research article ecto-nucleotidases activities in the...

Research ArticleEcto-nucleotidases Activities in the Contents of OvarianEndometriomas Potential Biomarkers of Endometriosis

Laura Texidoacute1 Claudia Romero1 August Vidal123 Joseacute Garciacutea-Valero4

M Eulalia Fernaacutendez Montoli25 Nuacuteria Baixeras23 Enric Condom123 Jordi Ponce25

Amparo Garciacutea-Tejedor25 and Mireia Martiacuten-Satueacute12

1 Departament de Patologia i Terapeutica Experimental Facultat de Medicina Universitat de Barcelona Campus de BellvitgeLrsquoHospitalet de Llobregat 08907 Barcelona Spain

2 Institut drsquoInvestigacio Biomedica de Bellvitge (IDIBELL) Barcelona Spain3 Servei drsquoAnatomia Patologica Hospital de Bellvitge Barcelona Spain4Departament de Biologia Celsdotlular Facultat de Biologia Universitat de Barcelona Barcelona Spain5 Servei de Ginecologia Hospital de Bellvitge LrsquoHospitalet de Llobregat 08907 Barcelona Spain

Correspondence should be addressed to Amparo Garcıa-Tejedor agarciatbellvitgehospitalcat andMireia Martın-Satue martinsatueubedu

Received 29 May 2014 Accepted 8 August 2014 Published 3 September 2014

Academic Editor Jean Sevigny

Copyright copy 2014 Laura Texido et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Endometriosis defined as the growth of endometrial tissue outside the uterus is a common gynecologic condition affectingmillionsof women worldwide It is an inflammatory estrogen-dependent complex disorder with broad symptomatic variability pelvic painand infertility being themain characteristics Ovarian endometriomas are frequently developed in womenwith endometriosis Latediagnosis is one of the main problems of endometriosis thus it is important to identify biomarkers for early diagnosis The aimof the present work is to evaluate the ecto-nucleotidases activities in the contents of endometriomas These enzymes through theregulation of extracellular ATP and adenosine levels are key enzymes in inflammatory processes and their expression has beenpreviously characterized in human endometrium To achieve our objective the echo-guided aspirated fluids of endometriomaswereanalyzed by evaluating the ecto-nucleotidases activities and compared with simple cysts Our results show that enzyme activitiesare quantifiable in the ovarian cysts aspirates and that endometriomas show significantly higher ecto-nucleotidases activitiesthan simple cysts (55-fold increase for ATPase and 20-fold for ADPase) thus being possible candidates for new endometriosisbiomarkers Moreover we demonstrate the presence of ecto-nucleotidases bearing exosomes in these fluids These results add upto the knowledge of the physiopathologic mechanisms underlying endometriosis and open up a promising new field of study

1 Introduction

Endometriosis is a chronic condition characterized by thepresence of endometrial tissue (stroma and glands) outsidethe uterine cavitymostly on pelvic peritoneum and ovaries Itaffects up to 15 of women of reproductive age and its preva-lence is rising It is an inflammatory estrogen-dependentcomplex disorder with broad symptomatic variability pelvicpain and infertility being the main characteristics Bothsymptoms are thought to be the result of an excessiveinflammatory environment not onlywithin the pelvis but alsoin the eutopic endometrium affecting implantation [1 2]

It is one of the principal causes of infertility in womenLate diagnosis is one of the main problems of this pathologytaking between five and ten years from detection of the firstsymptoms Therefore there is a need to identify biomarkersfor early diagnosis of endometriosis [3]

Ovarian endometriomas occur in 17ndash44 of patientsaffected by endometriosis [4] and account for 35 of benignovarian cysts Endometriomas are also called endometrioticor ldquochocolate cystsrdquo because of the internal fluid bloodderived dark color [5] Although the risk of malignanttransformation is low 06ndash08 [6] surgery has become thegold standard therapy Nowadays laparoscopic cystectomy is

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 120673 8 pageshttpdxdoiorg1011552014120673

2 Mediators of Inflammation

the most extended treatment for endometriomas althoughfertility could be afterwards compromised Some studieshave reported a lower ovarian reserve after excision ofovarian endometriomas due to incidental excision of normalovarian tissue together with the endometrioma wall [7 8]There is also evidence of a significant postoperative decreasein circulating Anti-Mullerian hormone a commonly usedbiomarker for ovarian reserve quantification suggesting anegative impact on ovarian reserve after the excision ofendometriomas [8] Therefore a new minimally invasiveinterventional technique was implemented to treat low riskmalignancy ovarian cysts including endometriomas theultrasound-guided aspiration followed by sclerosis whichseems to have even a low recurrence risk (0ndash15) [9ndash13] The sclerosis does not seem to have consequences onthe number of pregnancies term pregnancies abortionsextracted oocytes embryo quality and hormonal levels ifcompared with infertile women without ovarian cysts [11 14]

The analysis of the contents of endometriomas can helpidentify new biomarkers for this pathology For exampleendometriotic cysts contain significantly more iron thanother ovarian cysts this has been suggested as a possiblecause of carcinogenesis in endometriomas through iron-induced persistent oxidative stress [15] Proteomic analysisof follicular fluid of women with endometriosis has alsopointed to an altered immunologic function possibly relatedto infertility [16]

