• • •

MECCANISMO DI PLACENTAZIONE NORMALE

Lo stadio più precoce del rimodellamento vascolare (FASE 1) è rappresentato dalla vacuolizzazione endoteliale e dal rigonfiamento di alcune cellule muscolari della tunica media. Tale fase rappresenta il rimodellamento vascolare indipendente dal trofoblasto ed invece correlato alla decidualizzazione. Nella fase successiva (FASE 2) si verifica una disorganizzazione della tunica media con ampliamento degli spazi intercellulari determinando un’alterazione e perdita della tunica muscolare. Le modificazioni in questo stadio sono dipendenti dall’invasione del trofoblasto nel tessuto stromale e perivascolare, il quale determina cambiamenti nella matrice extracellulare tramite la secrezione di specifici enzimi.

La FASE 3 è caratterizzata dalla comparsa del trofoblasto endovascolare all’interno del lume vasale.

MECCANISMO DI PLACENTAZIONE NORMALE 2

• In un secondo tempo il trofoblasto endovascolare viene incorporato nella parete vasale, processo che risulta essere accompagnato dalla deposizione di materiale fibrinoide (FASE 4). L’originale strato muscolare liscio e la tunica elastica sono sostituiti da tale materiale fibrinoso, mentre le cellule trofoblastiche intercalate conferiscono, per la loro distribuzione scarsamente organizzata, un aspetto a ragnatela.

• L’ultimo stadio (FASE 5) è caratterizzato dalla riparazione endoteliale a volte associata all’ispessimento intimale legato alla proliferazione delle cellule miointimali di origine materna (Pijnenborg 2006). • l’assenza delle modificazioni fisiologiche, legate ad una alterata invasione trofoblastica o al fallimento della stessa, sembrerebbe essere associata allo sviluppo della preeclampsia

Circolazione uterina

(Bonnar et al: Pregnancy induced hypertension, 1980)

Arterie spirali

Utero non gravido sezione a livello della giunzione endometrio/miometrio

Arterie spirali

Secondo trimestre

Placentazione e PE

Redman e Sargent

Science

2005,308:1592

Invasione trofoblastica delle arterie utero-placentari

GRAVIDANZE NORMALI PRE-ECLAMPSIA IUGR

Art. spirale

Invasione endometrio e terzo interno miometrio Invasione limitata all’endometrio RIDUZIONE DELLA PORTATA EMATICA UTERO-PLACENTARE

Placentation and Preeclampsia: current hypotesis Imbalance of angiogenic factors sFlt1 VEGF, PIGF Disfunctional maternal endothelium

I punti deboli della teoria corrente

• La mancata invasione trofoblastica si ha solo dopo la 12^ settimana (Jauniaux,

Am J Pathol

2000,157:2111) • Nel 1^ trimestre sono già presenti nel sangue delle madre i markers sierici della PE

NUOVI MODELLI PATOGENETICI

• Le ipotesi attuali associano lo sviluppo della patologia a diverse alterazioni così riassunte: • effetti deleteri a livello del trofoblasto extracellulare; • fallimento dell’invasione trofoblastica nelle arterie spirali; • riduzione del flusso di sangue materno nello spazio intervilloso; • ipossia alternata ad episodi di riossigenazione della placenta; • danno ipossico al trofoblasto villoso; • rilascio di frammenti di membrana del sinciziotrofoblasto (STBM, SyncytioTrophoBlast Membrane fragments) nella circolazione materna; • risposta infiammatoria materna in seguito alla presenza degli STBM (Huppertz 2008a).

