07/03/11 CANDIDATE BIOCHEMICAL MARKERS FOR EARLY … · 07/03/11 1 candidate biochemical markers for early pregnancy screening of preeclampsia jeanjean--claude forest, md, phd, frcpcclaude

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CANDIDATE BIOCHEMICAL CANDIDATE BIOCHEMICAL MARKERS FOR EARLY PREGNANCY MARKERS FOR EARLY PREGNANCY

SCREENING OF PREECLAMPSIASCREENING OF PREECLAMPSIA

JEANJEAN--CLAUDE FOREST, MD, PhD, FRCPCCLAUDE FOREST, MD, PhD, FRCPC

Axe de recherche en reproduction, santé périnatale et santé de l’enfantCENTRE DE RECHERCHE DU CHUQ

Département de biologie moléculaire, biochimie médicale et pathologieUNIVERSITÉ LAVALUNIVERSITÉ LAVALQUÉBEC, CANADA

2nd IFCC-Ortho Clinical Diagnostics Conference : Pregnancy Related Disorders:

Present Perspectives and Emerging ChallengesParis, France

February 25th – 26th, 2011

PREECLAMPSIA AND OTHER HYPERTENSIVE DISORDERS OF PREGNANCY

• What is preeclampsia ?• What is known about its pathophysiology ?• What can be done for prevention/treatment ?• What can be done for prevention/treatment ?• What are the tools for detection ?

– Maternal risk factors– Biochemical markers– Ultrasonographic markers

PERSPECTIVES

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• What are the screening strategies (why and when)?• Criteria for effective predictive algorithms• Benefits and risks

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Terms and AbbreviationsHDP : Hypertensive Disorders of PregnancyPE : PreeclampsiaGH : Gestational HypertensionHELLP: Hemolysis Elevated Liver enzymes

Low Platelet

AFP : Alpha-FetoproteinhCG : human Chorionic GonadotropinPAPP-A: Pregnancy-Associated Plasma

Protein-AInhA : Inhibin A

CH : Chronic Hypertension PlGF : Placental Growth FactorsEndog (TGFβ receptor) : soluble

Endoglin VEGF : Vascular Endothelial Growth

FactorVEGFR1 (sFlt-1) : soluble Vascular

Endothelial Growth Factor Recceptor-1 (soluble fms-like tyrosine kinase-1)

BP : Blood Pressure (SBP: systolic; DBP: diastolic)MAP: Mean Arterial Blood Pressure

BMI : Body Mass IndexPI : Pulsatility Index (Doppler)UtA : Uterine Artery

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tyrosine kinase 1)PP-13 : Placental Protein 13IGF : Insulin-like Growth Factor

UtA : Uterine Artery

PTB: Premature BirthIUGR: Intra Uterine Growth Restriction SGA : Small-for-Gestational AgeGDM : Gestational DiabetesT21, T18, T13: Trisomies 21, 18, 13FL: Fetal Loss

H i f h 20 h k f

A Definition of Preeclampsia(National High Blood Pressure Education Program Working

Group USA 2000)

Hypertension after the 20th week of pregnancy, remit after delivery

Systolic BP ≥ 140 mmHg

Diastolic BP ≥ 90 mmHg

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Normal BP prior

With proteinuria

Early onset: ≤ 34 wks ; late onset: > 34 wks

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Quantitative measurement of total protein excretion over 24h period

Proteinuria

- Proteinuria: ≥ 300 mg protein / 24h - supersedes all dipstick values

If 24-h urine data not available:– Spot concentration of 0.3 g/L on single random

urine specimen

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urine specimen– Proteinuria on dipsticks in at least 2 random mid-

stream samples at least 4h apart (2+ = 1 g/L)

Chronic Hypertension Normal Pre-Pregnancy BP

CH PE Severe PE GH PE Severe

PE

Hypertensive Disorders of Pregnancy

PE PEBP before pregnancy DBP ≥ 90 mmHg Normal

BP during pregnancy

DBP ≥ 90

mmHg

DBP ≥ 90 mmHg

SBP ≥ 160 mmHg or

DBP ≥ 110 mmHg

DBP ≥ 90 mmHg

DBP ≥ 90 mmHg

SBP ≥ 160 mmHg or

DBP ≥ 110 mmHg

Onset of PE b f 34

Onset of PE b f 34

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Onset before ≥ 20 wks before 34 wks but may happen later