Purinergic signalling the group of biologic effects exertedby extracellular nucleotides such as ATP and nucleosidessuch as adenosine through specific receptors is an impor-tant regulatory mechanism in a wide range of inflamma-tory conditions [17 18] ATP is a ldquodanger signalrdquo releasedunder tissue stress conditions such as necrosis apoptosishypoxia and inflammation and is rapidly converted intoadenosine which protects cells and tissues from excessiveinflammation and immune-mediated damage The effectstransduced by both molecules are thus often opposite andthe resulting cellular responses are the consequence of theratio of ATP (and ADP) to adenosine concentrations whichis mainly controlled by ecto-nucleotidases key enzymes inthe purinergic signalling [19] The most well-characterizedectonucleotidase pathway involves the sequential action ofCD39 (ectonucleoside triphosphate diphosphohydrolase-1NTPDase1) which converts extracellular ATP (or ADP)to AMP and CD73 (ecto-51015840-nucleotidase) which generatesadenosine from AMP [20ndash23] We have previously char-acterized the expression of endometrial ecto-nucleotidasesdemonstrating changes along the cycle and in menopause[24 25] We have also demonstrated that CD39 and CD73are highly expressed in endometrial cancer [26] contributingto the increased immunosuppressive levels of extracellularadenosine found in tumor microenvironment

Exosomes are small secreted membrane vesicles (30ndash100 nm in diameter) of endocytic origin produced by manydifferent cell types that are formed by the fusion ofmultivesic-ular endosomeswith the plasmamembraneThey are thoughtto play an important role in intercellular communication forexample during immune cell-cell interaction and have beenidentified in biological fluids such as plasma and urine as

well as in cell culture supernatants [27 28] The presence ofthe ecto-nucleotidases CD39 and CD73 has been previouslydemonstrated in cancer exosomes [29ndash31] contributing tothe adenosine production that suppresses T-cell function

In the present work we aimed to determine whetherecto-nucleotidases activities (ATPase ADPase and AMPase)are present and measurable in the ultrasound-guided aspi-rated contents of ovarian cysts and whether these activitiesare altered in endometriotic cysts compared with simplecysts affecting the inflammatory condition of endometriosisOur goal is to evaluate their potential as biomarkers forendometriosis and to contribute to the knowledge of thephysiopathologic mechanisms underlying endometriosis

2 Materials and Methods

21 Samples The ethical principles of this study adhere to theDeclaration ofHelsinki and all the procedureswere approvedby the ethics committee for clinical investigation of BellvitgeHospital A prospective cohort of 27 adnexal cysts with alow risk of malignancy including 14 endometriomas and 13ovarian simple cysts was studied at the Gynecology Serviceof Bellvitge Hospital (Barcelona Spain) between March 2013and April 2014

Inclusion criteria in the study for patients were (1) agedgt18 years old (2) ultrasound features predictive of a lowrisk of cyst malignancy smooth walls no solid componentmaximum diameter between 40 and 100mm no more thanone thin septation (lt2-3mm) and no internal flow on colorDoppler imaging in the case of bilocular cysts according toIOTA criteria [32] (3) cyst persistence from diagnosis ge6months (4) tumor marker Ca 125 lt 200 IUmL and Ca199 lt 35 IUmL and (5) written informed consent from thepatients

For ultrasound-guided aspiration following the abdo-men or the vagina disinfection a sterilized 17G spinal needle(BD Medical Franklin Lakes NJ Becton Dickinson andCompany) was aimed at the center of the cyst under directultrasound guidance and the contents was aspirated Whenthick highly dense intracystic fluid was present in orderto facilitate the aspiration saline dilutions were performedDilutionsmadewere then considered in the final calculationsThen several intracyst saline washes were performed untilclearance followed by the instillation of ethanol as sclerosingagentThevolume of instilled ethanol was 23 of the aspiratedvolume of the endometrioma and always less than 100mLEthanol was left inside the cyst for 15min and then removedand washed out again with a saline solution The intracysticfluids were analyzed cytologically at the Pathology Service ofBellvitge Hospital to confirm the absence of atypical cells

Patients were divided into two groups following theultrasound features and internal color fluid (a) endometri-omas group that had homogeneous appearance involvingdiffuse internal echoes on a hypoechoic background andcontained blood color fluid and (b) simple cysts group thathad an anechoic structure and contained clear serous fluidTable 1 includes the descriptive statistics of the two groups of

Mediators of Inflammation 3

Table 1 Descriptive statistics of the two groups of patients and samples

Age (years) Size (mm) Volume (mL)Simple cyst (119873 = 13)

Mean (SD) 398 (135) 6954 (1402) 6985 (5936)Range 20ndash64 49ndash98 20ndash248

Endometrioma (119873 = 14)Mean (SD) 386 (141) 7371 (3326) 11250 (11177)Range 28ndash84 42ndash170 20ndash410

The size represents the largest cystic diameter measured on ultrasound The volume represents the total aspirated volume of intracystic fluid (SD standarddeviation)

patients including the age and the size and the volume of thecysts

An aliquot of each sample was maintained on ice untilusage for exosome enrichment The rest was aliquoted andstored at minus80∘C

22 Exosome Enrichment Endometriomas and simple cystsfluid samples were centrifuged at 3000timesg for 15min toremove cells and cell debris and supernatants were trans-ferred to sterile tubes 250 120583L of each supernatant was mixedwith 63 120583L of ExoQuick Exosome Precipitation Solution(SystemBiosciencesMountainView CAUSA) by inversionand the mixture was refrigerated overnight at 4∘C followingthe supplierrsquos instructions Samples were then centrifugedat 1500timesg for 30min After centrifugation the exosomesappeared as a beige or white pellet at the bottom of thetube Supernatant was aspirated and the residual ExoQuicksolution was spun down by centrifugation at 1500timesg for5min All traces of fluid were removed by aspiration andexosome pellets were resuspended in two different buffersFor Western blot analysis exosome pellets were resuspendedin 75 120583L of a buffer containing 10mM Tris-HCl pH 74150mMNaCl and 1 100 proteases inhibitor cocktail (Sigma-Aldrich Saint Louis MO USA) Alternatively for ecto-nucleotidases activity assays exosome pellets were resus-pended in 75120583Lof enzyme assay buffer (160mMTris-HCl pH74 10mMCaCl

