for HbF and total hemoglobin (Hb) by enzyme-linked immunosorbent assay and for A1M by radioimmunoassay. Sensitivity and specificity was calculated by logistic regression and receiver operating characteristic curve analysis. RESULTS The HbF/Hb ratio and A1M concentration were significantly elevated in serum from women with subsequent development of PE (P < .0001). The optimal sensitivity and specificity was obtained using the biomarkers in combination; 69% sensitivity for a 5% screen positive rate and 90% sensitivity for a 23% screen positive rate. CONCLUSION The study suggests that HbF/Hb ratio in combination with A1M is predictive biomarkers for PE.

The Human Endogenous Protection System against Cell-Free Hemoglobin and Heme Is Overwhelmed in Preeclampsia and Provides Potential Biomarkers and Clinical Indicators

Preeclampsia (PE) complicates 3–8% of all pregnancies and manifests clinically as hypertension and proteinuria in the second half of gestation. The pathogenesis of PE is not fully understood but recent studies have described the involvement of cell-free fetal hemoglobin (HbF). Hypothesizing that PE is associated with prolonged hemolysis we have studied the response of the cell-free Hb- and heme defense network. Thus, we have investigated the levels of cell-free HbF (both free, denoted HbF, and in complex with Hp, denoted Hp-HbF) as well as the major human endogenous Hb- and heme-scavenging systems: haptoglobin (Hp), hemopexin (Hpx), α1-microglobulin (A1M) and CD163 in plasma of PE women (n = 98) and women with normal pregnancies (n = 47) at term. A significant increase of the mean plasma HbF concentration was observed in women with PE. Plasma levels of Hp and Hpx were statistically significantly reduced, whereas the level of the extravascular heme- and radical scavenger A1M was significantly increased in plasma of women with PE. The Hpx levels significantly correlated with maternal blood pressure. Furthermore, HbF and the related scavenger proteins displayed a potential to be used as clinical biomarkers for more precise diagnosis of PE and are candidates as predictors of identifying pregnancies with increased risk of obstetrical complications. The results support that PE pathophysiology is associated with increased HbF-concentrations and an activation of the physiological Hb-heme defense systems.

Fetal hemoglobin, α1-microglobulin and hemopexin are potential predictive first trimester biomarkers for preeclampsia

OBJECTIVE Overproduction of cell-free fetal hemoglobin (HbF) in the preeclamptic placenta has been recently implicated as a new etiological factor of preeclampsia. In this study, maternal serum levels of HbF and the endogenous hemoglobin/heme scavenging systems were evaluated as predictive biomarkers for preeclampsia in combination with uterine artery Doppler ultrasound. STUDY DESIGN Case-control study including 433 women in early pregnancy (mean 13.7weeks of gestation) of which 86 subsequently developed preeclampsia. The serum concentrations of HbF, total cell-free hemoglobin, hemopexin, haptoglobin and α1-microglobulin were measured in maternal serum. All patients were examined with uterine artery Doppler ultrasound. Logistic regression models were developed, which included the biomarkers, ultrasound indices, and maternal risk factors. RESULTS There were significantly higher serum concentrations of HbF and α1-microglobulin and significantly lower serum concentrations of hemopexin in patients who later developed preeclampsia. The uterine artery Doppler ultrasound results showed significantly higher pulsatility index values in the preeclampsia group. The optimal prediction model was obtained by combining HbF, α1-microglobulin and hemopexin in combination with the maternal characteristics parity, diabetes and pre-pregnancy hypertension. The optimal sensitivity for all preeclampsia was 60% at 95% specificity. CONCLUSIONS Overproduction of placentally derived HbF and depletion of hemoglobin/heme scavenging mechanisms are involved in the pathogenesis of preeclampsia. The combination of HbF and α1-microglobulin and/or hemopexin may serve as a prediction model for preeclampsia in combination with maternal risk factors and/or uterine artery Doppler ultrasound.

Urinary Extracellular Vesicles of Podocyte Origin and Renal Injury in Preeclampsia

Renal histologic expression of the podocyte-specific protein, nephrin, but not podocin, is reduced in preeclamptic compared with normotensive pregnancies. We hypothesized that renal expression of podocyte-specific proteins would be reflected in urinary extracellular vesicles (EVs) of podocyte origin and accompanied by increased urinary soluble nephrin levels (nephrinuria) in preeclampsia. We further postulated that podocyte injury and attendant formation of EVs are related mechanistically to cellfree fetal hemoglobin (HbF) in maternal plasma. Our study population included preeclamptic (n=49) and normotensive (n=42) pregnant women recruited at delivery. Plasma measurements included HbF concentrations and concentrations of the endogenous chelators haptoglobin, hemopexin, and α1- microglobulin. We assessed concentrations of urinary EVs containing immunologically detectable podocyte-specific proteins by digital flow cytometry and measured nephrinuria by ELISA. The mechanistic role of HbF in podocyte injury was studied in pregnant rabbits. Compared with urine from women with normotensive pregnancies, urine from women with preeclamptic pregnancies contained a high ratio of podocin-positive to nephrin-positive urinary EVs (podocin+ EVs-to-nephrin+ EVs ratio) and increased nephrinuria, both of which correlated with proteinuria. Plasma levels of hemopexin, which were decreased in women with preeclampsia, negatively correlated with proteinuria, urinary podocin+ EVs-to-nephrin+ EVs ratio, and nephrinuria. Administration of HbF to pregnant rabbits increased the number of urinary EVs of podocyte origin. These findings provide evidence that urinary EVs are reflective of preeclampsia-related altered podocyte protein expression. Furthermore, renal injury in preeclampsia associated with an elevated urinary podocin+ EVs-to-nephrin+ EVs ratio and may be mediated by prolonged exposure to cellfree HbF.

