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Biological markers for the diagnosis and the Biological markers for the diagnosis and the prognosis of preterm prematureprognosis of preterm premature

rupture of membranesrupture of membranes

F. GoffinetF. GoffinetPortPort--Royal maternity, University ParisRoyal maternity, University Paris--DescartesDescartesINSERM U953 Epidemiological Research Unit on INSERM U953 Epidemiological Research Unit on

Perinatal Health and Women’s and Children HealthPerinatal Health and Women’s and Children Health

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2nd IFCC2nd IFCC--Ortho Clinical Diagnostics ConferenceOrtho Clinical Diagnostics Conference25th february 201125th february 2011

PPROM : implication for perinatal healthPPROM : implication for perinatal health

ManagementManagement

PPROMPPROMPreterm Premature Rupture Of MembranesPreterm Premature Rupture Of Membranes

ManagementManagementPositive impact on perinatal healthPositive impact on perinatal healthBut Invasive treatmentBut Invasive treatmentConsequences of false positive and false negative resultsConsequences of false positive and false negative results

Effective diagnosisEffective diagnosisClinicalClinical

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BiologicalBiological

Prognosis in cases of PPROMPrognosis in cases of PPROMPreterm delivery or infection ?Preterm delivery or infection ?Many biomarkers studied Many biomarkers studied Biomarkers used currentlyBiomarkers used currently

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PPROM : frequency and impact on PPROM : frequency and impact on prematurityprematurity

••PPROM: 3 % of pregnanciesPPROM: 3 % of pregnancies

––20 to 40 % of the PROM20 to 40 % of the PROM––30 to 40% of preterm deliveries 30 to 40% of preterm deliveries Mercer BM et al. Am J Obstet Gynecol 2000

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Am J Obstet Gynecol 2000

Goldenberg RL Goldenberg RL et al. Lancet 2008et al. Lancet 2008

PPROM, PPROM, laborlabor and infectionand infection

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PPROM, PPROM, laborlabor and infectionand infection

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inflammation

preterm delivery preterm delivery infection infection –– inflammationinflammation

Main cause of Main cause of preterm birthpreterm birth

Hervé R. et al. J Immunol. 2008Schmitz T. et al. J Immunol. 2007

Chorionic Chorionic cellscells

ProteinsProteinsreleasedreleased

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Rupture of Rupture of membranesmembranes

Myometrial Myometrial ContractionContraction

CervicalCervicalmaturationmaturation

Preterm birthPreterm birth

Preterm Preterm MarkersMarkers

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•• Maternal complications: Maternal complications: chorioamnionitis (2chorioamnionitis (2--8%), endometritis8%), endometritis

PPROMPPROMconsequencesconsequences

•• Perinatal complications = preterm birthPerinatal complications = preterm birthPerinatal mortalityPerinatal mortalityShort term complications: Enterocolitis, bronchodysplasia, Short term complications: Enterocolitis, bronchodysplasia, periventricular leukomalacia,periventricular leukomalacia,

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Long term outcome : cerebral palsy, sensorial deficit, Long term outcome : cerebral palsy, sensorial deficit, mental retardationmental retardation

Infection = additional pejorative factorInfection = additional pejorative factor

Management in cases of PPROM (1)Management in cases of PPROM (1)no signs of infectionno signs of infection

before 32before 32--34 Weeks34 Weeks after 32after 32--34 weeks34 weeks

HospitalisationHospitalisationAntibioticsAntibiotics

Maternal transferMaternal transfer

HospitalisationHospitalisationAntibioticsAntibiotics

Maternal transferMaternal transfer

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Tocolysis 24Tocolysis 24--48 hours48 hoursCorticosteroidsCorticosteroids

Search for infectionSearch for infection

Search for infectionSearch for infection

Induction of labor Induction of labor at 34at 34--36 weeks36 weeks

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Management in cases of PPROM (2)Management in cases of PPROM (2)use of a high CRP in decisionsuse of a high CRP in decisions

ClinicalClinicalCh i i itiCh i i iti

High CRPHigh CRPChorioamnionitisChorioamnionitis

11--2 % at admission2 % at admissionBefore 30Before 30--34 34

WksWksAfter 30After 30--34 34

WksWks

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« delivery»« delivery»

No tocolysisNo tocolysisTo gain time…To gain time…

deliverydelivery

History taking and clinicalHistory taking and clinical

Diagnostic methods and prognostic criteria in the case of premature rupture of the membranes

Verspyck E et Al. J Gynecol Obstet Biol Reprod. 1999 Verspyck E et Al. J Gynecol Obstet Biol Reprod. 1999