2) Protein concentration in exosome samples

was determined by the method of Bradford [33] Sampleswere stored at minus20∘C until usage

23 Western Blotting Analysis 15 120583g of protein was heated at95∘C for 3min loaded onto any kD Mini-PROTEAN TGXPrecast Gel (Bio-Rad Hercules CA USA) and transferred topolyvinylidene fluoride (PVDF)membranes using the Trans-Blot Turbo Transfer System (Bio-Rad) Blots were blockedwith Tris-buffered saline with 01 Tween-20 (TBS-T) con-taining 5 nonfat dry milk for 1 hour at room temperature(RT) Membranes were then incubated overnight at 4∘C withthe following antibodies exosomal marker antibodies CD9CD63 CD81 HSP70 (diluted 1 1000) from the ExoAB-KIT-1 Western blot antibody detection kit (System Biosciences)anti-CD39 (clone BU61 diluted 1 500) and anti-CD73 (clone4G4 fromAbcam diluted 1 50) Blots were then washed withTBS-T and incubated with the appropriate HRP-conjugatedsecondary antibody (System Biosciences dilution 1 20000)

for 1 hour at RT Membranes were then incubated withAmersham ECL Western Blotting Detection Reagents (GEHealthcare Pittsburgh PA USA) for 1min and exposed onX-ray films

24 Nucleotidase Activity Assays Nucleotidase activities(ATPase ADPase and AMPase) were determined in flat-bottom 96-well plates by measuring the amount of liberatedinorganic phosphate (Pi) using the malachite green colori-metric assay [34] All the samples were used for these assaysThe incubation mixture contained 160mM TrisndashHCl pH 7410mM CaCl

2 5mM levamisole as alkaline phosphatases

inhibitor and 1mM ATP ADP or AMP as substrates in afinal volume of 150 120583L The assay was initiated by addingthe sample either the fluid directly (5120583L for ATPase andADPase assays and 10 120583L for AMPase assay) or 50 120583g of theisolated exosome fraction Alternatively increasing amountsof exosomes were added to the well to assess if ectonucleoti-dase activity depended on the exosome dose (5 10 15 2030 and 50 120583g) After 20minutes of incubation at 37∘C 50 120583Lof malachite green solution was added to each well and 10and 20minutes later the OD

620was measured and recorded

Incubation times and protein concentrations were chosen toensure the linearity of the enzymatic reaction KH

2PO4was

used as a Pi standard Controls to determine nonenzymaticPi accumulation were performed by incubating either thesamples in the absence of the substrate or the substratealone Each experiment was performed three times and ineach experiment samples were analyzed in triplicate Enzymeactivity was expressed as pmols Pimin120583L in the case of thewhole fluids and as pmol Pimin120583g for exosomes

25 ElectronMicroscopy Studies For morphological analysiswhole-mount electron microscopy preparations of exosomeswere obtained as previously described by Thery et al [35]In brief exosome-containing pellets were resuspended in2 paraformaldehyde in 100mM phosphate-buffered saline(PBS) 5 120583L of resuspended pellets was deposited on formvar-coated grids and incubated at RT for 20min After thoroughrinses the samples were postfixed in 1 glutaraldehyde for5min and then contrasted in a solution of 2 uranyl acetate150mM oxalic acid pH 7Then the grids were transferred to adrop of 18 methyl-cellulose 04 uranyl acetate for 10minon ice Finally the grids were removed with stainless steelloops and air-dried for additional 10min


(a) (b)

Figure 1 Transvaginal ultrasound-guided aspiration of an endometrioma (a) Endometrioma measured before aspiration Dotted lineindicates one of the diameter measurements (b) Endometrioma being aspirated The arrow indicates the end of the needle inside the cystNote the reduction in the volume after aspiration

Alternatively and in order to obtain thin sectionsthe paraformaldehyde-fixed exosome-containing pellets werepostfixed in 25 glutaraldehyde and 2 paraformaldehydein 100mM PBS dehydrated in a graded ethanol series andembedded in the Spurr low viscosity resin Ultrathin sections(50ndash60 nm) from exosome-enriched pellets were cut witha diamond knife in an ultramicrotome UltracutE (LeicaMicrosystems) After being picked up on copper grids theultrathin sections were stained with uranyl acetate and leadcitrate Finally exosomes were observed with a JEM-1010transmission electron microscope (JEOL Japan) and theimages were recorded with a Bio Scan 792 camera (GATANGermany)

26 Statistical Analysis Statistical analysis was performedusing SigmaStat 32 software (SPSS Inc Chicago IL USA)Values are reported as the mean plusmn SEM Studentrsquos 119905-test wasused to compare the means of two independent groups ofnormally distributed data

3 Results and Discussion

Most of the attempts to identify peripheral biomarkers ofendometriosis have been focused on their presence in plasmaserum or urine [3] However the study of endometriomascontents has been proven to be very informative [15 16]In the present work we have evaluated the ectonucleotidaseenzyme activities present in endometriomas in comparisonwith ovarian simple cysts

