endometriosis is a chronic condition characterized by the presence of endometrial tissue ( stroma and glands ) outside the uterine cavity , mostly on pelvic peritoneum and ovaries . it affects up to 15% of women of reproductive age and its prevalence is rising . it is an inflammatory , estrogen - dependent complex disorder , with broad symptomatic variability , pelvic pain , and infertility being the main characteristics . both symptoms are thought to be the result of an excessive inflammatory environment not only within the pelvis but also in the eutopic endometrium , affecting implantation [ 1 , 2 ] . late diagnosis is one of the main problems of this pathology , taking between five and ten years from detection of the first symptoms . ovarian endometriomas occur in 1744% of patients affected by endometriosis   and account for 35% of benign ovarian cysts . endometriomas are also called endometriotic or  chocolate cysts  because of the internal fluid blood derived dark color . although the risk of malignant transformation is low , 0.60.8% , surgery has become the gold standard therapy . nowadays , laparoscopic cystectomy is the most extended treatment for endometriomas , although fertility could be afterwards compromised . some studies have reported a lower ovarian reserve after excision of ovarian endometriomas due to incidental excision of normal ovarian tissue together with the endometrioma wall [ 7 , 8 ] . there is also evidence of a significant postoperative decrease in circulating anti - mllerian hormone , a commonly used biomarker for ovarian reserve quantification , suggesting a negative impact on ovarian reserve after the excision of endometriomas . therefore , a new minimally invasive interventional technique was implemented to treat low risk malignancy ovarian cysts , including endometriomas , the ultrasound - guided aspiration followed by sclerosis , which seems to have even a low recurrence risk ( 015% ) [ 913 ] . the sclerosis does not seem to have consequences on the number of pregnancies , term pregnancies , abortions , extracted oocytes , embryo quality , and hormonal levels if compared with infertile women without ovarian cysts [ 11 , 14 ] . the analysis of the contents of endometriomas can help identify new biomarkers for this pathology . for example , endometriotic cysts contain significantly more iron than other ovarian cysts ; this has been suggested as a possible cause of carcinogenesis in endometriomas through iron - induced persistent oxidative stress . proteomic analysis of follicular fluid of women with endometriosis has also pointed to an altered immunologic function possibly related to infertility . purinergic signalling , the group of biologic effects exerted by extracellular nucleotides such as atp and nucleosides such as adenosine through specific receptors , is an important regulatory mechanism in a wide range of inflammatory conditions [ 17 , 18 ] . atp is a  danger signal  released under tissue stress conditions , such as necrosis , apoptosis , hypoxia , and inflammation and is rapidly converted into adenosine , which protects cells and tissues from excessive inflammation and immune - mediated damage . the effects transduced by both molecules are thus often opposite , and the resulting cellular responses are the consequence of the ratio of atp ( and adp ) to adenosine concentrations , which is mainly controlled by ecto - nucleotidases , key enzymes in the purinergic signalling . the most well - characterized ectonucleotidase pathway involves the sequential action of cd39 ( ectonucleoside triphosphate diphosphohydrolase-1 , ntpdase1 ) , which converts extracellular atp ( or adp ) to amp , and cd73 ( ecto-5-nucleotidase ) , which generates adenosine from amp [ 2023 ] . we have previously characterized the expression of endometrial ecto - nucleotidases , demonstrating changes along the cycle and in menopause [ 24 , 25 ] . we have also demonstrated that cd39 and cd73 are highly expressed in endometrial cancer , contributing to the increased immunosuppressive levels of extracellular adenosine found in tumor microenvironment . exosomes are small , secreted membrane vesicles ( 30100  nm in diameter ) of endocytic origin , produced by many different cell types that are formed by the fusion of multivesicular endosomes with the plasma membrane . they are thought to play an important role in intercellular communication , for example , during immune cell - cell interaction , and have been identified in biological fluids , such as plasma and urine , as well as in cell culture supernatants [ 27 , 28 ] . the presence of the ecto - nucleotidases cd39 and cd73 has been previously demonstrated in cancer exosomes [ 2931 ] , contributing to the adenosine production that suppresses t - cell function . in the present work , we aimed to determine whether ecto - nucleotidases activities ( atpase , adpase , and ampase ) are present and measurable in the ultrasound - guided aspirated contents of ovarian cysts and whether these activities are altered in endometriotic cysts compared with simple cysts , affecting the inflammatory