Tumorigenic Factor CRIPTO-1 Is Immunolocalized in ......2 BioMedResearchInternational...

Research ArticleTumorigenic Factor CRIPTO-1 Is Immunolocalized inExtravillous Cytotrophoblast in Placenta Creta

Carla Letiacutecia Bandeira1 Alexandre Urban Borbely1 Rossana Pulcineli Vieira Francisco2

Regina Schultz3 Marcelo Zugaib2 and Estela Bevilacqua1

1 Department of Cell and Developmental Biology Institute of Biomedical Sciences University of Sao PauloAvenida Professor Lineu Prestes No 1524 Cidade Universitaria 05508-900 Sao Paulo SP Brazil

2 Department of Obstetrics Clinics Hospital School of Medicine University of Sao Paulo Avenida Dr Eneas de Carvalho AguiarNo 255 Cerqueira Cesar 05403-000 Sao Paulo SP Brazil

3 Division of Pathology Clinics Hospital School of Medicine University of Sao Paulo Avenida Dr Eneas de Carvalho AguiarNo 255 Cerqueira Cesar 05403-000 Sao Paulo SP Brazil

Correspondence should be addressed to Estela Bevilacqua bevilacquspbr

Received 14 March 2014 Accepted 10 June 2014 Published 6 August 2014

Academic Editor Kosmas Paraskevas

Copyright copy 2014 Carla Letıcia Bandeira et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

CRIPTO-(CR)1 is a protein associated with tumorigenesis andmetastasis Here we demonstrate that CR-1 expression in normal andcreta placentas is associated with various degrees of uterine invasion Cytokeratin (CK) and CR-1 protein expression was visualizedby immunohistochemical staining of formalin-fixed paraffin-embedded placental specimens (control placentas 119899 = 9 accreta119899 = 6 increta 119899 = 10 percreta 119899 = 15) The pattern of extravillous trophoblast (EVT) cell morphology was distinctive increta placentas densely-compacted cell columns and large star-shaped cells with a typically migratory phenotype not commonamong third trimester control placentas Quantification revealed higher CR-1 immunoreactivities in accreta (119875 = 0001) increta(119875 = 00002) and percreta placentas (119875 = 0001) than in controls In contrast to controls there was a significant positiverelationship between CR-1 and CK reactivity in all creta placentas (accreta 119875 = 002 increta 119875 = 00001 and percreta 119875 = 0025)This study demonstrated CR-1 expression in the placental bed its increased expression in creta placentas and EVT cells asthe main CR-1-producing cell type Morphological examination revealed an immature and invasive trophoblast profile in cretaplacentas suggesting impairment of the trophoblast differentiation pathway These findings provide important new insights intothe pathophysiology of abnormal creta placentation and its gestational consequences

1 Introduction

Abnormal placentation is one of the most common preg-nancy complications and placenta creta is a common con-comitant it is closely associated with the need for hysterec-tomy and its consequences can lead to maternal death [1ndash5]Placenta creta was originally diagnosed in 1930 [6] and itsincidence has increased over the years (1 2510 in 1980 [7] and1 533 in 2002 [8]) closely associated with the incidence ofplacenta previa and increasing number of Cesarean sections[8ndash12]

Total or partial absence of decidual tissue reaction is acommon histological characteristic of placenta creta so the

placental villi insert directly into the myometrium [13ndash15]The pathogenesis of placenta creta remains to be elucidatedbut evidence indicates that it is primarily a maternal diseaseassociated with decidual deficiency which can be due touterine scarring and with secondary consequences for theregulatory mechanisms of trophoblast invasion and function[14 16 17]

Considered a fully iatrogenic pathology [18] placentacreta is currently classified according to the depth of abnor-mal adhesion and invasion of the chorionic villi to themyometrium in the absencedeficiency of decidualizationtaking into consideration whether the placental insertion issuperficial or deep and whether or not it transcends the

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 892856 9 pageshttpdxdoiorg1011552014892856

2 BioMed Research International

serous layer to reach adjacent structures such as the bladderand ureters [6 13 14 19] These descriptions characterize thesubtypes of creta placentas as accreta increta and percretarespectively [14ndash16] Abnormal invasion into the deeperlayers of the myometrium is accompanied by a distinctiveplacental neovascularization In consequence exacerbatedvascular remodeling usually reaches the radial arcuate andparametrial arteries increasing the caliber of these vesselswhich become barely capable of homeostatic response afterplacental abruption [20ndash23]

The factors responsible for invasive placental activity dur-ing normal and pathological placentation are not completelyunderstood at the cellular level Impairment of regulatorysignaling between these cells and the cellular and noncellulardecidual components has been strongly proposed along withmodulation of the expression of for example growth factorshormones cytokines adhesion molecules and oncogenes bythe components of the maternal-fetal interface [23ndash26]

Data obtained through cDNA microarray analysis ofmouse placentas have demonstrated that the CRIPTO-1oncogene is highly expressed at the maternal-fetal interface[27] CRIPTO-1 is a member of the epidermal growthfactor-CRIPTO-1FRL-1Cryptic (EGFCFC) family abun-dantly expressed in embryonic stem cells and tumor cells[28 29] Furthermore it is overexpressed in various primaryhuman carcinomas (breast lung colon gastric pancreasovary cervix endometrium and testis) [30 31] suggestinga role in tumorigenesis particularly in angiogenesis andinvasiveness [28 31]

Considering that creta placentas are characterized bya prominent deviation of villous invasion we hypothesizethat CRIPTO-1 is expressed by the invasive placental pop-ulation and we examine its expression at the maternal-fetalinterface using immunohistochemistry Creta placentas ofvarious degrees and placentas from healthy gestations werequantitatively and qualitatively analyzed and compared

