New predictive and diagnostic biomarkers for preeclampsia Ulrik Dolberg Anderson, Överläkare Kvinnokliniken Skånes UniversitetsSjukhus Malmö/Lund Jag har inget jäv att deklarera

Outline Ultra-short introduction to preeclampsia The HbF hypothesis Aims Papers I – V Conclusions Questions

Introduction to preeclampsia – a syndrome Symptoms Blurry vision Head ache Epigastric pain BP≥140/90 Preeclampsia Clinical signs Edema Hyper-reflexia PE as a syndrome The definition is changing – ACOG, ISSHP Lab findings Urate, creatinine, cystatin-c, thrombocytes, transaminases, albumin Significant proteinuria

Preeclampsia the two-stage model Genetic factors Environmental Factors Immunological factors Stage one First- and second trimester Impaired placentation – shallow invasion of maternal spiral arteries Uneven placental blood flow Placental oxidative stress – production and release of placental factors Maternal endotheliosis and glomerulo-endotheliosis Stage two After 20 weeks of gestation Maternal symptoms – Hypertension and proteinuria

Preeclampsia the two-stage model Powe et al Circulation 2011

Building the HbF hypothesis Gene analysis of term placentas. Up-regulation of the genes coding for fetal hemoglobin in PE placentas. Elevated levels of cell-free fetal hemoglobin(HbF) in placentas from women with preeclampsia. Centlow et al. Fertility and sterility, Nov. 2008

The HbF hypothesis – hemoglobin toxicity heme hemolysis Fe3+ auto-oxidation O2- radicals oxidation cell- and matrix damage inflammation vaso-spasm (NO scavenger)

Extracellular compartment Haptoglobin Hemoglobin Heme α1microglobulin Hemopexin CD163 receptor CD91 / LRP1 receptor Macrophage /hepatocyte cell membrane Macrophage cell membrane CO Hemoglobin Free iron Biliverdin HO-1 Lysosome Intracellular compartment Heme

The hemoglobin and heme scavenging systems Cell-free hemoglobin Haptoglobin Heme A1M Hemopexin Heme oxygenase 1

General aims of the thesis To study the role of cell-free fetal hemoglobin (HbF) and the specific scavenger systems for hemoglobin and heme in patients with preeclampsia. To study these proteins as predictive and diagnostic biomarkers of preeclampsia and to study HbF’s relation to renal injury.

Papers Paper I: Prediction study Paper II: Larger scale prediction study Paper III: Diagnosis - Late pregnancy Paper IV: Diagnosis -Late pregnancy Paper V: HbF/heme induced renal damages

Paper I – Predicting preeclampsia Anderson et. al 2011

Paper I – Study design First trimester 96 serum samples 60 preeclampsia 36 controls HbF, total Hb and A1M were measured blinded to groups Logistic regression analysis with ROC curves

Paper I – Results Serum concentrations

Paper I - Results AUC: HbF-ratio: 0.82 A1M: 0.75 Combination: 0.89

Paper I - Conclusions The HbF and A1M concentrations are significantly elevated in maternal serum from women who subsequently develop preeclampsia already at the end of the first trimester of pregnancy. HbF (HbF-ratio) in combination with A1M are potential first and second trimester biochemical markers of preeclampsia.

First trimester of pregnancy HbF Haptoglobin Heme Hemopexin A1M

Paper II Anderson et. al 2016

Paper II – Study design First trimester 433 serum samples 86 preeclampsia 347 controls HbF, total Hb, A1M, haptoglobin and hemopexin were measured blinded to groups All patients had uterine artery Doppler ultrasound Maternal risk factor characteristics were retrieved from patients charts Logistic regression analysis with ROC curves

Paper II - results HbF A1M Hemopexin Uterina PI p=0.02 p=0.03 p=0.05

Paper II – logistic regression and ROC-curve analyses Prediction model AUC 5 % false positive rate 10 % false positive rate Positive predictive value Negative predictive value Biomarkers + maternal risk factor characteristics 0.83 (0.75-0.91) 60% 62% 75% 91% Läg til ROC-curve

Paper II – Conclusions HbF and A1M concentrations are elevated and hemopexin is decreased in maternal serum. Potential predictive biomarkers for subsequent development of both early- and late-onset preeclampsia. Combining with uterine artery Doppler ultrasound and/or maternal characteristics, increases the sensitivity and specificity of prediction.

First trimester of pregnancy HbF Haptoglobin Heme A1M Hemopexin

Paper III Gram et. al 2015

Paper III – Study design 145 plasma samples 98 preeclampsia 47 controls HbF, total Hb, A1M, haptoglobin, hemopexin and CD 163 were measured blinded to groups Logistic regression analyses with ROC curves Subgroup analysis for early and late onset preeclampsia Correlation analysis to clinical severity parameters – blood pressure

Paper III - results HbF A1M Hemopexin Haptoglobin HbF-haptoglobin-complex

Paper III - results

Paper III - results

Paper III - results

Paper III - conclusions Increased circulating concentrations of HbF strain and slowly deplete the maternal hemoglobin- and heme scavenging proteins. HbF, A1M, hemopexin and haptoglobin in combination are clinical tools supporting the diagnosis, predicting the severity and specific clinical outcomes.

Third trimester of pregnancy HbF Haptoglobin Heme A1M Hemopexin Heme oxygenase 1

Extracellular compartment Haptoglobin Hemoglobin Heme α1microglobulin Hemopexin CD163 receptor CD91 / LRP1 receptor Macrophage /hepatocyte cell membrane Macrophage cell membrane CO Hemoglobin Free iron Biliverdin HO-1 Lysosome Intracellular compartment Heme

Paper IV Third trimester of pregnancy HbF Haptoglobin Heme A1M Hemopexin Hemopexin activity Heme oxygenase 1 Regulation of angiotensin II receptor Vasoconstriction Inflammation Vasoconstriction

Paper IV +V Third trimester of pregnancy HbF Endotheliosis Renal damage Haptoglobin Heme NO scavenging Reactive oxygen species A1M Hemopexin Heme oxygenase 1 Regulation of angiotensin II receptor Vasoconstriction Inflammation Vasoconstriction

Modified two-stage model Up-regulated placental synthesis of fetal hemoglobin Maternal hemoglobin- and heme-defense is overwhelmed Maternal hypertension and proteinuria Endotheliosis in maternal vessels and glomeruli – podocyte damage Stage one Stage two Powe et al Circulation 2011

Strain on scavenging systems Early onset Late onset Preeclampsia Normal pregnancy Weeks of gestation 34 weeks Stage one Stage two

Strain on scavenging systems Early onset Late onset Preeclampsia Normal pregnancy Weeks of gestation 34 weeks Treatment with scavengers Stage one Stage two

Acknowledgements Thanks to: Professor Stefan R. Hansson Professor Bo Åkerström Senior Consultant Karl Kristensen The lab at BMC C14 in Lund Bo Åkerström’s lab Statisticians: Per Erik Isberg Jonas Ranstam Collaborators: Basky Thilaganatan, London, UK Marijke Faas, Groningen, the Netherlands Vesna Garovic, Mayo Clinic, USA Funding: Region Skåne Department of Obstetrics and Gynecology, SUS

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