condition of endometriosis . our goal is to evaluate their potential as biomarkers for endometriosis and to contribute to the knowledge of the physiopathologic mechanisms underlying endometriosis . the ethical principles of this study adhere to the declaration of helsinki , and all the procedures were approved by the ethics committee for clinical investigation of bellvitge hospital . a prospective cohort of 27 adnexal cysts with a low risk of malignancy , including 14 endometriomas and 13 ovarian simple cysts , was studied at the gynecology service of bellvitge hospital ( barcelona , spain ) between march 2013 and april 2014 . inclusion criteria in the study for patients were ( 1 ) aged > 18 years old ; ( 2 ) ultrasound features predictive of a low risk of cyst malignancy : smooth walls , no solid component , maximum diameter between 40 and 100  mm , no more than one thin septation ( < 2 - 3  mm ) , and no internal flow on color doppler imaging in the case of bilocular cysts , according to iota criteria ; ( 3 ) cyst persistence from diagnosis 6 months ; ( 4 ) tumor marker ca 125 < 200  iu / ml and ca 19.9 < 35  iu / ml ; and ( 5 ) written informed consent from the patients . for ultrasound - guided aspiration , following the abdomen , or the vagina , disinfection , a sterilized 17 g spinal needle ( bd medical franklin lakes , nj becton , dickinson and company ) was aimed at the center of the cyst under direct ultrasound guidance and the contents was aspirated . when thick highly dense intracystic fluid was present , in order to facilitate the aspiration , saline dilutions were performed . then , several intracyst saline washes were performed until clearance , followed by the instillation of ethanol as sclerosing agent . the volume of instilled ethanol was 2/3 of the aspirated volume of the endometrioma and always less than 100  ml . ethanol was left inside the cyst for 15  min and then removed and washed out again with a saline solution . the intracystic fluids were analyzed cytologically at the pathology service of bellvitge hospital to confirm the absence of atypical cells . patients were divided into two groups following the ultrasound features and internal color fluid : ( a ) endometriomas group that had homogeneous appearance involving diffuse internal echoes on a hypoechoic background and contained blood color fluid and ( b ) simple cysts group that had an anechoic structure and contained clear serous fluid . table 1 includes the descriptive statistics of the two groups of patients , including the age and the size and the volume of the cysts . endometriomas and simple cysts fluid samples were centrifuged at 3000  g for 15  min to remove cells and cell debris , and supernatants were transferred to sterile tubes . 250  l of each supernatant was mixed with 63  l of exoquick exosome precipitation solution ( system biosciences , mountain view , ca , usa ) by inversion , and the mixture was refrigerated overnight at 4c , following the supplier 's instructions . after centrifugation , the exosomes appeared as a beige or white pellet at the bottom of the tube . supernatant was aspirated and the residual exoquick solution was spun down by centrifugation at 1500  g for 5  min . all traces of fluid were removed by aspiration and exosome pellets were resuspended in two different buffers . for western blot analysis , exosome pellets were resuspended in 75  l of a buffer containing 10  mm tris - hcl ph 7.4 , 150  mm nacl , and 1  :  100 proteases inhibitor cocktail ( sigma - aldrich , saint louis , mo , usa ) . alternatively , for ecto - nucleotidases activity assays , exosome pellets were resuspended in 75  l of enzyme assay buffer ( 160  mm tris - hcl ph 7.4 , 10  mm cacl2 ) . 15  g of protein was heated at 95c for 3  min , loaded onto any kd mini - protean tgx precast gel ( bio - rad , hercules , ca , usa ) , and transferred to polyvinylidene fluoride ( pvdf ) membranes using the trans - blot turbo transfer system ( bio - rad ) . blots were blocked with tris - buffered saline with 0.1% tween-20 ( tbs - t ) containing 5% nonfat dry milk for 1 hour at room temperature ( rt ) . membranes were then incubated overnight at 4c with the following antibodies : exosomal marker antibodies cd9 , cd63 , 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 ( clone 4g4 from abcam , diluted 1  :  50 ) . blots were then washed with tbs - t and incubated with the appropriate hrp - conjugated secondary antibody ( system biosciences , dilution 1  :  20,000 ) for 1 hour at rt . membranes were then incubated with amersham ecl western blotting detection reagents ( ge healthcare , pittsburgh , pa , usa ) for 1  min and exposed on x - ray films . nucleotidase activities ( atpase , adpase , and ampase ) were determined in flat - bottom 96-well plates by measuring the amount of liberated inorganic phosphate ( pi ) using the malachite green colorimetric assay . the incubation mixture contained 160  mm tris  hcl ph 7.4 , 10  mm cacl2 , 5  mm levamisole , as alkaline phosphatases inhibitor , and 1  mm atp , adp , or amp as substrates in a final volume of 150  l . the assay was