2 Materials and Methods

21 Sample Collection Paraffin blocks of formalin-fixed pla-centa samples were selected from the archives of the Divisionof Pathology at Clinics Hospital School of Medicine Univer-sity of Sao Paulo They included six maternal-fetal interfaceareas from placenta accreta (from 36 weeks of gestation) 10maternal-fetal interface areas from placenta increta and 15samples from placenta percreta (37-38 weeks of gestation)obtained from immediate postpartum hysterectomy Control(non-creta) cases consisted of nine third trimester placentas(119899 = 3 36 gestation weeks [gw] and 119899 = 6 38 gw) fromelective cesareans from healthy mothers and fetuses (withoutchronic hypertension renal disease vascular disease infec-tion fetal anomalies or any other pregnancy complications)Maternal risk factors for placentas creta are summarized inTable 1

The placentas were conventionally diagnosed as accreta(superficially implanted) increta (within the myometrium)and percreta (through the myometrium) by morphologicalexamination using clear evidence of loss of decidua and the

Table 1 Maternal risk factors for placentas creta incidence

Accreta Increta Percreta Normal119899 = 6 119899 = 10 119899 = 15 119899 = 9

Prior Gestation (number of gestations)

(1-2) 33 20 40 78(ge3) 67 80 60 11

Prior uterine surgery lowast 100 100 100 89C-section 83 90 93 89(number of surgerieslowast) (1-2) (2ndash5) (1ndash4) (1-2)

Age ge 35 yr 50 40 33 22Placenta praevia 66 70 80 0Praevia + C-section 66 60 80 0Prior abortion 66 70 33 0

(range) (1ndash3) (1ndash4) (1) 0lowastIncluding curettage

degree of myometrial adhesion as criteria The study wasapproved by the Ethics Committee for Human Research atthe School of Medicine University of Sao Paulo

Because the gestational age differed between the control(healthy) and pathological (accreta increta and percreta)placenta groups respective gestational age-matched groupswere used as controls (placentas of 36 gw for placenta accretaand placentas of 38 gw for placenta increta and percreta)

22 Immunohistochemistry The paraffin blocks were semise-rially sectioned at 5120583m intervals and mounted on slides andprocessed for immunohistochemical staining Standard con-ditions included immunostaining of three separate groupssubjected to the same experimental conditions (i) a groupcontaining the accreta placentas and age-matched normalplacentas (36 gw) (ii) a group containing increta and percretaplacentas and the age-matched controls (38 gw) and (iii)a group comprising healthy placentas from 36 and 38 gwThe primary antibodies were rabbit polyclonal IgGs againsthuman CRIPTO-1 protein (Santa Cruz Biotechnology SantaCruz CA) cytokeratin LMW and vimentin (Cell MarqueCorporation CA EUA) respectively diluted at 1 100 1 350and 1 100 Goat anti-rabbit and goat anti-mouse IgG (KPLKirkegaard amp Perry Laboratories Inc USA) were used assecond antibodies at 1 100 dilutions The antigens in the sec-tions were visualized using aDAB substrate kit for peroxidase(Vector Laboratories Inc CA) Slides were counterstainedwith Mayerrsquos hematoxylin Sections from each placentalgroup were used as negative controls with the primaryantibody replaced with Tris-buffered saline or nonimmunerabbit serum

23 Quantitative and Statistical Analysis Images of theimmunoreactions were acquired and captured using anAxioskop 2 Optical Microscope equipped with Axio Vision47 software (Carl Zeiss MicroImaging GmbH Jena Ger-many) Quantification was performed on images capturedusing a times 10 objective 1388 times 1040 pixels and a resolution of

BioMed Research International 3

150120583m

(a)

100120583m

(b)

150120583m

(c)

50120583m

(d)

200120583m

(e)

75120583m

(f)

75120583m

(g)

150120583m

(h)

50120583m

(i)

50120583m

(j)

Figure 1 Histological characterization of healthy and creta placentas Representative histological sections revealing cytokeratin-reactivecells in the placental bed of (andashd) term healthy placenta (36 gw) and (endashj) creta placentas (accreta (e) (g) and (i) percreta (f) (h) and(j)) Note cytokeratin-reactive cells (brownish color) disposed in healthy placentas as a group of large polygonal cells that apparently donot maintain contact with other cells (andashd) In (b) note a typical multinucleate trophoblast cell (endashj) In creta placenta samples cytokeratin-positive cells are organized as group of compact cells (endashg) or as isolated star-shaped cells (i-j) Arrows indicate multinucleate trophoblastcells (h) Immunoperoxidase Mayerrsquos hematoxylin counterstaining

four pixels120583m2 Five images from each slide from five paraf-fin blocks randomly selected for each group were capturedresulting in 25 images per group for comparison

Using computer-assisted image analysis (ImageJ NIHUSA) the density of cytokeratin and CRIPTO-1 immunore-activity wasmeasured in each field of each slice and averagedThe microscopic area was calculated as 90220120583m2 and theresults were recorded as pixels120583m2 The ratios of CRIPTO-1 to cytokeratin were calculated in each field and the meanswere statistically compared

The package PRISM Graph Pad for PC (version 50)was used for statistical analyses A nonparametric one-way

ANOVA (Kruskal-Wallis) with Dunnrsquos multiple comparisonposttest was used for all-group comparisons Results wereexpressed as mean plusmn SD The level of significance was set at119875 lt 005

