Abnormal Expression of Prostaglandins E2 and F2α Receptors ... · ClinicalStudy Abnormal...

Clinical StudyAbnormal Expression of Prostaglandins E2 and F2120572 Receptorsand Transporters in Patients with Endometriosis

Halima Rakhila12 Nathalie Bourcier1 Ali Akoum12 and Marc Pouliot12

1Centre Hospitalier Universitaire de Quebec Quebec Canada G1V 4G22Faculte de Medecine Universite Laval Quebec Canada G1V 0A6

Correspondence should be addressed to Halima Rakhila halimarakhila1ulavalca

Received 16 September 2014 Revised 25 November 2014 Accepted 8 December 2014

Academic Editor Andrea Tinelli

Copyright copy 2015 Halima Rakhila et alThis 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

Objective To investigate the level of expression of prostaglandin receptivity and uptake factors in eutopic and ectopic endometriumof women with endometriosisDesign Prospective study SettingHuman reproduction research laboratory Patients Seventy-eightpatients with endometriosis and thirty healthy control subjects Intervention(s) Endometrial and endometriotic tissue sampleswere obtained during laparoscopic surgery Main Outcome Measure(s) Real-time polymerase chain reaction assay of mRNAencoding prostaglandin E2 receptors (EP1 EP2 EP3 and EP4) prostaglandin F2120572 receptor (FP) prostaglandin transporter (PGT)and multidrug resistance-associated protein 4 (MRP4) immunohistochemical localization of expressed proteins ResultsMarkedincreases in receptors EP3 EP4 and FP and transporters PGT and MRP4 in ectopic endometrial tissue were noted withoutnoticeable change associated with disease stage An increase in EP3 expression and decreases in FP and PGT were observed inthe eutopic endometrium of endometriosis patients in conjunction with the phases of the menstrual cycle Conclusion(s) Thisstudy is the first to demonstrate a possible relationship between endometriosis and enhanced prostaglandin activity In view of thewide range of prostaglandin functions increasing cell receptivity and facilitating uptake in endometrial tissue could contribute tothe initial steps of overgrowth and have an important role to play in the pathogenesis and symptoms of this disease

1 Introduction

Endometriosis is a major health issue affecting nearly 10percent of women of childbearing age The main symptomsof this disease include chronic pelvic or abdominal painirregular bleeding and in 40ndash50 of cases infertility [1] Theamount of pain experienced correlates poorly with diseasestage Endometriotic tissue may settle and proliferate in thefallopian tubes or the ovaries or enter the peritoneal cavityand deposit in ectopic sites The causes and symptoms ofendometriosis are multifactorial Although knowledge of itsunderlying immunological and endocrine mechanisms isprogressing gray areas continue to obscure complete under-standing of its pathology Our studies were among the firstto highlight dysfunctions in eutopic endometrium includingelevated levels of the monocyte chemoattractant factorMCP-1 [2] In addition we have shown that ectopic endometrialtissue by itself is capable of producing growth-promoting

molecules such as vascular endothelial growth factor (VEGF)[3] as well as implantation-promoting integrins [4] whileinitiating peritoneal inflammationThis inflammation causesthe release of mediators such as prostaglandins E2 (PGE2)and F2120572 (PGF2120572) which play a role in reproductive functionssuch as ovulation luteolysis implantation parturition andlactation Excessive release of prostaglandins may affect peri-toneal function causing pain [5] and disrupting processessuch as oocyte maturation ovulation and fertilization [6 7]

Prostaglandins are lipid compounds derived enzymati-cally from arachidonic acid The reproductive system is theprincipal producer of PGE2 and PGF2120572 The involvementof prostaglandins in pain [5 8 9] infertility [10ndash12] angio-genesis [13 14] tissue remodeling [15] and cell proliferation[16] in association with various pathological conditions iswell documented We along with others have shown that anisoform of cyclooxygenase-2 (COX-2) is overexpressed inectopic endometrial cells [17ndash20] and that PGE2 PGF2120572 and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 808146 11 pageshttpdxdoiorg1011552015808146