The hemoglobin degradation pathway in patients with preeclampsia - Fetal hemoglobin, heme, heme oxygenase-1 and hemopexin - Potential diagnostic biomarkers?

OBJECTIVE The aim of this study was to investigate how maternal cell-free fetal hemoglobin and heme impact the scavenger enzyme systems Hemopexin and Heme Oxygenase-1 in patients with preeclampsia (PE). The secondary aims were to evaluate these proteins as biomarkers for severity of the clinical manifestation i.e. hypertension, in early- and late onset PE. MATERIAL AND METHODS Plasma samples taken within the last 24 h before delivery from 135 patients were analyzed, 89 PE and 46 normal pregnancies. All samples were analyzed for cell-free fetal hemoglobin (HbF), heme, hemopexin enzymatic activity (Hx activity), hemopexin concentration (Hx), and heme oxygenase 1 concentration (HO-1). Logistic regression analysis with ROC-curve analysis was performed to evaluate the possible use as biomarkers for preeclampsia. RESULTS There were significantly higher levels of HbF (p = 0.01) and heme (0.01) but significantly lower Hx activity (p = 0.02), Hx (p < 0.0001) and HO-1 (p = 0.03) in PE plasma as compared to plasma of normal pregnancies. The Hx activity was significantly inversely correlated (p = 0.04) to the diastolic blood pressure. The HO-1 concentration was significantly inversely correlated to both the systolic and diastolic blood pressure (p = 0.01 and p = 0.003). ROC-curve analysis showed a combined detection rate for these biomarkers of 84% at 10% false positive rate. CONCLUSIONS Increased maternal plasma levels of heme and HbF in PE are associated with decreased HO-1 and hemopexin protein levels as well as reduced hemopexin activity. By measuring the consumption of the scavenger protein Hx, and the proteins in the Hb degradation system, clinical information about the dynamics of the disease can be obtained.

PP006. Gene expression profiling of first trimester placentas from pregnancies at high risk of developing preeclampsia.

INTRODUCTION Preeclampsia (PE) is an important cause of fetal and maternal morbidity and mortality. It is considered a two-stage disease, the first stage characterized by a defect placentation and the second stage by maternal manifestations. Details of the patho-physiology behind the transition from stage one to stage two remain unclear. OBJECTIVE Was to study first trimester placental gene expression in patients identified as high risk for PE by either Doppler ultrasound or the biochemical markers cell free fetal hemoglobin (HbF) and alpha-1-microglobulin (A1M). METHODS Placental samples were obtained from seven women at highrisk of PE as determined by Doppler ultrasound of the uterine arteries and eight women with normal uterine artery resistance who for other reasons terminated their pregnancies surgically. Maternal serum samples were analyzed for HbF and A1M. The patients were risk stratified according to two risk classifications: (I) High vs. low uterine artery resistance and (II) High HbF and A1M vs. low HbF and A1M. Total RNA from the placentas was used for whole genome microarray. The results were analyzed by bioinformatics and genes of interest confirmed with qPCR. RESULTS A total of 453 and 332 significantly altered genes were identified in the two study groups. Bioinformatics revealed 12 genes of interest in study group I and 7 genes of interest in study group II. CONCLUSIONS Genes related to vascular tonus regulation and inflammatory response were identified in study group I suggesting that a lack of tonus regulation and increased inflammation might contribute to the high uterine artery resistance seen in this group. Genes related to regulation of hematopoiesis was found in group II suggesting dysfunctional hematopoiesis as a factor explaining the high levels of cell-free HbF seen.

P12. Placental gene expression analysis at the end of the first trimester of pregnancy in patients at high risk of subsequent development of preeclampsia

ments using the dual placental perfusion system. Perfusions were performed with medium only and with free Hb. Maternal perfusate was collected at four (4) time points during the dual perfusion and used for analysis of MPs from both controls and Hb perfusions. MPs were isolated using ultra-centrifugation at 10,000g and 150,000g, resulting in two pellets with different sized MPs referred to as 10 K ( 0.3 lm– 1.1 lm) and 150 K ( 0.3–0.6lm) MPs. Their protein concentration was determined spectrophotometrically using a Nanodrop. Small RNAs were extracted from the MPs and miRNA expression was determined using a miRNA specific real-time quantitative reverse transcriptase-polymerase chain reaction assay for nine selected miRNAs. Results: Placentas perfused with haemoglobin showed an increase in perfusion pressure as well as morphological changes, similar to what is seen in PE. The miRNAs; mir517b, mir-517a, mir-141 and mir-222 showed significantly decreased expression in MPs from Hb perfused placentas compared to controls. There was no difference in protein concentration in MPs from Hb and controls. The amount of total RNA per MP was higher in the 150 K compared to the 10 K MPs. Conclusion: Free Hb causes oxidative stress and PE like damage to the placenta. Our results show that the placenta specific mir-517b and mir-517a are decreased in MPs after perfusion with free Hb. Mir-517a has previously been found in exosomes from trophoblasts. We also showed that mir141 and mir-222 are down-regulated after perfusion with free Hb. Interestingly, decreased levels of mir-222 expression have been shown to increase synthesis of HbF, which would suggest mir-222 to be part of a possible mechanism behind the increased levels of HbF seen in PE. Future experiments need to focus on the relationship between miRNA expression in placental tissue and MPs in the different perfusion conditions. First Author <35 years: Yes.