3 Results

To assess similar areas of the maternal-fetal interfaceamong the samples each tissue was stained with hema-toxylineosin (HE) (data not shown) and further character-ized by vimentin cytokeratin and CRIPTO-1 immunostain-ing


Vm

36w

200120583m

lowastlowastlowast

(a)

lowastlowast

CK

(b)

lowast

CR-1

(c)

38w

(d) (e) (f)

38w

(g) (h) (i)

Figure 2 Expression of CRIPTO-1 and cell markers in healthy placentas (A) Representative histological sections demonstrating theimmunolocalization of vimentin (Vm a d) cytokeratin (CK b e) and CRIPTO-1 (CR-1 c f) in placental beds from healthy placentasat 36 (andashc) and 38 (dndashf) weeks of gestation Arrowheads indicate cells reactive to cytokeratin and CRIPTO-1 in semiserial histologicalsections Asterisks identify vascular areas stained for vimentin and CRIPTO-1 (a c) but not for cytokeratin (b) (gndashi) Negative control ofthe immunohistochemistry reactions in which the respective primary antibody has been omitted Immunoperoxidase Mayerrsquos hematoxylincounterstaining Bar in (a) = 200 120583m in all figures

31 Placental Bed inHealthy Placentas Deciduae of placentasfrom healthy gestations obtained at 36 and 38 weeks ofgestation were characterized by a face in which villi wereattached contiguously to connective tissue containing dif-ferent cell populations This region which denominated thebasal plaque comprises decidual cells leukocytes compo-nents of the endometrial vasculature and cytotrophoblastcells represented as interstitial and endovascular extravillouscells dispersed in a distinctive extracellular matrix Thedecidual vasculature was represented by a net of distendedlumen capillaries and more structured vessels in the deeperareas of the decidua No morphological differences could befound among samples from these three gestational periods(data not shown)

Immunolocalization of cytokeratin and vimentin inter-mediate filaments in tissue sections of the basal plaque

allowed us to characterize the populations of trophoblast(Figures 1-2) and nontrophoblast (Figure 2) cells

Extravillous cytokeratin-positive cytotrophoblast cellswere scattered throughout the decidua (Figures 1(a)ndash1(d))Although this antigen is expressed by all cells of epithelialorigin the absence of maternal luminalglandular epitheliumduring the final stages of gestation usually makes cytok-eratin reactivity a suitable marker for locating extravilloustrophoblast cells among the other decidual cells (decidualcells stromal cells macrophages lymphocytes and naturalkiller cells are cytokeratin-negative) Cytokeratin-reactivecells comprised a variable proportion of large-dimensionpolygonal cells (Figures 1(a)ndash1(d)) and occasional multinu-cleated cells dispersed in diffuse sheets or nests (Figure 1(b))These cells displayed euchromatic nuclei with prominentnucleoli Cytokeratin-positive cells lining thematernal vessels


100120583m

CK

CR-1

C

Increta Percreta

(A) (B) (C)

(D) (E) (F)

(G) (H) (I)

(J) (K) (L)

Vm

Accreta

(a)

16

12

8

4

0

C36w

CKCR1CR1CK

Accreta

a1 b2 c2a1 b1lowast c1

Imm

unos

tain

ing

(pix

els120583

m2)

(b)

24

18

12

6

0

C38w Increta Percreta

a2 b3lowastc2

a1 b1lowastc1

CKCR1CR1CK

a2 b2lowastc2

Imm

unos

tain

ing

(pix

els120583

m2)

(c)

Figure 3 Expression of CRIPTO-1 and cell markers in creta placentas (a) Representative histological sections demonstrating immunolocal-ization of cytokeratin (CK AndashC) CRIPTO-1 (CR-1 DndashF) and vimentin (Vm GndashI) in representative cases of accreta (A D G and J) increta(B E H and K) and percreta (C F I and L) placentas The arrowheads indicate cells reactive to cytokeratin and CRIPTO-1 in semiserialhistological sections Arrows depict vimentin-positive cells ((c) JndashL) Negative control of the immunohistochemistry reactions in which therespective primary antibody has been omitted Immunoperoxidase Mayerrsquos hematoxylin counterstaining Bar in ((a)(A)) = 100 120583m in allfigures (b-c) Quantification of the immunoreactivity (pixels120583m2) for cytokeratin (CK) and CRIPTO-1 (CR-1) proteins at the maternal-fetalinterface in placentas from healthy mothers (gestation week 36) and accreta placentas (b) and of healthy placentas (gestation week 38) andincreta and percreta placentas (c) Different superscript letters above the bars indicate the group statistically analyzed means with differentnumbers are significantly different 119875 lt 005 whereas means with similar numbers do not differ Asterisks indicate significant differences inrelation to CK in the same group (119875 lt 005) The results of the analysis are given in the text


were also common (Figure 1(a))mainly in deeper areas of thedecidua

Cells exhibiting morphological characteristics similar toCK-reactive extravillous cytotrophoblast cells (Figures2(b) and 2(e)) were the main intensely CRIPTO-1-immunoreactive cell type in decidua (Figures 2(c) and2(f)) at both 36 and 38 gw Some endothelial cells inthe deeper portions of the decidua were also CRIPTO-1immunoreactive (Figures 2(a) and 2(c))

Quantification of cytokeratin (CK)- and CRIPTO-1 (CR-1)-reactive cells in the placental bed from healthy gestations(Figures 3(b) and 3(c)) revealed a significant differencebetween CK and CR-1 immunointensities at gestation weeks36 (1185 plusmn 189 and 892 plusmn 078 resp 119875 = 0001) and 38(275 plusmn 043 and 222 plusmn 037 resp 119875 = 0002) Howeverthere was no significant difference in the CR-1CK ratio (36w077 plusmn 018 38 w 081 plusmn 016)