initiated by adding the sample , either the fluid directly ( 5  l for atpase and adpase assays and 10  l for ampase assay ) or 50  g of the isolated exosome fraction . alternatively , increasing amounts of exosomes were added to the well to assess if ectonucleotidase activity depended on the exosome dose ( 5 , 10 , 15 , 20 , 30 , and 50  g ) . after 20  minutes of incubation at 37c , 50  l of malachite green solution was added to each well and 10 and 20  minutes later , the od620 was measured and recorded . incubation times and protein concentrations were chosen to ensure the linearity of the enzymatic reaction . controls to determine nonenzymatic pi accumulation were performed by incubating either the samples in the absence of the substrate or the substrate alone . each experiment was performed three times and in each experiment samples were analyzed in triplicate . enzyme activity was expressed as pmols pi / min/l in the case of the whole fluids and as pmol pi / min/g for exosomes . for morphological analysis , whole - mount electron microscopy preparations of exosomes were obtained as previously described by thry et al . . in brief , exosome - containing pellets were resuspended in 2% paraformaldehyde in 100  mm phosphate - buffered saline ( pbs ) . 5  l of resuspended pellets was deposited on formvar - coated grids and incubated at rt for 20  min . after thorough rinses , the samples were postfixed in 1% glutaraldehyde for 5  min and then contrasted in a solution of 2% uranyl acetate , 150  mm oxalic acid ph 7 . then the grids were transferred to a drop of 1.8% methyl - cellulose , 0.4% uranyl acetate for 10  min on ice . finally , the grids were removed with stainless steel loops and air - dried for additional 10  min . alternatively , and in order to obtain thin sections , the paraformaldehyde - fixed exosome - containing pellets were postfixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 100  mm pbs , dehydrated in a graded ethanol series , and embedded in the spurr low viscosity resin . ultrathin sections ( 5060  nm ) from exosome - enriched pellets were cut with a diamond knife in an ultramicrotome ultracute ( leica microsystems ) . after being picked up on copper grids , the ultrathin sections were stained with uranyl acetate and lead citrate . finally , exosomes were observed with a jem-1010 transmission electron microscope ( jeol , japan ) , and the images were recorded with a bio scan 792 camera ( gatan , germany ) . student 's t - test was used to compare the means of two independent groups of normally distributed data . most of the attempts to identify peripheral biomarkers of endometriosis have been focused on their presence in plasma , serum , or urine . however , the study of endometriomas contents has been proven to be very informative [ 15 , 16 ] . in the present work , we have evaluated the ectonucleotidase enzyme activities present in endometriomas in comparison with ovarian simple cysts . echo - guided aspiration was the technique used to remove the fluid contained in endometriomas and simple cysts . immediate ethanol sclerosis was performed to chemically destroy the cyst lining and to prevent its regrowth . figure 1 shows the ultrasound picture of an endometrioma before and during the aspiration of its contents . ecto - nucleotidases activities ( atpase , adpase , and , to a lesser extent , ampase ) were present and quantifiable in the aspirated fluid of both endometriomas and ovarian simple cysts ( figure 2 ) . endometriomas aspirates showed significantly higher ( p < 0,001 ) atpase and adpase activities ( mean  sem , 38,96  5,11 and 33,68  3,95  pmol / min/l , resp . ) than simple cysts ( 7,19  1,88 and 1,55  0,4  pmol / min/l , resp . ) . low activity rates were detected with amp as substrate in the aspirated fluid and although endometriomas displayed higher ampase activity ( 1,28  0,46  pmol / min/l ) than simple cysts ( 0,87  0,25  pmol / min/l ) , differences were not significant . the same results were also obtained when the activities were calculated per g of protein in the fluid , with even higher activities in endometriomas ( data not shown ) ; however , since it is known that endometriotic cysts differ from simple cysts in protein contents , results are shown here as pi generation per unit of volume . this is the first time that ecto - nucleotidases have been studied in ovarian cysts , although these enzyme activities have been previously characterized in other fluids such as pancreatic juice   and plasma , where there is a correlation between their activity and different inflammatory pathologies . since to perform the enzyme activity assays , aspirated fluids were previously centrifuged to remove cells and cell debris , the resulting activities could be attributed either to soluble enzymes or to membrane bound ecto - nucleotidases present in extracellular membrane vesicles such as microvesicles and exosomes that were not removed by the initial centrifugation . therefore , we decided to perform exosome enrichment to assess their presence and to determine if they had ectonucleotidase activities . to