Echo-guided aspirationwas the technique used to removethe fluid contained in endometriomas and simple cystsImmediate ethanol sclerosis was performed to chemicallydestroy the cyst lining and to prevent its regrowth Figure 1shows the ultrasound picture of an endometrioma before andduring the aspiration of its contents

Aspirated fluids were then used for enzyme activitystudies Ecto-nucleotidases activities (ATPase ADPase andto a lesser extent AMPase) were present and quantifi-able in the aspirated fluid of both endometriomas andovarian simple cysts (Figure 2) Endometriomas aspirates

Enzy

me a

ctiv

ity (p

mol

Pim

in120583

L)

50

40

30

20

10

0

ATP ADP AMP

lowastlowastlowast

lowastlowastlowast

Simple cystsEndometriomas

Figure 2 Enzyme activity in fluids from simple cysts and fromendometriomas Ectonucleotidase activity represented as pmoles ofgenerated Pi in fluids from simple cysts (white columns) and fromendometriomas (gray columns) in the presence of ATP ADP orAMP as substrate ATPase and ADPase activities are significantlyhigher in endometriomas than in simple cysts (lowastlowastlowast

119875lt 0 001) while

AMPase activity was barely detectable and differences could not bedetermined

showed significantly higher (119875 lt 0 001) ATPase andADPase activities (mean plusmn SEM 3896 plusmn 511 and 3368 plusmn395 pmolmin120583L resp) than simple cysts (719 plusmn 188 and155 plusmn 04 pmolmin120583L resp) Low activity rates weredetected with AMP as substrate in the aspirated fluid andalthough endometriomas displayed higher AMPase activ-ity (128 plusmn 046 pmolmin120583L) than simple cysts (087 plusmn025 pmolmin120583L) differences were not significant Thesame results were also obtained when the activities werecalculated per 120583g of protein in the fluid with even higheractivities in endometriomas (data not shown) however sinceit is known that endometriotic cysts differ from simplecysts in protein contents [16] results are shown here as Pi


(A) (B)

(a)

Hsp70CD63

CD81 CD925

3750

75

25

37

50

75

EMSC EMSC

EMSC EMSC

(B)(A)

(C) (D)

25

37

50

75

(53kDa)(53ndash70kDa)

(26kDa) (28kDa)

25

3750

75

(b)

Figure 3 Exosome characterization (a) Representative TEM images of exosomes isolated from endometriomas (bar = 25 nm) (b) Westernblot with four different exosome markers CD63 (A) Hsp70 (B) CD81 (C) and CD9 (D) of exosome-enriched fractions from simple cysts(SC) and from endometriomas (EM) The figure shows one representative experiment of each type of the sample Molecular weight markersin kDa are indicated at the left of each image

generation per unit of volumeThis is the first time that ecto-nucleotidases have been studied in ovarian cysts althoughthese enzyme activities have been previously characterizedin other fluids such as pancreatic juice [36] and plasma[37] where there is a correlation between their activity anddifferent inflammatory pathologies [38]

Since to perform the enzyme activity assays aspiratedfluids were previously centrifuged to remove cells and celldebris the resulting activities could be attributed either tosoluble enzymes or to membrane bound ecto-nucleotidasespresent in extracellularmembrane vesicles such asmicrovesi-cles and exosomes that were not removed by the initialcentrifugation Therefore we decided to perform exosomeenrichment to assess their presence and to determine if theyhad ectonucleotidase activities To our knowledge this isthe first study demonstrating the presence of exosomes in

the fluids contained in ovarian cysts Electron microscopyanalysis confirmed the size and morphology of exosomes(Figure 3(a)) Western blots using antibodies against fourexosome markers (CD9 CD63 CD81 and Hsp70) demon-strated the presence of these structures in 100 of endometri-omas and in 46 of simple cysts aspirates (Figure 3(b)) Fromthese experiments we conclude that among the markersused CD9 is the most suitable for exosome demonstrationin the aspirated fluids of ovarian cysts since it was presentin all exosome samples analyzed CD9 has been alreadydemonstrated to be a good exosome marker for epithelialendometrial cells [39 40] All the samples expressed togetherwith CD9 at least one more of the other exosome markersassayed Interestingly using the anti-CD63 antibody the pre-dominant band in endometriomas was of a lower molecularweight than in simple cysts A possible explanation is the


(nm

ols P

i)4

3

2

1

00 10 20 30 40 50

ATPADP

Exosome dose (120583gwell)

(a)

Enzy

me a

ctiv

ity (p

mol

Pim

in120583

g)

5

4

3

2

1

0

ATP ADP AMP

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


(b)

37

50

75

100

(kDa)

CD39

37

50

75100

(kDa)

CD73

(sim56kDa)(sim63kDa)

(c)

Figure 4 Enzyme activity in exosome-enriched fractions from simple cysts and from endometriomas (a) ATPase (black circles) and ADPase(white circles) activities of increasing amounts of exosomes from endometriomas (0 5 10 20 30 and 50120583g) (b) Ectonucleotidase activityrepresented as pmoles of generated Pi in exosome-enriched fractions from simple cysts (white columns) and from endometriomas (graycolumns) in the presence of ATP ADP or AMP as substrate All the activities are significantly higher in endometriomas than in simple cysts(lowastlowastlowast119875lt 0 001) (c)Western blot with anti-CD39 and anti-CD73 antibodies of exosome-enriched fractions from an endometriomaMolecular

weight markers in kDa are indicated at the left of each image

alternative splicing already described for the gene encodingCD63 that results in multiple transcript variants encodingdifferent proteins Since this result was consistent in all thesamples studied we propose to further study its presence inexosomes from other origins in endometriosis patients toestablish if it could be used as biomarker for the pathology