Diagnosis of PPROM : clinicalDiagnosis of PPROM : clinical

80% 80%

History taking and clinical History taking and clinical examination are often sufficientexamination are often sufficient

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Sterile speculum examination: Sterile speculum examination: leakage of amniotic fluid leakage of amniotic fluid

from cervical os continued from cervical os continued and expanded by the and expanded by the

mobilization mobilization

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Biomarkers: Quality requiredBiomarkers: Quality required

Strictly specific molecule of the AF Strictly specific molecule of the AF Present at all gestational agesPresent at all gestational agesNO «NO « GOLD STANDARDGOLD STANDARD » »

To confirm the diagnosis of To confirm the diagnosis of rupture of membranesrupture of membranes

Present at all gestational ages Present at all gestational ages Slow degradation Slow degradation Easily detectable at low concentrationEasily detectable at low concentrationThe test should be rapid and available The test should be rapid and available 24 h a day24 h a day

•• prevent false positive resultsprevent false positive resultsC t i ti (bl d i l li id)C t i ti (bl d i l li id)

No perfect No perfect biomarkersbiomarkers

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•• Contamination (blood, seminal liquid)Contamination (blood, seminal liquid)

•• prevent false negative resultsprevent false negative results•• Rupture of membranes very early in the pregnancyRupture of membranes very early in the pregnancy•• Disappearance of the markerDisappearance of the marker•• Concentration too lowConcentration too low

N G ld t d d f di i f PPROMN G ld t d d f di i f PPROM

Verspyck E, Landman T, Marpeau L J Gynecol Obstet Biol Reprod 1999

Published results Published results methodological drawbacksmethodological drawbacks

Very few studies compared diagnostic value of different testsVery few studies compared diagnostic value of different tests

CutCut--offs to define an abnormal test could be different in the studiesoffs to define an abnormal test could be different in the studies

No Gold standard for diagnosis of PPROM No Gold standard for diagnosis of PPROM

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Comparison groups : No rupture of membranes versus cases with clear Comparison groups : No rupture of membranes versus cases with clear liquid flow liquid flow Target population : suspicion of PPROM (intermittent or no leakage of Target population : suspicion of PPROM (intermittent or no leakage of fluid) fluid) -- cases in which clinical diagnosis is not evidentcases in which clinical diagnosis is not evident

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Localization of the biomarkers usedLocalization of the biomarkers used

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amniotic fluid crystallization testing amniotic fluid crystallization testing –– fern testfern test

SensSens SpecSpec PPVPPV NPVNPV

Fern testFern test

42%42% 76%76%

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--Gibbs, 1982Gibbs, 1982 42%42% 76%76% -- --

Contamination with cervical mucus (false positive result)Contamination with cervical mucus (false positive result)Learning required (microscope)Learning required (microscope)

subjectivesubjective

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NbNb SensSens SpecSpec VPPVPP VPNVPN

A lot of biomarkers studiedA lot of biomarkers studied

DAO (DAO (DiAmineDiAmine Oxydase)Oxydase)

‐‐ GaucherandGaucherand, 1997, 1997 100100 83,783,7 100100 100100 8989

‐‐ De Meeus, 1997De Meeus, 1997 7171 90,990,9 100100 100100 98,398,3

AFP (AFP (AphaApha FetoFeto‐‐ProteinProtein))

‐‐ KishidaKishida 19951995 103103 100100 97 497 4 92 592 5 100100

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‐‐ KishidaKishida, 1995, 1995 103103 100100 97,497,4 92,592,5 100100

‐‐ Gaucherand, 1995Gaucherand, 1995 131131 8888 8484 8686 8787

Others : hCG, Prolactin, urea, creatinin, lactates …Others : hCG, Prolactin, urea, creatinin, lactates …

amniotic fluid/serum ratio lowamniotic fluid/serum ratio low

FIBRONECTINEFIBRONECTINE

NbNb SensSens SpecSpec VPPVPP VPNVPN

FibronectineFibronectine

‐‐Gaucherand, 1995Gaucherand, 1995‐‐Rutanen, 1993Rutanen, 1993

1311315454

94949292

97978080

97977979

9494‐‐

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Extracellular matrix protein of membranes > AFExtracellular matrix protein of membranes > AFReleased in the case of preterm labor or labor with intact membranesReleased in the case of preterm labor or labor with intact membranes

Protein present in seminal fluidProtein present in seminal fluidamniotic fluid/serum ratio lowamniotic fluid/serum ratio low

Guibourdenche J et al. Ann Clin Biochem 1999

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NITRAZINE paper testingNITRAZINE paper testingsimple, cheapsimple, cheap

cervical pH= 5cervical pH= 5‐‐66Amniotic fluid : alkalineAmniotic fluid : alkalineDi i k l i iDi i k l i i