32 Maternal-Fetal Interface Areas in Creta Placentas Thematernal-fetal interface in creta placentas (Figure 3) wascharacterized by endometrialmyometrialperimetrial hem-orrhage leukocyte infiltration areas of leakage and necrosisand almost total absence of decidual cells The examinationswere mainly performed on the transitional area between theatrophic endometrium and myometrium in accreta placentaand in the myometrium in increta and percreta placentas

In all specimens the vimentin antibody stained endothe-lial cells leukocytes and fibroblasts (Figures 3(a) (G)ndash(I))Cytokeratin-positive cytotrophoblast cells permeated musclecells and were morphologically different from those found inhealthy placentas They were either organized as a compactgroup of histologically and immunophenotypically homoge-nous cells (resembling tightly packed colonies Figures 1(e)ndash1(g)) or were sparsely distributed (Figures 1(h)ndash1(j)) Isolatedcells displayed migratory characteristics exhibiting star-shaped cytoplasm and long projections (Figures 1(h)ndash1(j))

The increta placentas had similar characteristicsalthough the cytokeratin-reactive cytotrophoblast invasionreached deeper layers of the myometrium In placentapercreta cytokeratin-reactive cells were also found liningthe perimetrium Cytokeratin-reactive cell aggregates oftensurrounded andor lined the uterine vessels

CRIPTO-1 colocalized withmost of the large cytokeratin-reactive cells in the placental bed (Figure 3(a)) at all levelsin the creta placentas here analyzed However CRIPTO-1expression was not exclusive to this cell population Endothe-lial and myometrial cells were also immunoreactive to theanti-CRIPTO-1 antibody

Quantification of cytokeratin- and CRIPTO-1-reactivecells in the placental bed demonstrated significantly higherimmunointensity for CR-1 (1367 plusmn 155 119875 = 0001) and forthe ratio CR-1CK (161 plusmn 053 119875 = 002) but not for CK(1046 plusmn 497) in accreta placentas than in the age-matchedcontrol group (Figure 3(b))

The intensity of CR-1-reactive cells was higher in incretaand percreta placentas (Figure 3(c)) than in the respectiveCK-reactive cell population (1254 plusmn 2 versus 909 plusmn 311119875 = 0008 and 1822 plusmn 426 119875 = 004) and higher than

in the age-matched control (119875 lt 005) The CR-1CK ratiowas approximately 2-fold higher in the pathological placentas(increta 147 plusmn 035 and percreta 165 plusmn 054 119875 lt 005) thanin the controls

4 Discussion

Abnormal placentation is one of the most common preg-nancy complications and creta placentas appear extensivelyamong them they are closely associated with the need forhysterectomy with consequences that can lead to maternaldeath [1ndash5] Creta placentas are becoming more commonwith their incidence increasing over the years (1 2510 in1980 [7] and 1 533 in 2002 [8]) Several factors have beenimplicated in this augmentation primarily the increasingincidence of placenta previa the increasing proportion ofdeliveries by caesarean and the rising maternal age atdelivery (gt35 years) [8ndash12 16 18] In this study our selectedpathological groups exhibited many of the risk factors singlyor in association all had some kind of prior uterine surgeryand almost all (60ndash80) had cesarean sections and placentaprevia

Despite the factors or combination of factors that increasethe risk for placenta creta its exact etiology is still unknownIn the present study we found using immunohistochemistryincreasedCRIPTO-1 expression in the termplacental bed andin creta placentas exhibiting different degrees of abnormalimplantation relative to normal placentation Moreover wedescribed for the first time that this expression is particularlyassociatedwith cellsmorphologically characterized as extrav-illous cytotrophoblast cells

In the placental bed CRIPTO-1 expression colocalizedwith cytokeratin-reactive cells in the semiserial sectionssuggesting that extravillous cytotrophoblast cells are themainCRIPTO-producers at this site We believe that our findingscould underscore the specific roles of trophoblast cells at thematernal-fetal interface CRIPTO-1 signaling within tumorcells has previously been demonstrated to modulate cellulargrowth survival and invasion in several human cancers [3032 33] and this could be especially relevant to the biology oftrophoblast cells In particular extravillous cytotrophoblastcells are nonproliferative and exhibit a low apoptotic indexduring the late stages of gestation which suggests the hypoth-esis that CRIPTO-1 contributes to trophoblast invasiveness orcell survival mechanisms [34] As a survival factor CRIPTO-1 acts through a phosphoinositol-3 kinase (PI3 K-) dependentsignaling pathway involving AKT and GSK-3120573 [35] whichcould be an active mechanism in trophoblast cells [36]Further studies are required to elucidate the mechanismsunderlying CRIPTO-dependent responses in trophoblastcells

The ratio of CRIPTO-1cytokeratin reactive cells inhealthy placentas indicates that not all CK+ trophoblast cellsexpress this factor however this relationship is significantlydifferent in creta placentas In line with this we foundCRIPTO-1 to be expressed in these placentas extrapolatingthe reactivity to the trophoblast cell population and alsoin the endothelial and myometrial cells CRIPTO-1 was


abundant in percreta and accreta placentas and less abundantin increta placentaThese data suggest a relationship betweenCRIPTO-1 and the overall degree of placental invasiveness inwhich trophoblast cells are of pivotal importance Moreoverthis finding adds one more item to the list of similaritiesbetween trophoblast and cancer cells