Enzyme experiments performed with exosome-enrichedfractions demonstrated that these samples retain the ectonu-cleotidase activity (ATPase ADPase and AMPase) displayedby the whole fluid Moreover these activities are dependenton the sample dose as shown in Figure 4(a) in line withprevious results obtained with exosomes from other originsthat express active CD39 and CD73 [29] For ATPase andADPase maximal activity was found with 50120583g of exosomesAMPase activity which was barely detectable in the wholefluid was present in purified exosomes though only at highconcentrations (50120583g) indicating that this activity in thewhole fluid is mainly due to the enzymes bearing exosomesOn the contrary ATPase and ADPase activities would berelated to both exosome membrane bound and soluble

enzymes Figure 4(b) shows the enzyme activity using ATPADP and AMP as substrates of exosomes from simple cystsand from endometriomas These activities were significantlyhigher in endometriomas than in simple cysts Western blotanalysis demonstrated the presence of CD39 and CD73 inthese samples (Figure 4(c))

Our results reinforce the emerging evidence linkinginflammation to endometriosis pathophysiology where eva-sion of the immune surveillance seems to be crucial in the ini-tial establishment and subsequent development of displacedendometrial tissues [41] In this sense increased sequentiallyacting ectonucleotidase activities would contribute to themaintenance of the suppression of local immune responsesnecessary for the disease progression by regulating extra-cellular ATP and rising extracellular adenosine levels Wepropose to detect their presence in other more accessiblebiological fluids a fact that together with the simplicity oftheir detection would make them promising endometriosisbiomarkers


4 Conclusions

Our work contributes to the knowledge of endometriosis byproviding new data on the fluid composition of endometri-omas Ecto-nucleotidases activities are quantifiable in theovarian cysts aspirates and they are significantly increased inthe contents of endometriomas compared with simple cystsMoreover the identification of ecto-nucleotidases bearingexosomes in ovarian cyst contents opens the field for a broadrange of proteomics and genomics studies

Conflict of Interests

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

Authorsrsquo Contribution

Laura Texido Claudia Romero andAugustVidal contributedequally to this work

Acknowledgments

The authors thank the Tumour Bank of Hospital Universitaride Bellvitge (IDIBELLrsquos Biobank) for their technical assis-tanceThey also thank Inmaculada Gomez de Aranda for herexcellent technical support andGloria Gannaway for her helpwith English editing This study was supported by Institutode Salud Carlos III (Grant FIS-PI1000305) to MMS and byGeneralitat de Catalunya (Grant SGR2009152)

References

[1] S E Bulun ldquoEndometriosisrdquoNew England Journal of Medicinevol 360 no 3 pp 268ndash279 2009

[2] J A Maybin H O D Critchley and H N Jabbour ldquoInflamma-tory pathways in endometrial disordersrdquoMolecular andCellularEndocrinology vol 335 no 1 pp 42ndash51 2011

[3] K E May S A Conduit-Hulbert J Villar S Kirtley SH Kennedy and C M Becker ldquoPeripheral biomarkers ofendometriosis a systematic reviewrdquo Human ReproductionUpdate vol 16 no 6 pp 651ndash674 2010

[4] D B Redwine ldquoOvarian endometriosis a marker for moreextensive pelvic and intestinal diseaserdquo Fertility and Sterilityvol 72 no 2 pp 310ndash315 1999

[5] J A Sampson ldquoMetastatic or embolic endometriosis due to themenstrual dissemination of endometrial tissue into the venouscirculationrdquoAmerican Journal of Pathology vol 3 no 2 pp 93ndash11043 1927

[6] M Nishida K Watanabe N Sato and Y Ichikawa ldquoMalignanttransformation of ovarian endometriosisrdquo Gynecologic andObstetric Investigation vol 50 no 1 pp 18ndash25 2000

[7] L Muzii A Bianchi C Croce N Manci and P B PanicildquoLaparoscopic excision of ovarian cysts is the stripping tech-nique a tissue-sparing procedurerdquo Fertility and Sterility vol 77no 3 pp 609ndash614 2002

[8] F Raffi M Metwally and S Amer ldquoThe impact of excisionof ovarian endometrioma on ovarian reserve a systematicreview andmeta-analysisrdquo Journal of Clinical Endocrinology andMetabolism vol 97 no 9 pp 3146ndash3154 2012

[9] N Akamatsu T Hirai H Masaoka K Sekiba and TFujita ldquoUltrasonically guided puncture of endometrial cyst -Aspiration of contents and infusion of ethanolrdquo Nihon SankaFujinka Gakkai Zasshi vol 40 no 2 pp 187ndash191 1988

[10] E M Messalli G Cobellis E Pecori et al ldquoAlcohol sclerosisof endometriomas after ultrasound-guided aspirationrdquoMinervaGinecologica vol 55 no 4 pp 359ndash362 2003

[11] J D Fisch and G Sher ldquoSclerotherapy with 5 tetracycline isa simple alternative to potentially complex surgical treatmentof ovarian endometriomas before in vitro fertilizationrdquo Fertilityand Sterility vol 82 no 2 pp 437ndash441 2004

[12] C Yazbeck PMadelenat J P Ayel et al ldquoEthanol sclerotherapya treatment option for ovarian endometriomas before ovarianstimulationrdquo Reproductive BioMedicine Online vol 19 no 1 pp121ndash125 2009