NbNb SensSens SpecSpec VPPVPP VPNVPN

NitrazineNitrazine

-- Gaucherand, 1997Gaucherand, 1997 100100 90,790,7 77,277,2 7575 91,791,7-- De Meeus, 1997De Meeus, 1997 7171 81,181,1 83,383,3 52,652,6 96,196,1

Dipstick colorimetricDipstick colorimetric

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bacterial bacterial vaginosisvaginosiscervicitiscervicitissemen, alkaline urine, semen, alkaline urine, blood, soap and blood, soap and antiseptic solutionsantiseptic solutions

false false positivepositive

IGFBPIGFBP--1 (ActimProm1 (ActimProm®®))insulininsulin--like growth factor binding proteinlike growth factor binding protein--II

PProduced by decidual cells (placental protein)roduced by decidual cells (placental protein)Immunoenzymometric assay for quantitation Immunoenzymometric assay for quantitation Concentrations = 100 to 1000 fold higher than those in serumConcentrations = 100 to 1000 fold higher than those in serumVery low concentration in urine cervical mucus and seminal fluidVery low concentration in urine cervical mucus and seminal fluid

ActimProm ActimProm –– IGFBPIGFBP--11 NbNb SensSens SpecSpec VPPVPP VPNVPN

Lockwood et al. 1994Lockwood et al. 1994 105105 74,474,4 92,692,6 96,796,7 55,655,6Rutanen et al. 1996Rutanen et al. 1996 130130 100100 94,794,7 93,293,2 100100Gaucherand et al. 1997Gaucherand et al. 1997 100100 95,395,3 98,298,2 97,697,6 96,596,5Marcellin et al 2011Marcellin et al 2011 9090 95 395 3 95 695 6 97 797 7 93 693 6

Very low concentration in urine, cervical mucus and seminal fluidVery low concentration in urine, cervical mucus and seminal fluidRapid strip test (5 min) = > 95th percentile of serum levelsRapid strip test (5 min) = > 95th percentile of serum levels

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Marcellin et al. 2011Marcellin et al. 2011 9090 95,395,3 95,695,6 97,797,7 93,693,6

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PAMGPAMG--1 (1 (AmniSureAmniSure®®))

placental alphaplacental alpha--microglobulinmicroglobulin--11PProduced by decidual cells (placental protein)roduced by decidual cells (placental protein)Concentrations = 1 000 to 10 000 fold higher than Concentrations = 1 000 to 10 000 fold higher than those in those in cervical mucuscervical mucus

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Amnisure Amnisure –– PAMGPAMG--11 NbNb SensSens SpecSpec VPPVPP VPNVPN

Cousin et al . 2005 Cousin et al . 2005 203203 98,998,9 100100 100100 99,199,1

Marcellin et al. 2011Marcellin et al. 2011 9090 92,892,8 91,691,6 90,790,7 93,693,6

Prognosis in case of PPROMPrognosis in case of PPROM

Prognosis of what ?Prognosis of what ?

Preterm Preterm birthbirth

InfectionInfectionNeonatal ?Neonatal ?Maternal ?Maternal ?

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Neonatal outcome ?Neonatal outcome ?Long term outcome ?Long term outcome ?

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Intra uterine inflammation or infectionIntra uterine inflammation or infection

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Clinical chorioamnionitis:Clinical chorioamnionitis:Admission : 1Admission : 1--2 %2 %Subsequently: 3Subsequently: 3--8 %8 %

Positive culture by Positive culture by amniocentesis: 25amniocentesis: 25--40 %40 %

Neonatal infection Neonatal infection 55--10%10%

Natural history of PPROMNatural history of PPROM((SavitzSavitz et al, Am J et al, Am J ObstetObstet GynecolGynecol 1997 et Johnson, 1997 et Johnson, ObstetObstet GynecolGynecol 1981)1981)

100

30405060708090

100 delivery < 7 dy delivery < 48 h

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01020

24-28 weeks 29-32 weeks 33-36 weeks

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Relationship between PROM and infectionRelationship between PROM and infectioncause and consequencecause and consequence

maternomaterno fetalfetal

ChorioChorio--decidual decidual Infection or Infection or

inflammationinflammation Non Non infectiousinfectious

intraintra--amniotic amniotic Infection or Infection or

inflammationinflammationPPROMPPROM

maternomaterno--fetalfetalinfection and infection and

deliverydelivery

causescauses

1/2 ?1/2 ?

1/2 ?1/2 ?

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BacteriaBacteria fromfromcervicocervico--vaginal vaginal

tractustractus

DeliveryDelivery withoutwithoutinfectioninfection

Most WomenMost WomenProportion ?Proportion ?