Prime fasi dello sviluppo embrionale

Morula

Massa cellulare interna

Blastocisti

Progenitrici dello stroma placentare Progenitrici dell’embrione Progenitrici del trofoblasto

Lo sviluppo precoce del trofoblasto

Huppertz

Hypertension

2008,51:970

Morula

Prima differenziazione verso la linea trofoblastica

Blastocisti

fallimento

PE & FGR

Differenziazione in citotrofoblasto e sinciziotrofoblasto fallimento

PE & FGR

Precoce citotrofoblasto e sinciziotrofoblasto Differenziazione verso Trofoblasto villoso

PE

fallimento

Figura 3.1

Citotrofoblasto villoso e sinciziotrofoblasto villoso Differenziazione verso Trofoblasto extravilloso fallimento Citotrofoblasto extravilloso

FGR

Due vie differenti alla PE e all’IUGR

Huppertz

Hypertension

2008,51:970

Trophoblast lineage and PE\IUGR origin

Towards trophoblast lineage Failure •Abortion •PE+IUGR Towards cytotrophoblast and syncytiotrophoblast Towards villous trophoblast Failure PREECLAMPSIA Towards extravillous trophoblast Failure IUGR Huppertz

Hypertension

2008,51:970, mod

Due forme di Pre-eclampsia

• • • •

EARLY ONSET

Inadequate trophoblast invasion Abnormal uterine arteries blod flow Abnormal umbelical arteries blood flow Intrauterine growth restriction (IUGR)

LATE ONSET

• Normally grown baby • Normal uterine spiral arteries • Normal umbelical arteries blood flow • Enlarged placental mass

Il passaggio di materiale trofoblastico nel sangue materno Huppertz e Kingdom

J Soc Gynecol Investig

2004,11:353 Burton, 2009 Placental debris

Stress or necrosis in maternal-fetal surface Increased expression of Damage Associated Molecular Pattern (DAMPs) (DNA, heat shock proteins, uric acid, etc) Activation of dendritic cells The danger model suggests that, if preeclampsia is an immune disease, the initial factor is not the fetal antigen recognition, but rather recognition of DAMPs generated by poor placentation

L’ipotesi sulla origine placentare della PE

Huppertz

Hypertension

2008,51:970, mod INTRINSIC PLACENTAL FACTORS Increased Placental Mass Surface Inadequate Maternal Response or Removal increased adequate Apoptosis Overload of Apoptotic Removal

Syncytial Knots

Secondary Necrosis of Apoptotic Particles Engulfment of Apoptotic Particles in the Lung

PREECLAMPSIA NORMAL PREGNANCY

inadequate Aponecrosis Non-apoptotic Trophoblast Fragments Sistemic Effects of Necrotic Material

PREECLAMPSIA

Fattori intrinseci placentari Fattori estrinseci Fattori materni Aumento della massa/superficie Placentare: -diabete -gravidanza multipla -ipossia (anemia, elevate altitudini) aumentano Inadeguata risposta o rimozione materna Sovraccarico della rimozione apoptotica Secondaria necrosi delle particelle apoptotiche Citotrofoblasto Villoso differenziazione adeguata inadeguata Sinciziotrofoblasto Villoso differenziazione adeguata Apoptosi Syncytial Knots inadeguata Aponecrosi/Necrosi STBM Eliminazione delle particelle apoptotiche a livello polmonare Effetti sistemici del materiale necrotico PREECLAMPSIA Gravidanza fisiologica PREECLAMPSIA

Figura 3.2

HANDBUCH DER GEBURTSHILFE Doderlin A. (Editor), Wiesbaden, 1916 Eklampsie ZWEIFEL P.

“Eclampsia is a disease of theories”

OGGI: fallimento della differenziazione del trofoblasto villoso con successivo rilascio esagerato di materiale trofoblastico all’interno della circolazione materna.

STBM responsabili di una risposta infiammatoria materna esagerata con conseguente esordio della preeclampsia

• Nella patologia preeclamptica il rilascio del materiale proveniente dal sinciziotrofoblasto non segue le normali regole, infatti, a causa di una alterazione precoce nel processo di differenziazione del trofoblasto villoso, il rilascio dei “syncytial knots” non rappresenta più il principale meccanismo di eliminazione a disposizione, ma predominano altri processi come la necrosi e l’aponecrosi (Formigli 2000). • L’aumento dei frammenti necrotici del trofoblasto si riscontra esclusivamente nella preeclampsia, mentre nelle gravidanze complicate da FGR i livelli sono normali (Goswami 2006). • I frammenti trofoblastici necrotici rilasciati, chiamati STBM, hanno dimensioni tali (200-600 nm) (Gupta 2005) da poter oltrepassare il filtro polmonare indenni, in modo tale da persistere nel sangue periferico materno, determinando alterazioni sistemiche a carico dell’endotelio materno e del processo infiammatorio.