[13] G Gatta V Parlato G Di Grezia et al ldquoUltrasound-guidedaspiration and ethanol sclerotherapy for treating endometrialcystsrdquo Radiologia Medica vol 115 no 8 pp 1330ndash1339 2010

[14] T Koike H Minakami M Motoyama S Ogawa H Fujiwaraand I Sato ldquoReproductive performance after ultrasound-guided transvaginal ethanol sclerotherapy for ovarianendometriotic cystsrdquo European Journal of Obstetrics Gynecologyand Reproductive Biology vol 105 no 1 pp 39ndash43 2002

[15] K Yamaguchi M Mandai S Toyokuni et al ldquoContents ofendometriotic cysts especially the high concentration of freeiron are a possible cause of carcinogenesis in the cysts throughthe iron-induced persistent oxidative stressrdquo Clinical CancerResearch vol 14 no 1 pp 32ndash40 2008

[16] E G Lo Turco F B Cordeiro P H de Carvalho Lopeset al ldquoProteomic analysis of follicular fluid from womenwith and without endometriosis new therapeutic targets andbiomarkersrdquoMolecular Reproduction and Development vol 80no 6 pp 441ndash450 2013

[17] H K Eltzschig M V Sitkovsky and S C Robson ldquoPurinergicsignaling during inflammationrdquo The New England Journal ofMedicine vol 367 no 24 pp 2322ndash2333 2012

[18] W G Junger ldquoImmune cell regulation by autocrine purinergicsignallingrdquo Nature Reviews Immunology vol 11 no 3 pp 201ndash212 2011

[19] F Kukulski S A Levesque and J Sevigny ldquoImpact of ectoen-zymes on p2 and p1 receptor signalingrdquo Advances in Pharma-cology vol 61 pp 263ndash299 2011

[20] G G Yegutkin ldquoNucleotide- and nucleoside-convertingectoenzymes important modulators of purinergic signallingcascaderdquoBiochimica et BiophysicaActaMolecular Cell Researchvol 1783 no 5 pp 673ndash694 2008

[21] M Martın-Satue E G Lavoie M Fausther et al ldquoHigh expres-sion and activity of ecto-5rsquo-nucleotidaseCD73 in the malemurine reproductive tractrdquoHistochemistry and Cell Biology vol133 no 6 pp 659ndash668 2010

[22] M Fausther J Lecka E Soliman et al ldquoCoexpression of ecto-5∘-nucleotidaseCD73 with specific NTPDases differentiallyregulates adenosine formation in the rat liverrdquo The AmericanJournal of PhysiologymdashGastrointestinal and Liver Physiologyvol 302 no 4 pp 447ndash459 2012

[23] L Antonioli P Pacher E S Vizi and G Hasko ldquoCD39 andCD73 in immunity and inflammationrdquo Trends in MolecularMedicine vol 19 no 6 pp 355ndash367 2013

[24] E Aliagas B Torrejon-Escribano E G Lavoie et al ldquoChangesin expression and activity levels of ecto-51015840-nucleotidase CD73along the mouse female estrous cyclerdquo Acta Physiologica vol199 no 2 pp 191ndash197 2010


[25] E Aliagas A Vidal B Torrejon-Escribano et al ldquoEcto-nucleotidases distribution in human cyclic and postmenopausicendometriumrdquo Purinergic Signalling vol 9 no 2 pp 227ndash2372013

[26] E Aliagas A Vidal L Texido J Ponce E Condom and MMartin-Satue ldquoHigh expression of ecto-nucleotidases CD39and CD73 in human endometrial tumorsrdquoMediators of Inflam-mation vol 2014 Article ID 509027 8 pages 2014

[27] C Thery L Zitvogel and S Amigorena ldquoExosomes composi-tion biogenesis and functionrdquoNature Reviews Immunology vol2 no 8 pp 569ndash579 2002

[28] CGutierrez-Vazquez CVillarroya-BeltriMMittelbrunn andF Sanchez-Madrid ldquoTransfer of extracellular vesicles duringimmune cell-cell interactionsrdquo Immunological Reviews vol 251no 1 pp 125ndash142 2013

[29] A Clayton S Al-Taei J Webber M D Mason and Z TabildquoCancer exosomes express CD39 and CD73 which suppress Tcells through adenosine productionrdquo Journal of Immunologyvol 187 no 2 pp 676ndash683 2011

[30] L A Smyth K Ratnasothy J Y Tsang et al ldquoD73 expressionon extracellular vesicles derived from CD4+ CD25+ Foxp3+ Tcells contributes to their regulatory functionrdquo European Journalof Immunology vol 43 no 9 pp 2430ndash2440 2013

[31] P J Schuler Z Saze C SHong et al ldquoHumanCD4(+)CD39(+)regulatory T cells produce adenosine upon co-expression ofsurface CD73 or contact with CD73(+) exosomes or CD73(+)cellsrdquo Clinical amp Experimental Immunology vol 177 no 2 pp531ndash543 2014

[32] D Timmerman L Valentin T H Bourne W P Collins HVerrelst and I Vergote ldquoTerms definitions and measurementsto describe the sonographic features of adnexal tumors aconsensus opinion from the International Ovarian TumorAnalysis (IOTA) grouprdquoUltrasound in Obstetrics ampGynecologyvol 16 no 5 pp 500ndash505 2000

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] P A Lanzetta L J Alvarez P S Reinach and O A Candia ldquoAnimproved assay for nanomole amounts of inorganic phosphaterdquoAnalytical Biochemistry vol 100 no 1 pp 95ndash97 1979