≥ 20 wks ≥ 20 wks before 34 wks but may happen later

BP after pregnancy DBP ≥ 90 mmHg Normal

Adapted from Magee et al. (2008) JOGC 30 (suppl1):1-52

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CH PE Severe PE GH PE Severe

PE

Proteinuria None

≥ 2+ dipstick ≥ 300 mg/d≥ 30 mg/ mmol creatinine

3-5 g/d None

≥ 2+ dipstick ≥ 300 mg/d≥ 30mg/ mmol creatinine

3-5 g/d

Onset of PE Onset of PE

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Onset before ≥ 20 wks before 34 wks but may happen later

≥20 wks ≥ 20 wks before 34

wks but may happen later



CH PE Severe GH PE Severe


CH PE PE GH PE PE

Other adverse conditions(variable

• Maternal symptoms• Vision, Headache• Dyspnea

• Maternal signs of end-organ dysfunction

• Edema, eclampsia• Abnormal maternal

laboratory testing

• Maternal symptoms• Vision, Headache• Dyspnea

• Maternal signs of end-organ dysfunction

• Edema, eclampsia• Abnormal maternal

laboratory testing

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extent)laboratory testing

• HELLP : Liver (ALT, AST, LDH) + Platelet Count

• Fetal Morbidity

laboratory testing• HELLP : Liver (ALT, AST, LDH) + Platelet Count

• Fetal Morbidity


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Worldwide, hypertensive disorders of pregnancy affect up to 15% of pregnancies (specific reports of > 20%).

Preeclampsia affects between 2 and 8 % of pregnancies

Epidemiology of Preeclampsia

Preeclampsia affects between 2 and 8 % of pregnancies in developed countries.

Wide geographical variationSevere PE : 10 – 20 % of all PEEarly-onset PE ( ≤ 34 weeks): 10-20% of all PE

Is largely a disease of the first pregnancy.

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Is a major cause of maternal and perinatal morbidity and mortality (5-fold increase)

Is a cause of iatrogenic prematurity (15 % of preterm births are secondary to delivery for preeclampsia)Is a cause of intra uterine growth restriction (IUGR)

Maternal constitutional factors• Genetic predisposition

• Immunological maladaptation• Pre-existing vascular disease

Environmental factors

Pathophysiology of Preeclampsia

Environmental factors

Defective placentation

Placental ischemia

Oxidative stress

Cytotoxic factors

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Systemic maternal endothelial dysfunction

VasospasmHypertensionHypovolemia

Abnormal coagulationThrombosis

Endothelial damageIncreased permeability

Oedema Proteinuria

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– Couple-related risk factors– Limited sperm exposure– Primiparity / Primipaternity– In vitro fertilization

Some Risk Factors of Preeclampsia

– Maternal risk factors– Extremes of maternal age– Women’s own birth weight– Previous PE (or family history of PE)– Chronic hypertension or renal disease– Inflammatory diseases – Obesity and insulin resistance

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y

– Pregnancy-associated risk factors– Multiple pregnancy– Gestational diabetes– Hydrops fetalis / Hydatiform mole– Chromosomal anomalies (trisomy 13,…)– Various infections (urinary tract, gingivitis, …)

Adapted from Sibai et al. (2005) Lancet 365: 785.

Normal Placentation

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• Invasion of the decidua and the myometrium by the cytotrophoblastic cells occurs early in pregnancy.

• Transformation of the walls of the uterine spiral arteries: replacement of endothelial cells by cytotrophoblastic cells, creation of high-flow, low-resistance arteriolar system, and lost of maternal vasomotor control.

http://www.sgul.ac.uk/depts/immunology/~dash/troph/repro.pdf

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Endovascular Trophoblast Invasion

Interstitialtrophoblastic invasion

al b

edB

asal

Pla

te

Dec

idua

13

Plac

enta

Myo

met

rium

After Kaufman P et al. Biol Reprod 2003; 69: 1-7

Anatomopathology of Preeclampsia

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Abnormal uterine vascular remodelling during early pregnancy reduces blood flow from uterus to placenta

From: Bell (2004)