THE RELATION BETWEEN TROPHOBLAST DEVELOPMENT AND MARKER APPEARANCE

 Biochemical markers of preeclampsia are circulating factors, whose measurement could potentially aid its diagnosis or prediction  Some are products of trophoblast or of the adjacent decidua and reflect the placental dysfunction which is an important aspect of the pathogenesis of pre-eclampsia  Others reflect the systemic responses of maternal systems to abnormal pregnancy that may be inflammatory or metabolic.

Trophoblast and Villous Development Pregnancy Implantation Delivery Maternal blood flow through the placenta Mesenchymal Villi (4 to 23 weeks) Immature Intermediate Villi (8 to 23 weeks) Villous growth Villous stabilization Nutritional exchange Stem Villi (17/20 to 40 weeks) Mature Intermediate Villi (25 to 40 weeks) Terminal Villi (27 to 40 weeks) from 34 w the dominating type of villi By courtesy of P. Kaufmann

Angiogenic Factors • PlGF • (VEGF) • sFlt-1 • sEndoglin where do they come from?

Trophoblast Development and Marker Appearance

Villous Trophoblast

-

Cytotrophoblast

- PlGF - Flt-1 - Endoglin

Extravillous Trophoblast

- PlGF - sFlt-1 - sEndoglin (?) -

Syncytiotrophoblast

- PlGF - sFlt-1 - sEndoglin

Maternal Tissues

- PlGF - sFlt-1 - sEndoglin - VEGF

What about marker appearance in maternal blood?

sFlt-1 in maternal blood

Changes in circulating level of angiogenic factors from the first to second trimester as predictors of preeclampsia.

Vatten LJ, Eskild A, Nilsen TI, Jeansson S, Jenum PA, Staff AC Am J Obstet Gynecol. 2007 Mar;196(3):239.e1-6.

PlGF in maternal blood

Changes in circulating level of angiogenic factors from the first to second trimester as predictors of preeclampsia.

Vatten LJ, Eskild A, Nilsen TI, Jeansson S, Jenum PA, Staff AC Am J Obstet Gynecol. 2007 Mar;196(3):239.e1-6.

PP13 in the placenta

Galectin, highly expressed in syncitiotrophoblast.

Involved in placental implantation and maternal artery remodelling

Trophoblast Development and Marker Appearance

Villous Trophoblast

-

Cytotrophoblast

- PlGF - Flt-1 - Endoglin

Extravillous Trophoblast

- PlGF - sFlt-1 - sEndoglin (?) -

Syncytiotrophoblast

- PlGF - sFlt-1 - sEndoglin - PP13

Maternal Tissues

- PlGF - sFlt-1 - sEndoglin - VEGF

PP13 in maternal blood 5.1 – 56.6 weeks after LMP maternal serum Nutritional exchange

Longitudinal determination of serum placental protein 13 during development of preeclampsia.

Huppertz B, Sammar M, Chefetz I, Neumaier-Wagner P, Bartz C, Meiri H.

Fetal Diagn Ther 24:230-6 (2008) Weakness: Only 4 late-PE cases -> 51 blood samples with 41 normal cases -> 440 blood samples and 18 unaffected cases -> 169 blood samples

Other Placental Markers for Preeclampsia in Maternal Blood

• Cell-free fetal DNA (fetal): elevated levels starting at 17 weeks • ADAM12 (maternal/fetal; low in trisomies 18 and 21): decreased levels in the first trimester, interfers with adhesion and implantation • PAPP-A (maternal/fetal?): widely used in trisomy screening, a protease for IGFBP-4, decreased levels cause decreased levels of free IGF.