[35] C Thery S Amigorena G Raposo and A Clayton ldquoIsolationand characterization of exosomes fromcell culture supernatantsand biological fluidsrdquo Current Protocols in Cell Biology vol 32006

[36] GG Yegutkin S S Samburski S Jalkanen and I Novak ldquoATP-consuming andATP-generating enzymes secreted by pancreasrdquoThe Journal of Biological Chemistry vol 281 no 40 pp 29441ndash29447 2006

[37] G G Yegutkin B Wieringa S C Robson and S JalkanenldquoMetabolism of circulatingADP in the bloodstream ismediatedvia integrated actions of soluble adenylate kinase-1 and NTP-Dase1CD39 activitiesrdquo The FASEB Journal vol 26 no 9 pp3875ndash3883 2012

[38] M R Schetinger V M Morsch C D Bonan and A T SWyse ldquoNTPDase and 5∘-nucleotidase activities in physiologicaland disease conditions new perspectives for human healthrdquoBiofactors vol 31 no 2 pp 77ndash98 2007

[39] K R Park T InoueMUeda et al ldquoCD9 is expressed on humanendometrial epithelial cells in association with integrins 1205726 1205723and 1205731rdquoMolecular Human Reproduction vol 6 no 3 pp 252ndash257 2000

[40] Y H Ng S Rome A Jalabert et al ldquoEndometrial exo-somesmicrovesicles in the uterine microenvironment a newparadigm for embryo-endometrial cross talk at implantationrdquoPLoS ONE vol 8 no 3 Article ID e58502 2013

[41] K L Bruner-Tran J L Herington A J DulebaH S Taylor andK G Osteen ldquoMedical management of endometriosis emerg-ing evidence linking inflammation to disease pathophysiologyrdquoMinerva Ginecologica vol 65 no 2 pp 199ndash213 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the most extended treatment for endometriomas althoughfertility could be afterwards compromised Some studieshave reported a lower ovarian reserve after excision ofovarian endometriomas due to incidental excision of normalovarian tissue together with the endometrioma wall [7 8]There is also evidence of a significant postoperative decreasein circulating Anti-Mullerian hormone a commonly usedbiomarker for ovarian reserve quantification suggesting anegative impact on ovarian reserve after the excision ofendometriomas [8] Therefore a new minimally invasiveinterventional technique was implemented to treat low riskmalignancy ovarian cysts including endometriomas theultrasound-guided aspiration followed by sclerosis whichseems to have even a low recurrence risk (0ndash15) [9ndash13] The sclerosis does not seem to have consequences onthe number of pregnancies term pregnancies abortionsextracted oocytes embryo quality and hormonal levels ifcompared with infertile women without ovarian cysts [11 14]

The analysis of the contents of endometriomas can helpidentify new biomarkers for this pathology For exampleendometriotic cysts contain significantly more iron thanother ovarian cysts this has been suggested as a possiblecause of carcinogenesis in endometriomas through iron-induced persistent oxidative stress [15] Proteomic analysisof follicular fluid of women with endometriosis has alsopointed to an altered immunologic function possibly relatedto infertility [16]

Purinergic signalling the group of biologic effects exertedby extracellular nucleotides such as ATP and nucleosidessuch as adenosine through specific receptors is an impor-tant regulatory mechanism in a wide range of inflamma-tory conditions [17 18] ATP is a ldquodanger signalrdquo releasedunder tissue stress conditions such as necrosis apoptosishypoxia and inflammation and is rapidly converted intoadenosine which protects cells and tissues from excessiveinflammation and immune-mediated damage The effectstransduced by both molecules are thus often opposite andthe resulting cellular responses are the consequence of theratio of ATP (and ADP) to adenosine concentrations whichis mainly controlled by ecto-nucleotidases key enzymes inthe purinergic signalling [19] The most well-characterizedectonucleotidase pathway involves the sequential action ofCD39 (ectonucleoside triphosphate diphosphohydrolase-1NTPDase1) which converts extracellular ATP (or ADP)to AMP and CD73 (ecto-51015840-nucleotidase) which generatesadenosine from AMP [20ndash23] We have previously char-acterized the expression of endometrial ecto-nucleotidasesdemonstrating changes along the cycle and in menopause[24 25] We have also demonstrated that CD39 and CD73are highly expressed in endometrial cancer [26] contributingto the increased immunosuppressive levels of extracellularadenosine found in tumor microenvironment

Exosomes are small secreted membrane vesicles (30ndash100 nm in diameter) of endocytic origin produced by manydifferent cell types that are formed by the fusion ofmultivesic-ular endosomeswith the plasmamembraneThey are thoughtto play an important role in intercellular communication forexample during immune cell-cell interaction and have beenidentified in biological fluids such as plasma and urine as

well as in cell culture supernatants [27 28] The presence ofthe ecto-nucleotidases CD39 and CD73 has been previouslydemonstrated in cancer exosomes [29ndash31] contributing tothe adenosine production that suppresses T-cell function

In the present work we aimed to determine whetherecto-nucleotidases activities (ATPase ADPase and AMPase)are present and measurable in the ultrasound-guided aspi-rated contents of ovarian cysts and whether these activitiesare altered in endometriotic cysts compared with simplecysts affecting the inflammatory condition of endometriosisOur goal is to evaluate their potential as biomarkers forendometriosis and to contribute to the knowledge of thephysiopathologic mechanisms underlying endometriosis