our knowledge , this is the first study demonstrating the presence of exosomes in the fluids contained in ovarian cysts . electron microscopy analysis confirmed the size and morphology of exosomes ( figure 3(a ) ) . western blots using antibodies against four exosome markers ( cd9 , cd63 , cd81 , and hsp70 ) demonstrated the presence of these structures in 100% of endometriomas and in 46% of simple cysts aspirates ( figure 3(b ) ) . from these experiments , we conclude that , among the markers used , cd9 is the most suitable for exosome demonstration in the aspirated fluids of ovarian cysts , since it was present in all exosome samples analyzed . cd9 has been already demonstrated to be a good exosome marker for epithelial endometrial cells [ 39 , 40 ] . all the samples expressed , together with cd9 , at least one more of the other exosome markers assayed . interestingly , using the anti - cd63 antibody , the predominant band in endometriomas was of a lower molecular weight than in simple cysts . a possible explanation is the alternative splicing already described for the gene encoding cd63 that results in multiple transcript variants encoding different proteins . since this result was consistent in all the samples studied , we propose to further study its presence in exosomes from other origins in endometriosis patients , to establish if it could be used as biomarker for the pathology . enzyme experiments performed with exosome - enriched fractions demonstrated that these samples retain the ectonucleotidase activity ( atpase , adpase , and ampase ) displayed by the whole fluid . moreover , these activities are dependent on the sample dose , as shown in figure 4(a ) , in line with previous results obtained with exosomes from other origins that express active cd39 and cd73 . for atpase and adpase , ampase activity , which was barely detectable in the whole fluid , was present in purified exosomes , though only at high concentrations ( 50  g ) , indicating that this activity in the whole fluid is mainly due to the enzymes bearing exosomes . on the contrary , atpase and adpase activities figure 4(b ) shows the enzyme activity using atp , adp , and amp as substrates of exosomes from simple cysts and from endometriomas . western blot analysis demonstrated the presence of cd39 and cd73 in these samples ( figure 4(c ) ) . our results reinforce the emerging evidence linking inflammation to endometriosis pathophysiology , where evasion of the immune surveillance seems to be crucial in the initial establishment and subsequent development of displaced endometrial tissues . in this sense , increased sequentially acting ectonucleotidase activities would contribute to the maintenance of the suppression of local immune responses necessary for the disease progression , by regulating extracellular atp and rising extracellular adenosine levels . we propose to detect their presence in other more accessible biological fluids , a fact that , together with the simplicity of their detection , would make them promising endometriosis biomarkers . our work contributes to the knowledge of endometriosis by providing new data on the fluid composition of endometriomas . ecto - nucleotidases activities are quantifiable in the ovarian cysts aspirates , and they are significantly increased in the contents of endometriomas compared with simple cysts . moreover , the identification of ecto - nucleotidases bearing exosomes in ovarian cyst contents opens the field for a broad range of proteomics and genomics studies .
endometriosis , defined as the growth of endometrial tissue outside the uterus , is a common gynecologic condition affecting millions of women worldwide . 
 it is an inflammatory , estrogen - dependent complex disorder , with broad symptomatic variability , pelvic pain , and infertility being the main characteristics . 
 ovarian endometriomas are frequently developed in women with endometriosis . 
 late diagnosis is one of the main problems of endometriosis ; thus , it is important to identify biomarkers for early diagnosis . 
 the aim of the present work is to evaluate the ecto - nucleotidases activities in the contents of endometriomas . 
 these enzymes , through the regulation of extracellular atp and adenosine levels , are key enzymes in inflammatory processes , and their expression has been previously characterized in human endometrium . to achieve our objective , 
 the echo - guided aspirated fluids of endometriomas were analyzed by evaluating the ecto - nucleotidases activities and compared with simple cysts . 
 our results show that enzyme activities are quantifiable in the ovarian cysts aspirates and that endometriomas show significantly higher ecto - nucleotidases activities than simple cysts ( 5.5-fold increase for atpase and 20-fold for adpase ) , thus being possible candidates for new endometriosis biomarkers . 
 moreover , we demonstrate the presence of ecto - nucleotidases bearing exosomes in these fluids . these results 
 add up to the knowledge of the physiopathologic mechanisms underlying endometriosis and , open up a promising new field of study .