No Pregnancy NormalPregnancy with PE Pregnancy

Moffet-King Nature Reviews Immunology 2002; 2: 656-663

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15Adapted from Mosby’s Color Atlas and Text of Obstetrics and Gynecology, Greer et al., fig 7.8, p.160

Impaired trophoblast invasion of the myometrium (limited at the decidua);30 to 50 % of the placental bed’s spiral arteries escape

Placenta and Preeclampsia

entirely from endovascular trophoblastic invasion;Myometrial segments remain anatomically intact and adrenergic nerve supply to the spiral arteries remains also intact;Increased responsiveness of spiral arteries to vasoconstrictor mediators results in…

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placental hypoperfusion, local hypoxia,overproduction of various mediators,oxidative stress.

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Initial mechanisms

Pathogenesis of Preeclampsia The “Angiogenic Imbalance” Theory

– Inflammatory challenges (disorders);– Alterations in the regulation and signalling of

angiogenesis pathways contribute to inadequate cytotrophoblastic invasion;

– Up-regulation of anti-angiogenic factors (sFlt-1, s-Endog);

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g)– Down-regulation of circulating angiogenic factors

(VEGF, PlGF).Young et al. Annu Rev Pathol Mech Dis 2010; 5: 173-192

Abnormal Uterine Vascular Remodelling during early PregnancyAbnormal Uterine Vascular Remodelling during early Pregnancy

Immune / Inflammatory FactorsGenetic Susceptibility Environment / Maternal

Characteristics

Pathophysiology of Preeclampsia

Foetal Pain / Distress : Intrauterine Growth Restriction

Intrauterine Foetal Death

Placental Hypotrophia,Infarctus

Reduced Placental PerfusionReduced Placental Perfusion

Hypoxia (Oxidative Stress)Hypoxia (Oxidative Stress)

Placental DysfunctionPlacental Dysfunction

Release of Placental Mediators in Maternal CirculationRelease of Placental Mediators in Maternal Circulation

18Adapted from Giguère et al (2011) submitted

HypertensionProteinuria

Glomerular EndotheliosisHELLP syndrome

Eclampsia

Systemic Maternal Endothelial DysfunctionSystemic Maternal Endothelial Dysfunction

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Abnormal profile of adhesion molecules (∆ VE-cadherin, VCAM-1, PECAM-1)Decrease of plasminogen activation (∆ urokinase, MMP-9, PAI-1)

Abnormal Uterine Vascular Remodelling during early PregnancyAbnormal Uterine Vascular Remodelling during early Pregnancy

Pathophysiology of PE - Biomarkers

p g ( , , )Modification of uterine angiogenesis (∆ sFlt-1, sEndog, PlGF, VEGF, hCG )

Growth Factors : EGF, IGF-II, TGF α/β, VEGF, PlGF, sFlt-1, sEndog;C ki TNF I l ki 1 6 10 IF I f P l di

Placental DysfunctionPlacental Dysfunction

Release of Placental Mediators in Maternal CirculationRelease of Placental Mediators in Maternal Circulation

AFP, hCG, PAPP-A, inhibin A, activin A, PP-13

19Adapted from Giguère et al (2011) submitted

Cytokines : TNFα; Interleukins 1, 6, 10; LIF; Interferon; Prostaglandins;Enzymes : PAI 1, PAI 2, MMP-2, MMP-9;Others : Peroxylated Lipids, Endothelins, Apoptotic/Necrotic debris, Cell-free foetal nucleic acids

Systemic Maternal Endothelial DysfunctionSystemic Maternal Endothelial Dysfunction

Clinical (maternal history and

Altered Parameters before Occurrence of Clinical Symptoms of PE

Clinical (maternal history and characteristics, BMI, MAP, …)Blood and Urine ParametersDoppler Uterine Artery Blood Flow Velocity Measurements

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3-Dimensional Doppler Ultrasonography (placental volume)

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– Maternal risk factors (maternal history)– Biochemical markers– Ultrasonographic markers

– PERSPECTIVES

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PERSPECTIVES • What are the screening strategies (why and when)?• Criteria for effective predictive algorithms• Benefits and risks

Objectives:

Early Prediction of PE

Object es• To identify women at high risk of developing

preeclampsia later in pregnancy, and to offer specific preventive measures;

• To apply prophylactic interventions at optimal time;T l f ll

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• To propose closer follow-up.