Decreased levels in the first trimester? only 8-23% of PE cases can be detected, not specific for PE (IUGR)

Trophoblast Development and Marker Appearance

Villous Trophoblast

-

Cytotrophoblast

- PlGF - Flt-1 - Endoglin - fetal DNA/RNA - ADAM12 - PAPP-A

Extravillous Trophoblast

- PlGF - sFlt-1 - sEndoglin (?) - fetal DNA/RNA - ADAM12 (?) - PAPP-A (?)

Maternal Tissues

-

Syncytiotrophoblast

- PlGF - sFlt-1 - sEndoglin - PP13 - fetal DNA/RNA - ADAM12 - PAPP-A - PlGF - sFlt-1 - sEndoglin - VEGF

Trophoblast Development and Marker Appearance

Villous Trophoblast

-

Cytotrophoblast

- PlGF - Flt-1 - Endoglin - fetal DNA/RNA - ADAM12 - PAPP-A

Extravillous Trophoblast

- PlGF - sFlt-1 - sEndoglin (?) - fetal DNA/RNA - ADAM12 (?) - PAPP-A (?) -

Syncytiotrophoblast

- PlGF - sFlt-1 - sEndoglin - PP13 - fetal DNA/RNA - ADAM12 - PAPP-A

Maternal Tissues

- P-selectin - Pentraxin-3 - PlGF - sFlt-1 - sEndoglin - VEGF

PTX3: belongs to famiy of pentraxins, involved in inflammation, like CRP A multifunctional soluble Pattern Recognition Receptor

(Garlanda C., Ann. Rev. Immunol. 2005)

CELLULAR SOURCES OF PTX3

(Garlanda C., Ann. Rev. Immunol. 2005)

PTX3 as a novel diagnostic tool: a working hypothesis

A new marker for inflammatory disorders reflecting in particular the involvement of the vascular bed.

(Peri G, Circulation 2000) (Klouche M, Atherosclerosis 2004)

Pre-existing maternal endothelial dysfunction.

Excessive proinflammatory status (cytokines)

Metabolic syndrome Obesity Insulin resistance Hyperglycemia Hyperlipidemia

PREECLAMPSIA

Placental insufficiency

IUGR

PTX3 levels throughout the three trimesters in normal pregnancies compared to non-pregnant women.

PTX3 levels increase in pregnancy, but do not change significantly throughout the three trimesters of normal pregnancies.

PTX3 single values with respective median levels. p<0.001

Cetin I. et al. Am.J.Obstet.Gynecol. 2006

PTX3 maternal levels in preeclampsia and IUGR vs normal pregnancies.

*** * **

PTX3 levels in preeclampsia are significantly higher compared to normal pregnancies of corresponding gestational age. IUGR showed intermediate levels.

n=26 n=16 n=20

Maternal plasma median levels and interquartile range.

***p<0.001; **p<0.01; *p<0.05

Cetin I. et al. Am.J.Obstet.Gynecol. 2006

Patrizia Rovere-Querini et al.

Plasma and Tissue Expression of the Long Pentraxin 3 During Normal Pregnancy and Preeclampsia

Obstet Gynecol 2006;108:148 –55)

Fig. 2 - Higher circulating levels of PTX3 in patients with preeclampsia.

16 14 12 10 4 2 8 6 0

PTX3 maternal levels in relation to severity of preeclampsia

The severity of the

***

disease is associated

**

to maternal PTX3 levels.

***

AGA mild PE severe PE

n=32 n=15 n=30

Maternal plasma median levels. °°° p<0.001; **p<0.01

Cetin I. et al. manuscript in preparation

Placenta: stroma of stem villi and anchoring villi Normal Preeclampsia

NO DIFFERENCES for immunostaining (brown) for PTX3 localization in the stroma of the stem villi (panel a-c) and in the anchoring villi (panels b-d) in normal (a-b) and preeclamptic (c-d) placentas.

Maternal peritoneum

PTX3 localization in the parietal peritoneum in a case of IUGR. Strong

endothelial signal

a preeclamptic case.

in the peritoneum of

HYPOTHESIS PTX3: New

marker

of altered endothelial function, typical of preeclampsia.