Our quantitative data also highlight variations in theCK-reactive cell population in the placental bed duringthe last weeks of a healthy gestation which is corrob-orated by previous studies [14] and the proportionalitybetween CK and CRIPTO-1 reactivities during those finalgestational stages Interestingly our previous study demon-strated that the extravillous trophoblast cells retained somecapacity for migration and invasion though it was lessthan in first trimester placentas [37] These findings rein-force the conclusion that CRIPTO-1 mostly expressed inpotentially invasive cells is associated with this cellularactivity

Our quantitative analysis demonstrated increased inten-sity of CK+ cells in increta and percreta placental bedsperhaps because there are more trophoblast cells Few studieshave addressed this possibility Ki-67 staining is rarely seenin the extravillous trophoblast indicating low proliferation[38] though a significantly thicker layer of implantation-site intermediate trophoblast and more extravillous cytotro-phoblast cells have also been reported [39] An increasednumber of cells could result from a critical imbalancebetween trophoblast cell proliferation and death leading tothe accumulation of this specific cell population over timeand this could explain our results at least in part Howeveranother possible explanation is the distribution of trophoblastcells in these pathologies The absence of decidua could leadto an atypical invasion process in which trophoblast cellswould form a more compact front of invasion [39] unlike thesituation in a normal pregnancy when these cells are betterdistributed along the endometrial maternal-fetal interface

Our histological study also revealed different patterns ofCK+ trophoblast cell distribution and morphology in cretaplacentas Overall these placentas had cells organized asconfluent groups resembling epithelium-like cells growingcohesively as compact islands These arrangements are sim-ilar to the cytotrophoblast cell column organization foundduring the first trimester in normal placentas [14] but notin the third trimester The cohesive arrangement of thesecells could suggest a coordinate organization of daughtercells following division though they could also result frompost-division aggregations In any case cell-cell interactionscould be supported by the expression of distinctive adhesiveproteins or specific intercellular junctions which is atypicalbehavior for the third trimester extravillous cytotrophoblastGiven the severity and outcome complications of thesepathologies more detailed studies should be conducted toclarify the behavior of these cells at cellular subcellular andmolecular levels

Cells from accreta placentas also include multinucleategiant cells and cells with invasive morphological charac-teristics Large star-shaped cells presenting long projectionsdistributed among the myometrial fibers seem to replace thepolygonal cells found in normal placentas Maintenance of

the invasive phenotype in accreta placentas was suggestedby Kim et al [39] and again reveals characteristics normallyfound during very early pregnancy

In summary the morphological features of the extravil-lous cell population in the placental bed of accreta placentassuggest that the differentiation characteristics of earlier stageshave been maintained Under this perspective regardless ofthe factors contributing to this invasive profile (absence ofdecidual regulatory factors eg) it could partially explain theabnormal invasion by creta placentas

The mechanisms underlying the expression of CR-1 inplacentas and particularly in extravillous trophoblast cellsare still to be studied However experimental studies usingtumor cells have demonstrated that CR-1 is closely regu-lated by transforming growth factor (TGF)-120573 superfamilymembers and particularly by TGF-1205731 and bone morpho-genetic protein (BMP)-4 both expressed by endometrial cells[40] TGF-1205731 upregulates CR-1 expression whereas BMP-4 downregulates it [41] Therefore control of the balancebetween these two factors is relevant to CR-1 expressionand activity and could be markedly changed by endometrialimpairment with absencedefect of decidua as seen in cretaplacentas

Taking these findings together we suggest that CRIPTO-1 is part of the mechanism that leads to abnormal placentaldevelopment Furthermore these data provide importantnew insights into the pathophysiology of creta placentationaffording possibilities for studying its underlying mecha-nisms and gestational consequences

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] WHO World Health organization ldquoMaternal mortalityrdquo FactSheet 348 WHO 2012

[2] G Daskalakis E Anastasakis N Papantoniou S MesogitisMTheodora and A Antsaklis ldquoEmergency obstetric hysterec-tomyrdquoActaObstetricia et Gynecologica Scandinavica vol 86 no2 pp 223ndash227 2007

[3] S T Bauer and C Bonanno ldquoAbnormal Placentationrdquo Seminarsin Perinatology vol 33 no 2 pp 88ndash96 2009

[4] C S Shellhaas S Gilbert M B Landon et al ldquoThe frequencyand complication rates of hysterectomy accompanying cesareandeliveryrdquoObstetrics and Gynecology vol 114 no 2 pp 224ndash2292009

[5] M S Hoffman R A Karlnoski D Mangar et al ldquoMorbidityassociated with nonemergent hysterectomy for placenta acc-retardquo The American Journal of Obstetrics and Gynecology vol202 no 6 pp 628e1ndash628e5 2010

[6] C Irvinig and A Hertig ldquoA study of placenta accretardquo SurgeryGynecology amp Obstetrics vol 64 pp 178ndash200 1937

[7] G Pridjian J U Hibbard and A H Moawad ldquoCesareanchanging the trendsrdquo Obstetrics and Gynecology vol 77 no 2pp 195ndash200 1991


[8] S Wu M Kocherginsky and J U Hibbard ldquoAbnormal placen-tation twenty-year analysisrdquoAmerican Journal of Obstetrics andGynecology vol 192 no 5 pp 1458ndash1461 2005

[9] D A Miller J A Chollet and T M Goodwin ldquoClinicalrisk factors for placenta previa-placenta accretardquoThe AmericanJournal of Obstetrics and Gynecology vol 177 no 1 pp 210ndash2141997