2 BioMed Research International

other specific prostaglandins are present at abnormally highlevels in uterine tissues of women suffering from menorrha-gia dysmenorrhoea or endometriosis [21ndash23] It is also wellknown that PGE2 and PGF2120572 are more concentrated in theperitoneal fluid of endometriosis patients [5 10 12 24 25]Such evidence has led us to investigate a possible role of theseprostaglandins in the pathogenesis of endometriosis Ourrecent comparison of endometrium (eutopic and ectopic)from endometriosis patients to healthy eutopic endometriumshowed overproduction of PGE2 and PGF2120572 apparentlypromoted by increased expression of enzymes such as COX-2with cPGES or AKR1C3 [26]

Regulation of production is not the only means bywhich the body modulates the action of PGE2 and PGF2120572Expression of prostaglandin receptors and transporters mayalso be regulated Once released as messenger moleculesprostaglandins act locally on receptors in an autocrine orparacrine manner The four main subtypes of PGE2 receptorare designated as EP1 EP2 EP3 and EP4 while the PGF2120572receptor is called FP [27 28] The receptor subtype deter-mines the nature of the physiological response Receptioneither elicits the intracellular calcium-inositol triphosphatepathway or increasesdecreases cyclic adenosine monophos-phate (cAMP) activity Engagement of some receptors mayelicit both pathways depending on cell type and receptorsplice variety

Prostaglandins were originally believed to exit fromproducer cells via passive diffusion because of their stronglylipophilic characterThediscovery of the prostaglandin trans-porter protein PGT (SLCO2A1) which mediates prostagl-andin uptake and release [29 30] demonstrated that diffu-sion alone did not explain the penetration of prostaglandinsthrough the cell membrane Furthermore a specific trans-porter namely multidrug resistance protein 4 (MRP4ABCC4) of the ATP-binding cassette transporter superfam-ily has been shown to mediate prostaglandin release [31]Whether or not MRP4 is the only transporter that does thisis still unclear

Although it is clear that PGE2 and PGF2120572 play importantroles in a number of female reproductive physiological proc-esses as well as in endometriosis-associated infertility andpain [5 10 12 32] current understanding of these rolesremains incomplete In the present study we analyzed theexpression of EP1 EP2 EP3 EP4 FP PGT and MRP4 inendometriosis patients in comparison to their expression innormal eutopic endometrium We observed marked differ-ences between eutopic and ectopic endometria in terms ofprostaglandins receptivity and transport readiness

2 Materials and Methods

21 Patients and Tissue Collection The study received ap-proval from the Human Research Ethics Committee atSaint-Francois drsquoAssise Hospital and informed consent wasobtained from all participants who were recruited betweenFebruary 2002 and March 2007 Endometriosis patients wereaged 342 plusmn 36 years (119899 = 78) and were consulting forpelvic pain andor infertility They were diagnosed usinglaparoscopy and the disease stage (IndashIV) was determined

according to the Revised American Fertility Society classifi-cation system Endometriotic tissue samples were collectedfrom 28 of these patients We also recruited healthy womenaged 353 plusmn 38 (119899 = 30) scheduled for tubal ligation Theseparticipants had no pelvic pathology or sign of endometrialhyperplasia or neoplasia and had not received any anti-inflammatory or hormonal medication for at least 3 monthsMenstrual cycle dating was determined using the cyclehistory

Endometrial and endometriotic biopsies were obtainedduring laparoscopy Tissue was placed immediately at 4∘C insterile Hankrsquos balanced salts solution (HBSS) (GIBCO Invit-rogen Corp Burlington ON Canada) containing 100 IUmLpenicillin 100 120583gmL streptomycin and 025 120583gmL ampho-tericin and transported to the laboratory After washingin HBSS at 4∘C samples were frozen at minus80∘C in Eppen-dorf tubes for quantitative real-time PCR (qRT-PCR) orembedded in paraffin and stored at room temperature forimmunohistochemical analysis

22 Quantitative Real-Time PCR Total RNA was extractedfrom endometrial tissue using the TRIzol reagent accordingto the manufacturerrsquos instructions (Invitrogen Life Technolo-gies Inc Grand Island NY USA) and reverse-transcribedin the presence of random hexamers The qRT-PCR reactionwas carried out in an ABI 7000 Thermal Cycler (AppliedBiosystems Foster City CA USA) The standard reactionmixture contained 2120583L of RT product 05 120583L of each primer(final concentration 01mM) 125 120583L SYBR Green PCRMaster Mix (Invitrogen Life technologies Inc Grand IslandNY USA) consisting of Taq DNA polymerase reaction bufferdNTP mix SYBR green I MgCl

