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

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

3. 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

4. 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

5. Preeclampsia the two-stage model
Powe et al Circulation 2011

6. 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

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

8. 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

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

10. 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.

11. 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

12. Paper I – Predicting preeclampsia
Anderson et. al 2011

13. 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

14. Paper I – Results
Serum concentrations

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

16. 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.

17. First trimester of pregnancy
HbF
Haptoglobin
Heme
Hemopexin
A1M

18. Paper II
Anderson et. al 2016

19. 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

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

21. 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 ( )
60%
62%
75%
91%
Läg til ROC-curve

22. 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.

23. First trimester of pregnancy
HbF
Haptoglobin
Heme
A1M
Hemopexin

24. Paper III
Gram et. al 2015

25. 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

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

27. Paper III - results

28. Paper III - results

29. Paper III - results

30. 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.

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

32. 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

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

34. 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

35. 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

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

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

38. 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

39. Thank you for your attention