[10] S Sofiah and Y C Fung ldquoPlacenta accreta clinical riskfactors accuracy of antenatal diagnosis and effect on pregnancyoutcomerdquo Medical Journal of Malaysia vol 64 no 4 pp 298ndash302 2009

[11] E A S Clark and R M Silver ldquoLong-term maternal morbidityassociated with repeat cesarean deliveryrdquoThe American Journalof Obstetrics amp Gynecology vol 205 pp S2ndashS10 2011

[12] IM Usta EMHobeika A A AbuMusa G E Gabriel andAH Nassar ldquoPlacenta previa-accreta risk factors and complica-tionsrdquo The American Journal of Obstetrics and Gynecology vol193 no 3 pp 1045ndash1049 2005

[13] H Fox and N J Sebire Eds Pathology of the Placenta WBSaunders Philadelphia Pa USA 3rd edition 2007

[14] K Benirschke P Kaufmann and R N Baergen Pathology ofthe Human Placenta Springer New York NY USA 5th edition2006

[15] F T Kraus R W Redline D J Gerseil D M Nelson and JM Dicke ldquoDisordesrs of placental developmentrdquo in Atlas ofNontumor Pathology Placental Pathology D W King Ed pp47ndash74 Armed Forces Institute of Pathology Washington DCUSA 2004

[16] T Y Khong ldquoThe pathology of placenta accreta a worldwideepidemicrdquo Journal of Clinical Pathology vol 61 no 12 pp 1243ndash1246 2008

[17] A Al-Khan I L Aye I Barsoum et al ldquoIFPA Meeting2010 Workshops Report II placental pathology trophoblastinvasion fetal sex parasites and the placenta decidua andembryonic or fetal loss trophoblast differentiation and syncy-tialisationrdquo Placenta vol 32 no 2 pp S90ndashS99 2011

[18] E Jauniaux and D Jurkovic ldquoPlacenta accreta pathogenesis ofa 20th century iatrogenic uterine diseaserdquo Placenta vol 33 no4 pp 244ndash251 2012

[19] M A Belfort ldquoPlacenta accretardquo The American Journal ofObstetrics amp Gynecology vol 203 pp 430ndash439 2010

[20] T Y Khong and W B Robertson ldquoPlacenta creta and placentapraevia cretardquo Placenta vol 8 no 4 pp 399ndash409 1987

[21] M M Chou J J Tseng and E S C Ho ldquoThe applicationof three-dimensional color power Doppler ultrasound in thedepiction of abnormal uteroplacental angioarchitecture in pla-centa previa percretardquo Ultrasound in Obstetrics amp Gynecologyvol 19 no 6 pp 625ndash627 2002

[22] J Shih W Cheng M Shyu C Lee and F Hsieh ldquoPowerDoppler evidence of placenta accreta appearing in the firsttrimesterrdquo Ultrasound in Obstetrics and Gynecology vol 19 no6 pp 623ndash625 2002

[23] J J Tseng M M Chou Y T Hsieh M C Wen E S C Hoand S L Hsu ldquoDifferential expression of vascular endothelialgrowth factor placenta growth factor and their receptors inplacentae from pregnancies complicated by placenta accretardquoPlacenta vol 27 no 1 pp 70ndash78 2006

[24] R Ohlsson ldquoGrowth factors protooncogenes and human pla-cental developmentrdquo Cell Differentiation and Development vol28 no 1 pp 1ndash16 1989

[25] J Muhlhauser C Crescimanno P Kaufmann H Hofler DZaccheo and M Castellucci ldquoDifferentiation and proliferationpatterns in human trophoblast revealed by c-erbB-2 oncogeneproduct and EGF-Rrdquo Journal of Histochemistry and Cytochem-istry vol 41 no 2 pp 165ndash173 1993

[26] D Marzioni L Capparuccia T Todros A Giovannelli and MCastellucci ldquoGrowth factors and their receptors fundamentalmolecules for human placental developmentrdquo Italian Journal ofAnatomy and Embryology vol 110 no 2 pp 183ndash187 2005

[27] M S Hoshida R Gorjao C Lima S Daher R Curi andE Bevilacqua ldquoRegulation of gene expression in mouse tro-phoblast cells by interferon-gammardquo Placenta vol 28 no 10pp 1059ndash1072 2007

[28] K Watanabe M J Meyer L Strizzi et al ldquoCripto-1 is a cell sur-face marker for a tumorigenic undifferentiated subpopulationin human embryonal carcinoma cellsrdquo Stem Cells vol 28 no 8pp 1303ndash1314 2010

[29] M C Rangel H Karasawa N P Castro T Nagaoka D SSalomon and C Bianco ldquoRole of Cripto-1 during epithelial-to-mesenchymal transition in development and cancerrdquo TheAmerican Journal of Pathology vol 180 no 6 pp 2188ndash22002012

[30] D S Salomon C Bianco A D Ebert et al ldquoThe EGF-CFC family novel epidermal growth factor-related proteins indevelopment and cancerrdquo Endocrine-Related Cancer vol 7 no4 pp 199ndash226 2000

[31] E D Adamson G Minchiotti and D S Salomon ldquoCripto atumor growth factor and morerdquo Journal of Cellular Physiologyvol 190 no 3 pp 267ndash278 2002

[32] C Bianco L Strizzi A Ebert et al ldquoRole of human cripto-1in tumor angiogenesisrdquo Journal of the National Cancer Institutevol 97 no 2 pp 132ndash141 2005

[33] K Watanabe C Bianco L Strizzi et al ldquoGrowth factorinduction of cripto-1 shedding by glycosylphosphatidylinositol-phospholipase D and enhancement of endothelial cell migra-tionrdquo Journal of Biological Chemistry vol 282 no 43 pp 31643ndash31655 2007