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• Improved socio-economic conditions

Interventions for the Prevention of Preeclampsia

p(perinatal policy)

• Structured prenatal counselling and follow-up

• Potential prophylactic therapies– Antiplatelet agents (aspirin)

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Antiplatelet agents (aspirin)• Inhibit thromboxane-mediated vasoconstriction

and pathological blood coagulation in the placenta

– Low-molecular weight heparin

Early recognition, key to prevention and treatment.– The objective is to prolong pregnancy whenever

Clinical Intervention and Treatment

The objective is to prolong pregnancy whenever possible to permit fœtal lung maturity while preventing progression to severe disease;

– Antiplatelet prophylactic therapy (aspirin);– Antihypertensive therapy (>160/100 mmHg:

methyldopa, labetalol, nifedipine);

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– The definitive treatment is delivery;

– Eclampsia: emergency; High-risk morbidity/mortality.

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PE can be Prevented by ASA

• 27 trials including 11,348 women

“ … current evidence indicates that low-dose aspirin started in

Trials using ASA

before 16 weeksRR: 0.47

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pearly pregnancy may reduce the incidence of PE, IUGR and PTB in women identified at moderate or high risk for PE.”Bujold et al. (2010) Obstet Gynecol 116: 402-414

Trials using ASA after

16 weeksRR: 0.81



– Maternal risk factors (maternal history)– Biochemical markers – Ultrasonographic markers

PERSPECTIVES

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Recommendations for determination of HDP risk at 1st visit• Intensity of monitoring

– Ex.: Maternal age > 40 yrs; nulliparity, woman’s own birth

Clinical Maternal Risk Factors

g y ; p y,weight, pre-existing chronic hypertension, diabetes, previous PE, BMI > 30.

• Very few published data of performance– In one recent study by Poon et al. (J Hum Hypert (2010) 24 : 104-10),

logistic regression analysis of maternal characteristics shows:•At a false positive rate of 5 %, detection rates:f l PE 37% l t PE 29 % GH 20%

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for early PE: 37%; late PE: 29 %; GH: 20%.•Screening suggested by NICE (Natl. Inst. for Clinical Excellence):(essentially treats each factor as a separate screening test) false positive rate of 64 %; detection rate of 89, 93 and 85 % , respectively.

• Elements from maternal history may serve to

Maternal Risk Factors for HDP as a priori Risk

determine a priori risk using a multivariate approach.– Ethnicity, maternal age, woman’s own birth weight, prior PE,

BMI…

• As for screening for Trisomy 21, patient specific risk may be derived by multiplying a priori risk by the likelihood ratio associated with selected biochemical

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likelihood ratio associated with selected biochemical and biophysical parameters.

Cuckle (2011) Placenta 32: S42-S48

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– Maternal risk factors (maternal history)– Biochemical markers – Ultrasonographic markers

PERSPECTIVES

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Marker Altered levels during Assessed in combination with Associated withBefore onset

of PE (1st /2nd trim.)

After onset of PE

(3rd trim.)

Other biochem. markers

Ultrasound markers

Maternal characteristics

VEGF UtA PI GH, GDM, HELLP, T21

Potential Biomarkers for PE – Summary

PlGFsFlt-1;PlGF;PAPP-A

UtA PI

Ethnicity, MAP,weight, history CH, smoking, Conception

GH, GDM, HELLP, PTB, IUGR, T21, T18, T13, triploidy, Turner

sFlt-1 PlGF;sEng; UtA PI GH, GDM, HELLP, FD, IUGR, stillbirth, T13

sEndog PlGF;sFlt-1; PAPP-A UtA PI weight GH, HELLP, FD, IUGR,

SGA,

AFP hCG UtA PIGDM, GH, HELLP, PTB, FL, SGA, T21, Neural t be defectstube defects

hCG AFP, PAPP-A UtA PI GDM, GH, PTB, T21

Inhibin A PAPP-A, activin A UtA PI Ethnicity, parity,

BMIGDM, GH, HELLP, PTB, SGA, T21

PAPP-AsEng, PlGFPP-13, inhibinA

UtA PI

Weight, BMI, MAP, ethnicity, CH, parity, age conception, fetal CRL, smoking

GDM, GH, HELLP, PTB,FD, SGA, T21, T18,Turner

From Giguère et al. 2011 submitted

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Marker Altered levels during Assessed in combination with Associated with

Before onset of PE

(1st /2nd trim.)