 Causal or consequential role in pregnancy complications?

 Early biomarker for the prediction of adverse pregnancy outcome?

I Cetin, V Cozzi, AT Papageorghiou, V Maina, A Montanelli, C Garlanda, B Thilaganathan

First trimester PTX3 levels in women who subsequently develop preeclampsia and fetal growth restriction

Acta Obstetricia et Gynecologica Scandinavica,2009; 88:7,846 — 849

Prospective maternal plasma collection from pregnant women at 11-14 week of gestation. Outcome: preeclampsia, IUGR

Table I - Maternal demographic characteristics and pregnancy outcomes in the study groups.

I Cetin, V Cozzi, AT Papageorghiou, V Maina, A Montanelli, C Garlanda, B Thilaganathan

First trimester PTX3 levels in women who subsequently develop preeclampsia and fetal growth restriction

Acta Obstetricia et Gynecologica Scandinavica,88:7,846 — 849

PTX3 levels are elevated in women who subsequently develop preeclampsia, but are normal in those with IUGR .

PTX3 levels as mean (diamonds) ± SD (box) and respective range (whiskers). *** p<0.001.

I Cetin, V Cozzi, AT Papageorghiou, V Maina, A Montanelli, C Garlanda, B Thilaganathan

First trimester PTX3 levels in women who subsequently develop preeclampsia and fetal growth restriction

Acta Obstetricia et Gynecologica Scandinavica,88:7,846 — 849

The median CRP levels did not vary significantly between the three groups (p=0.26)

R Akolekar, D Casagrandi, P Livanos, A Tetteh and K H Nicolaides

Maternal plasma pentraxin 3 at 11 to 13 weeks of gestation in hypertensive disorders of pregnancy

Prenat Diagn (2009) Table 2 —Median (interquartile range) for plasma pentraxin 3 and uterine artery pulsatility index (PI) in the outcome groups

R Akolekar, D Casagrandi, P Livanos, A Tetteh and K H Nicolaides

Maternal plasma pentraxin 3 at 11 to 13 weeks of gestation in hypertensive disorders of pregnancy

Prenat Diagn (2009) Fig.1 - Association between log pentraxin 3 (PTX3) multiple of the median (MoM) and log uterine artery pulsatility index (PI) MoM in the preeclampsia group

Baschat A et al

PTX-3 levels at 11 to 14 weeks' gestation: association with maternal and placental characteristics

Am J Obstet Gynecol (2009)

• 111 pregnant women at 11-14 weeks • PTX 3 levels range: 0.2 – 13.8 ng/ml • Correlation analyses failed to show significant relationship with gestational age or CRL • no relationship with maternal age or BMI • no relationship with uterine artery Doppler or umbilical artery Doppler

I trimester

Pre-existing maternal endothelial dysfunction.

II-III trimester

Leukocytes/platelets activation Decidualisation and Implantation (Popovici 2006,Tranguch 2007, Hess 2007), Excessive inflammatory response to pregnancy (TNFa-IL-1, IL-6) Complement

PTX3-C1q

activation Coagulation activation Apoptosys

PTX3 Θ FGF-2

PTX3 gene locally upregulated

Angiogenesis disregulation (s-flt-1 VEGF) PREECLAMPSIA

Placental insufficiency

IUGR

Unbalanced vasodilating/ vasoconstrictor molecules Clinical manifestations

Conclusions

•

Only very few markers are available that show significant changes already in the first trimester of pregnancy: PP13, PTX3, ADAM12, PAPP-A .

–

sFlt-1, PlGF ?

•

These markers include proteins released from maternal cells as well as from fetal trophoblast.

•

Angiogenic markers do not show consistent alterations early in pregnancy. However, they may have a value to assess severity of the disorder later in pregnancy.

•

Some markers such as PP13 and maybe sFlt-1 and sEndoglin show a biphasic concentration pattern in preeclampsia:

–

1. Low in the first trimester

–

2. Reaching normal levels at about mid gestation

–

3. Overshooting at the onset of clinical symptoms