[34] S L Straszewski-Chavez V M Abrahams and G Mor ldquoTherole of apoptosis in the regulation of trophoblast survival anddifferentiation during pregnancyrdquo Endocrine Reviews vol 26no 7 pp 877ndash897 2005

[35] A D Ebert C Wechselberger S Frank et al ldquoCripto-1 inducesphosphatidylinositol 31015840-kinase-dependent phosphorylation ofAKT and glycogen synthase kinase 3120573 in human cervicalcarcinoma cellsrdquoCancer Research vol 59 no 18 pp 4502ndash45051999

[36] J Pollheimer T Loregger S Sonderegger et al ldquoActivationof the canonical winglessT-cell factor signaling pathway pro-motes invasive differentiation of human trophoblastrdquoTheAmer-ican Journal of Pathology vol 168 no 4 pp 1134ndash1147 2006

[37] A U Borbely S Sandri I R Fernandes et al ldquoThe term basalplate of the human placenta as a source of functional extravil-lous trophoblast cellsrdquo Reproductive Biology and Endocrinologyvol 12 article 7 2014

[38] P Tantbirojn C P Crum and M M Parast ldquoPathophysi-ology of placenta creta the role of decidua and extravilloustrophoblastrdquo Placenta vol 29 no 7 pp 639ndash645 2008

[39] K Kim S Jun J Kim and J Y Ro ldquoImplantation site interme-diate trophoblasts in placenta cretasrdquoModern Pathology vol 17no 12 pp 1483ndash1490 2004


[40] R L Jones C Stoikos J K Findlay and L A Salam-onsen ldquoTGF-120573 superfamily expression and actions in theendometrium and placentardquo Reproduction vol 132 no 2 pp217ndash232 2006

[41] M Mancino L Strizzi C Wechselberger et al ldquoRegulationof human Cripto-1 gene expression by TGF-1205731 and BMP-4 in embryonal and colon cancer cellsrdquo Journal of CellularPhysiology vol 215 no 1 pp 192ndash203 2008


serous layer to reach adjacent structures such as the bladderand ureters [6 13 14 19] These descriptions characterize thesubtypes of creta placentas as accreta increta and percretarespectively [14ndash16] Abnormal invasion into the deeperlayers of the myometrium is accompanied by a distinctiveplacental neovascularization In consequence exacerbatedvascular remodeling usually reaches the radial arcuate andparametrial arteries increasing the caliber of these vesselswhich become barely capable of homeostatic response afterplacental abruption [20ndash23]

The factors responsible for invasive placental activity dur-ing normal and pathological placentation are not completelyunderstood at the cellular level Impairment of regulatorysignaling between these cells and the cellular and noncellulardecidual components has been strongly proposed along withmodulation of the expression of for example growth factorshormones cytokines adhesion molecules and oncogenes bythe components of the maternal-fetal interface [23ndash26]

Data obtained through cDNA microarray analysis ofmouse placentas have demonstrated that the CRIPTO-1oncogene is highly expressed at the maternal-fetal interface[27] CRIPTO-1 is a member of the epidermal growthfactor-CRIPTO-1FRL-1Cryptic (EGFCFC) family abun-dantly expressed in embryonic stem cells and tumor cells[28 29] Furthermore it is overexpressed in various primaryhuman carcinomas (breast lung colon gastric pancreasovary cervix endometrium and testis) [30 31] suggestinga role in tumorigenesis particularly in angiogenesis andinvasiveness [28 31]

Considering that creta placentas are characterized bya prominent deviation of villous invasion we hypothesizethat CRIPTO-1 is expressed by the invasive placental pop-ulation and we examine its expression at the maternal-fetalinterface using immunohistochemistry Creta placentas ofvarious degrees and placentas from healthy gestations werequantitatively and qualitatively analyzed and compared

2 Materials and Methods

21 Sample Collection Paraffin blocks of formalin-fixed pla-centa samples were selected from the archives of the Divisionof Pathology at Clinics Hospital School of Medicine Univer-sity of Sao Paulo They included six maternal-fetal interfaceareas from placenta accreta (from 36 weeks of gestation) 10maternal-fetal interface areas from placenta increta and 15samples from placenta percreta (37-38 weeks of gestation)obtained from immediate postpartum hysterectomy Control(non-creta) cases consisted of nine third trimester placentas(119899 = 3 36 gestation weeks [gw] and 119899 = 6 38 gw) fromelective cesareans from healthy mothers and fetuses (withoutchronic hypertension renal disease vascular disease infec-tion fetal anomalies or any other pregnancy complications)Maternal risk factors for placentas creta are summarized inTable 1

The placentas were conventionally diagnosed as accreta(superficially implanted) increta (within the myometrium)and percreta (through the myometrium) by morphologicalexamination using clear evidence of loss of decidua and the

Table 1 Maternal risk factors for placentas creta incidence

Accreta Increta Percreta Normal119899 = 6 119899 = 10 119899 = 15 119899 = 9

Prior Gestation (number of gestations)

(1-2) 33 20 40 78(ge3) 67 80 60 11

Prior uterine surgery lowast 100 100 100 89C-section 83 90 93 89(number of surgerieslowast) (1-2) (2ndash5) (1ndash4) (1-2)

Age ge 35 yr 50 40 33 22Placenta praevia 66 70 80 0Praevia + C-section 66 60 80 0Prior abortion 66 70 33 0