After onset of PE

(3rd trim.)

Other biochem. markers

Ultrasound markers

Maternal characteristics

Cell free

Potential Biomarkers for PE – Summary

Cell freefetal DNA IUGR

Cell freefetal RNA

P-Selectin UtA PI

Ethnicity, weight, history CH, smoking, MAP,conception

GH, PTB, SGA

VCAM GDM, GH, HELLP, SGA

HELLP PTB T18 T13

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PP-13 PAPP-A UtA PI Weight, smoking HELLP, PTB, T18, T13,Turner

ADAM-12 SGA

Visfatin GDM, PTB, SGA

PTX3 GH, PPROM, PTB, IUGR

From Giguère et al. 2011 submitted

Challenges with Measurement of Biochemical Markers to Predict PE Early in Pregnancy

• Concentration of marker changes during pregnancy;• Necessity to express results in Multiple of Median – MoM

• Lack of standardized methods and reference materials, commercial availability and automation, CE label;

• Overlap of results between normal pregnancy and HDP.

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Which of the putative biomarkers

Potential Biomarkers for PE

Which of the putative biomarkers – Are predictive of preeclampsia and other

adverse pregnancy outcomes ?– Have demonstrated clinical utility ?

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Condition • Important health problem;• Natural history understood;• Recognizable latent or early symptomatic stage;

Summary of Criteria for ScreeningJungner & Wilson WHO (1968); Holland et al. WHO (2006)

• Recognizable latent or early symptomatic stage;

Diagnosis • Suitable diagnostic test available, safe and acceptable to the population concerned;

• Agreed policy, based on respectable test findings and national standards, as to whom to regard as patients;

• Whole process, continuing;

Treatment • Accepted and established treatment / intervention for

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individuals identified as having the disease or pre-disease condition;

• Facilities for treatment should be available;

Cost • Cost of case-finding (including diagnosis and treatment) should be economically balanced in relation to possible expenditure on medical care.

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Summary of Criteria for Evaluation of Screening Tests (Holland et al. WHO (2006))

Simplicity The test should be simple to perform, easy to interpret and, where possible, capable of use by paramedics and other personnel.

Acceptability Since participation in screening is voluntary, the test must be acceptable to those undergoing it.

Accuracy The test must give a true measurement of the condition or symptom under investigation.

Cost The expense of the test must be considered in relation to the benefits of early detection of the disease.

Repeatability The test should give consistent results in repeated

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Repeatability e test s ou d g e co s ste t esu ts epeatedtrials.

Sensitivity The test should be capable of giving a positive finding when the individual being screened has the condition being sought.

Specificity The test should be capable of giving a negative finding when the individual being screened does not have the condition being sought.

• Until now, numerous systematic reviews have been unable to demonstrate a single biochemical marker capable of detecting, early in pregnancy, those women susceptible to develop PE.

Systematic Reviews of Combinations of Biomarkers

Conde-Argudelo et al. Obstet Gynecol. 2004; 104: 1367-1391.Cnossens et al. Health Techn. Assess. 2008; 12(6):1-128.

• Because of the heterogeneous nature of PE, a combination of independent biomarkers, each reflecting a different pathophysiological process, should increase the likelihood to derive suitable predictive algorithms.

• Indeed, combinations of biochemical and ultrasonographic markers into algorithms derived by multivariate analysis improve

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markers into algorithms derived by multivariate analysis improve the performance of early prediction of PE.

• From the perspective of integrative medicine, there is a clear need for prospective large-scale studies with rigorous study design criteria to determine the clinical usefulness of combinations of biomarkers in different geographic and healthcare environments.

Giguère et al. Clin. Chem. 2010; 56(3): 361-374.Cetin et al. Placenta 2011; 32:S4-S16.

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Clinical Screening Strategies for PEExisting Clinical Investigations

Dating, Nuchal translucencyTrisomy screening

Trisomy screening

Fetal Development(Ultrasound)

Screening for

Fetal Development(Ultrasound)

Trisomy screening(Biochemistry)

Screening forGestational Diabetes

0 4010 -13(1st trim.)