Best way : diagnosis of inflammation or infection Best way : diagnosis of inflammation or infection in amniotic fluid (amniocentesis)in amniotic fluid (amniocentesis)

•• Culture could be the gold standard for the diagnosis ofCulture could be the gold standard for the diagnosis of•• Culture could be the gold standard for the diagnosis of Culture could be the gold standard for the diagnosis of infectioninfection–– Direct diagnosis of the inflammation or the infectionDirect diagnosis of the inflammation or the infection–– Identification of the germaIdentification of the germa

•• Many short term tests studiedMany short term tests studied

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–– Gram stain, white blood cell count, leucocyte Gram stain, white blood cell count, leucocyte esterase, glucose concentrationesterase, glucose concentration

–– Interleukines, metalloproteinaseInterleukines, metalloproteinase–– Etc etcEtc etc

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Disadvantages of amniocentesisDisadvantages of amniocentesis

•• Disadvantages of the procedureDisadvantages of the procedure–– Results of the culture (48 hours) after the delivery frequentlyResults of the culture (48 hours) after the delivery frequently–– Short term test : low specificity and meaning ?Short term test : low specificity and meaning ?–– Success of amniocentesis is associated with amount of amniotic fluid Success of amniocentesis is associated with amount of amniotic fluid

remaining (oligohydramnios in PPROM)remaining (oligohydramnios in PPROM)–– Complications associated with amniocentesis ?Complications associated with amniocentesis ?

•• No evaluation of the use of test No evaluation of the use of test

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–– Decision ? : antibiotics, Cesarean section, corticosteroid before Decision ? : antibiotics, Cesarean section, corticosteroid before extractionextraction

•• Few team published (many, many) papers on the diagnostic Few team published (many, many) papers on the diagnostic value of theses tests, many methodological weaknesses value of theses tests, many methodological weaknesses

Cytokines in serum or vaginal secretions ?Cytokines in serum or vaginal secretions ?

•• Parturition: ILParturition: IL--6, TNF6, TNFαα, IL, IL--11ββ, IL, IL--88

•• Glycoproteins involved in inflammationGlycoproteins involved in inflammationl d i l ft ti ti f hl d i l ft ti ti f h–– released mainly after activation of macrophagesreleased mainly after activation of macrophages

–– result in the production of prostaglandins and proteases (MMP)result in the production of prostaglandins and proteases (MMP)–– measurement difficult and costly (ELISA in most studiesmeasurement difficult and costly (ELISA in most studies))

•• Prospective study (73 women with PPROM) (Kayem et al 2005)Prospective study (73 women with PPROM) (Kayem et al 2005)–– diagnostic value of ILdiagnostic value of IL--6 in vaginal secretions for neonatal infection in cases of 6 in vaginal secretions for neonatal infection in cases of

PPROMPPROM–– Immunochromatographic bed side test (20 minutes)Immunochromatographic bed side test (20 minutes)

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IL-6control

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Odds of neonatal infection as function of maternalOdds of neonatal infection as function of maternalmarkers, based on logistic regressionmarkers, based on logistic regression

Congenital proven or probable

Without

congenital sepsis

OR

Adjusted OR

probable sepsis n =16

sepsis

n = 68

Vaginal IL6 positive

12 (75.0)

29 (42.6)

4.0 (1.3-13)

5.5 (1.2-17.7)

Maternal serum C-Reactive Protein

> 5 mg/dl > 20 mg /dl

9 (52.9) 4 (23 5)

31 (41.3) 5 (7 4)

1.2 (0.5-4.6) 3 4 (1 0 16 5)

5 6 (0 95 32 6)

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> 20 mg /dl

4 (23.5) 5 (7.4) 3.4 (1.0-16.5) 5.6 (0.95-32.6)

Maternal WBC (x 1000 : ) > 15 > 20

3 (18.9) 1 (6.2)

21 (30.9) 4 (5.9)

0.6 (0.13-1.97)1.0 (0.1-10.2)

ConclusionConclusion•• DiagnosisDiagnosis

–– Clinical Clinical Biomakers necessary in 10Biomakers necessary in 10 20 %20 %–– Biomakers necessary in 10Biomakers necessary in 10--20 %20 %

•• Nitrazine +Nitrazine +-- PROM test or Amnisure testPROM test or Amnisure test•• Perspective : Biomarker sPerspective : Biomarker strictly specific of the AF trictly specific of the AF and and

eeasily detectable at low concentrationasily detectable at low concentration

•• PrognosisPrognosis

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–– To predict subclinical infection before onset of To predict subclinical infection before onset of labor, butlabor, but

•• Which kind of management ?Which kind of management ?•• No simple biomarkers availableNo simple biomarkers available