(range) (1ndash3) (1ndash4) (1) 0lowastIncluding curettage

degree of myometrial adhesion as criteria The study wasapproved by the Ethics Committee for Human Research atthe School of Medicine University of Sao Paulo

Because the gestational age differed between the control(healthy) and pathological (accreta increta and percreta)placenta groups respective gestational age-matched groupswere used as controls (placentas of 36 gw for placenta accretaand placentas of 38 gw for placenta increta and percreta)

22 Immunohistochemistry The paraffin blocks were semise-rially sectioned at 5120583m intervals and mounted on slides andprocessed for immunohistochemical staining Standard con-ditions included immunostaining of three separate groupssubjected to the same experimental conditions (i) a groupcontaining the accreta placentas and age-matched normalplacentas (36 gw) (ii) a group containing increta and percretaplacentas and the age-matched controls (38 gw) and (iii)a group comprising healthy placentas from 36 and 38 gwThe primary antibodies were rabbit polyclonal IgGs againsthuman CRIPTO-1 protein (Santa Cruz Biotechnology SantaCruz CA) cytokeratin LMW and vimentin (Cell MarqueCorporation CA EUA) respectively diluted at 1 100 1 350and 1 100 Goat anti-rabbit and goat anti-mouse IgG (KPLKirkegaard amp Perry Laboratories Inc USA) were used assecond antibodies at 1 100 dilutions The antigens in the sec-tions were visualized using aDAB substrate kit for peroxidase(Vector Laboratories Inc CA) Slides were counterstainedwith Mayerrsquos hematoxylin Sections from each placentalgroup were used as negative controls with the primaryantibody replaced with Tris-buffered saline or nonimmunerabbit serum

23 Quantitative and Statistical Analysis Images of theimmunoreactions were acquired and captured using anAxioskop 2 Optical Microscope equipped with Axio Vision47 software (Carl Zeiss MicroImaging GmbH Jena Ger-many) Quantification was performed on images capturedusing a times 10 objective 1388 times 1040 pixels and a resolution of


150120583m

(a)

100120583m

(b)

150120583m

(c)

50120583m

(d)

200120583m

(e)

75120583m

(f)

75120583m

(g)

150120583m

(h)

50120583m

(i)

50120583m

(j)






3 Results



Vm

36w

200120583m

lowastlowastlowast

(a)

lowastlowast

CK

(b)

lowast

CR-1

(c)

38w

(d) (e) (f)

38w

(g) (h) (i)







100120583m

CK

CR-1

C

Increta Percreta

(A) (B) (C)

(D) (E) (F)

(G) (H) (I)

(J) (K) (L)

Vm

Accreta

(a)

16

12

8

4

0

C36w

CKCR1CR1CK

Accreta


Imm

unos

tain

ing

(pix

els120583

m2)

(b)

24

18

12

6

0


a2 b3lowastc2

a1 b1lowastc1

CKCR1CR1CK

a2 b2lowastc2

Imm

unos

tain

ing

(pix

els120583

m2)

(c)













4 Discussion

















References













































150120583m

(a)

100120583m

(b)

150120583m

(c)

50120583m

(d)

200120583m

(e)

75120583m

(f)

75120583m

(g)

150120583m

(h)

50120583m

(i)

50120583m

(j)






3 Results



Vm

36w

200120583m

lowastlowastlowast

(a)

lowastlowast

CK

(b)

lowast

CR-1

(c)

38w

(d) (e) (f)

38w

(g) (h) (i)







100120583m

CK

CR-1

C

Increta Percreta

(A) (B) (C)

(D) (E) (F)

(G) (H) (I)

(J) (K) (L)

Vm

Accreta

(a)

16

12

8

4

0

C36w

CKCR1CR1CK

Accreta


Imm

unos

tain

ing

(pix

els120583

m2)

(b)

24

18

12

6

0


a2 b3lowastc2

a1 b1lowastc1

CKCR1CR1CK

a2 b2lowastc2

Imm

unos

tain

ing

(pix

els120583

m2)

(c)













4 Discussion

















References













































Vm

36w

200120583m

lowastlowastlowast

(a)

lowastlowast

CK

(b)

lowast

CR-1

(c)

38w

(d) (e) (f)

38w

(g) (h) (i)







100120583m

CK

CR-1

C

Increta Percreta

(A) (B) (C)

(D) (E) (F)

(G) (H) (I)

(J) (K) (L)

Vm

Accreta

(a)

16

12

8

4

0

C36w

CKCR1CR1CK

Accreta


Imm

unos

tain

ing

(pix

els120583

m2)

(b)

24

18

12

6

0


a2 b3lowastc2

a1 b1lowastc1

CKCR1CR1CK

a2 b2lowastc2

Imm

unos

tain

ing

(pix

els120583

m2)

(c)













4 Discussion

















References













































100120583m

CK

CR-1

C

Increta Percreta

(A) (B) (C)

(D) (E) (F)

(G) (H) (I)

(J) (K) (L)

Vm

Accreta

(a)

16

12

8

4

0

C36w

CKCR1CR1CK

Accreta


Imm

unos

tain

ing

(pix

els120583

m2)

(b)

24

18

12

6

0


a2 b3lowastc2

a1 b1lowastc1

CKCR1CR1CK

a2 b2lowastc2

Imm

unos

tain

ing

(pix

els120583

m2)

(c)













4 Discussion

















References























































4 Discussion

















References
























































References












































Tumorigenic Factor CRIPTO-1 Is Immunolocalized in ......2 BioMedResearchInternational...

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Transcript of Tumorigenic Factor CRIPTO-1 Is Immunolocalized in ......2 BioMedResearchInternational...