14 -17 20 24 -28(2nd trim.)

30, 32, 36 (depending)

(3rd trim.)

PREGNANCY

Development of Preeclampsia

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Screening Strategies

1st trimester:Prediction of HDP / PE

(Preventive treatment for high-risk women)

2nd trimester:Late identification of imminent

HDP / PE(Closer follow-up of high-risk

women)

Benefits• To develop an approach using biomarkers to

detect, as early as possible, women at risk of d l i l i ith t

Clinical screening strategies for PE

developing preeclampsia with a greater performance than that of conventional methods in order to offer, in a targeted manner, prophylactic interventions, specific surveillance and / or closer management.

Risks

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• False positive results creating anxiety and possibly unnecessary interventions

• False negative results causing false reassurance or reduction of surveillance.

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• What should be the expected performance to reach clinical utility?

Challenges

• Will the same factors for calculation of a priori risk and biomarkers (biochemical / biophysical) perform uniformly in different environments?

• Commercial availability of best performing markers?C t ff ti t di ?

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• Cost-effectiveness studies?• Optimal time to screen?

• No individual biochemical or biophysical markers have met the criteria for a screening test.

In Summary

• Maternal history & risk factors, have as a whole poor predictive value; with some guidelines, 2/3 of the patients would be falsely classified as high risk.

• A growing body of evidence suggests that the combination of maternal factors (BMI age parity

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combination of maternal factors (BMI, age, parity, ethnicity, past history,...) with serum markers and UtA Doppler indices may identify early in pregnancy women at risk of developing preeclampsia.

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In Summary

There is a need for large-scale prospective studies to determine the usefulness ofstudies to determine the usefulness of multivariate risk-prediction algorithms early in pregnancy, in different populations.

This may lead to the implementation of an efficient screening procedure to identify

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efficient screening procedure to identify women at risk for PE who would benefit from early targeted interventions.

• Bujold et al. (2010) “Prevention of Preeclampsia and Intrauterine Growth Restriction With Aspirin Started in Early Pregnancy - A Meta-Analysis” Obstet Gynecol 116:402–14)

• Cetin et al. (2011) “Pregenesys pre-eclampsia markers consensus meeting: What do we require from markers, risk assessment and model systems to tailor preventive strategies?” Placenta 32: S4-S16.

References

• Cuckle, H. (2011) “Screening for Pre-eclampsia - Lessons from Aneuploidy Screening” Placenta 32: S40-S48.

• Giguère et al. (2010) “Combining Biochemical and Ultrasonographic Markers in Predicting Preeclampsia: A Systematic Review” Clin Chem. 56 : 361-374.

• Grill et al. (2009) “Potential markers of preeclampsia – a review” Reproductive Biology and Endocrinology 7:70.

• Lindheimer et al. (2009) “ASH Position Paper: Hypertension in Pregnancy” J Clin Hypertens (Greenwich) 11:214–225.

• Silasi et al. (2010) “Abnormal Placentation, Angiogenic Factors, and the Pathogenesis of P l i ” Ob t t G l Cli N A 37 239 253

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Preeclampsia” Obstet Gynecol Clin N Am 37 : 239–253.• Steegers et al (2010) “Preeclampsia” Lancet 2010; 376: 631–44.

• Wang et al. (2009) “Preeclampsia: The role of angiogenic factors in its pathogenesis”. Physiology 24:147-158.

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• Yves Giguère, Jacques Massé, Emmanuel Bujold, Julie Lafond, Jean-Marie Moutquin, François Rousseau

• Aziz Aris Joël Girouard Sébastien Lévesque

Acknowledgements

Aziz Aris, Joël Girouard, Sébastien Lévesque, Patrice Savard, Nathalie Bernard, Mélissa Laroche

• Marc Charland, Véronique Goulet, Mylène Badeau, Nathalie Bernard, Francine Déchenes, Monique Longpré, Pierre DeGrandpré, nurses and laboratory technicians

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MERCI!MERCI!

THANK YOU!THANK YOU!

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Documents

07/03/11 CANDIDATE BIOCHEMICAL MARKERS FOR EARLY … · 07/03/11 1 candidate biochemical markers for early pregnancy screening of preeclampsia jeanjean--claude forest, md, phd, frcpcclaude