2 Materials and Methods

21 Samples The ethical principles of this study adhere to theDeclaration ofHelsinki and all the procedureswere approvedby the ethics committee for clinical investigation of BellvitgeHospital A prospective cohort of 27 adnexal cysts with alow risk of malignancy including 14 endometriomas and 13ovarian simple cysts was studied at the Gynecology Serviceof Bellvitge Hospital (Barcelona Spain) between March 2013and April 2014

Inclusion criteria in the study for patients were (1) agedgt18 years old (2) ultrasound features predictive of a lowrisk of cyst malignancy smooth walls no solid componentmaximum diameter between 40 and 100mm no more thanone thin septation (lt2-3mm) and no internal flow on colorDoppler imaging in the case of bilocular cysts according toIOTA criteria [32] (3) cyst persistence from diagnosis ge6months (4) tumor marker Ca 125 lt 200 IUmL and Ca199 lt 35 IUmL and (5) written informed consent from thepatients

For ultrasound-guided aspiration following the abdo-men or the vagina disinfection a sterilized 17G spinal needle(BD Medical Franklin Lakes NJ Becton Dickinson andCompany) was aimed at the center of the cyst under directultrasound guidance and the contents was aspirated Whenthick highly dense intracystic fluid was present in orderto facilitate the aspiration saline dilutions were performedDilutionsmadewere then considered in the final calculationsThen several intracyst saline washes were performed untilclearance followed by the instillation of ethanol as sclerosingagentThevolume of instilled ethanol was 23 of the aspiratedvolume of the endometrioma and always less than 100mLEthanol was left inside the cyst for 15min and then removedand washed out again with a saline solution The intracysticfluids were analyzed cytologically at the Pathology Service ofBellvitge Hospital to confirm the absence of atypical cells

Patients were divided into two groups following theultrasound features and internal color fluid (a) endometri-omas group that had homogeneous appearance involvingdiffuse internal echoes on a hypoechoic background andcontained blood color fluid and (b) simple cysts group thathad an anechoic structure and contained clear serous fluidTable 1 includes the descriptive statistics of the two groups of



















2PO4was




(a) (b)








Enzy

me a

ctiv

ity (p

mol

Pim

in120583

L)

50

40

30

20

10

0

ATP ADP AMP

lowastlowastlowast

lowastlowastlowast



119875lt 0 001) while




(A) (B)

(a)

Hsp70CD63

CD81 CD925

3750

75

25

37

50

75

EMSC EMSC

EMSC EMSC

(B)(A)

(C) (D)

25

37

50

75


(26kDa) (28kDa)

25

3750

75

(b)






(nm

ols P

i)4

3

2

1

00 10 20 30 40 50

ATPADP


(a)

Enzy

me a

ctiv

ity (p

mol

Pim

in120583

g)

5

4

3

2

1

0

ATP ADP AMP

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


(b)

37

50

75

100

(kDa)

CD39

37

50

75100

(kDa)

CD73


(c)








4 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































2PO4was




(a) (b)








Enzy

me a

ctiv

ity (p

mol

Pim

in120583

L)

50

40

30

20

10

0

ATP ADP AMP

lowastlowastlowast

lowastlowastlowast



119875lt 0 001) while




(A) (B)

(a)

Hsp70CD63

CD81 CD925

3750

75

25

37

50

75

EMSC EMSC

EMSC EMSC

(B)(A)

(C) (D)

25

37

50

75


(26kDa) (28kDa)

25

3750

75

(b)






(nm

ols P

i)4

3

2

1

00 10 20 30 40 50

ATPADP


(a)

Enzy

me a

ctiv

ity (p

mol

Pim

in120583

g)

5

4

3

2

1

0

ATP ADP AMP

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


(b)

37

50

75

100

(kDa)

CD39

37

50

75100

(kDa)

CD73


(c)








4 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)








Enzy

me a

ctiv

ity (p

mol

Pim

in120583

L)

50

40

30

20

10

0

ATP ADP AMP

lowastlowastlowast

lowastlowastlowast



119875lt 0 001) while




(A) (B)

(a)

Hsp70CD63

CD81 CD925

3750

75

25

37

50

75

EMSC EMSC

EMSC EMSC

(B)(A)

(C) (D)

25

37

50

75


(26kDa) (28kDa)

25

3750

75

(b)






(nm

ols P

i)4

3

2

1

00 10 20 30 40 50

ATPADP


(a)

Enzy

me a

ctiv

ity (p

mol

Pim

in120583

g)

5

4

3

2

1

0

ATP ADP AMP

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


(b)

37

50

75

100

(kDa)

CD39

37

50

75100

(kDa)

CD73


(c)








4 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(A) (B)

(a)

Hsp70CD63

CD81 CD925

3750

75

25

37

50

75

EMSC EMSC

EMSC EMSC

(B)(A)

(C) (D)

25

37

50

75


(26kDa) (28kDa)

25

3750

75

(b)






(nm

ols P

i)4

3

2

1

00 10 20 30 40 50

ATPADP


(a)

Enzy

me a

ctiv

ity (p

mol

Pim

in120583

g)

5

4

3

2

1

0

ATP ADP AMP

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


(b)

37

50

75

100

(kDa)

CD39

37

50

75100

(kDa)

CD73


(c)








4 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(nm

ols P

i)4

3

2

1

00 10 20 30 40 50

ATPADP


(a)

Enzy

me a

ctiv

ity (p

mol

Pim

in120583

g)

5

4

3

2

1

0

ATP ADP AMP

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


(b)

37

50

75

100

(kDa)

CD39

37

50

75100

(kDa)

CD73


(c)








4 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4 Conclusions






Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article ecto-nucleotidases activities in the...

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