2 and Taq DNA polymerase

Following denaturing for 2min at 95∘C the reactions werecycled 45 times with denaturing for 15 sec at 95∘C and anneal-ing for 60 sec at 60∘C The primer sequences are listed inTable 1 The primers were designed using Primer Express 20(Applied Biosystems Foster City CA USA) to span intron-exon boundaries to avoid amplification of genomic DNA andselected to have compatible Tm values (59ndash61∘C) A relativequantification method was used Expression of mRNA ofEP1 EP2 EP3 EP4 FP PGT total MRP4 and MRP4 variant1 was normalized to that of the gene GAPDH After eachrun melting curve analysis (55ndash95∘C) was performed toverify the specificity of the PCR reaction All samples weretested in duplicate and each run included a template controlBaseline curves melting curves melting points crossingpoints slopes and errors were monitored for each gene

23 Immunohistochemical Probe Endometrium was fixedin 10 formalin (Fisher Scientific New Jersey USA) andthen embedded in paraffin Serial tissue sections 4120583m thickwere rinsed in phosphate buffered saline (PBS) and treatedwith 3 hydrogen peroxide to block endogenous peroxidaseactivity All antibodies were diluted in PBS containing 02bovine serum albumin and 01 Tween 20 Sections wereincubated for twohours at room temperaturewith the specificantibody (Cayman Ann Arbor USA) Rabbit polyclonalanti-human EP2 was diluted 1 800 and rabbit polyclonalantibodies directed against human EP1 EP3 EP4 FP and

BioMed Research International 3

Table 1 List of primers used for qRT-PCR

Gene Forward ReverseEP1 51015840-ATGGTGGGCCAGCTTGTC-31015840 51015840-GCCACCAACACCAGCATTG-31015840

EP2 51015840-TCCTTGCCTTTCACGATTT-31015840 51015840-AGAGCTTGGAGGTCCCATT-31015840

EP3 51015840-TGGTCTCCGCTCCTGATAA-31015840 51015840-TGCATTCTTTCTGCTTCTCC-31015840

EP4 51015840-TGCTCTTCTTCAGCCTGTCC-31015840 51015840-GAGCTACCGAGACCCATGTT-31015840

FP 51015840-TCTGGTCTGTGCCCACTTC-31015840 51015840-GACTCCAATACACCGCTCAAT-31015840

PGT 51015840-CTGGTGGATTTCATTAAACGG-31015840 51015840-GGCTGCTGAGGTGCCATAC-31015840

MRP4 total 51015840-AAAGTGCCAAAGTAATCCAGC-31015840 51015840-GTTCAAAGCCACAGAATCCA-31015840

MRP4 variant 1 51015840-CGGGCATACAAAGCAGAA-31015840 51015840-GGACCCAAAGGCAACG-31015840

PGT were all diluted 1 200 Rat monoclonal anti-humantotal MRP4 (Abcam Cambridge USA) was diluted 1 50Sections were then held for 45 minutes at room temper-ature with peroxidase-conjugated goat anti-rabbit antibody(Jackson ImmunoResearch Laboratories Inc West GroveUSA) diluted 1 2000 or peroxidase-conjugated rabbit anti-rat antibody (Jackson ImmunoResearch Laboratories Mis-sissauga Canada) diluted 1 500 and then for 40 minuteswith the peroxidase substrate 331015840-diaminobenzidine for 5minutes at room temperature followed by rinsing in PBScounterstaining with hematoxylin and mounting in Mowiol

24 Statistical Analysis Data that followed a normal (Gaus-sian) distribution were subjected to one-way analysis of vari-ance (ANOVA) and Bonferronirsquos post hoc test for multiplecomparisons while data that were not normally distributedwere analyzed using the Kruskal-Wallis test and Dunnrsquosmultiple comparison post hoctest for multiple comparisonsComparison of the two groupswas performed using the para-metric unpaired 119905-test or the nonparametric Mann-Whitneytest All statistical analyses were performed using GraphPadPrism 50 Software (San Diego CA USA) Differences wereconsidered to be statistically significant at 119875 lt 005

3 Results

31 Expression of PGE2 and PGF2120572 Receptors andTransporters in Eutopic and Ectopic Endometria inthe Diseased State

311 Prostaglandins E2 and F2120572 Receptors Neither EP1 norEP2 was expressed differentially to any appreciable degreewhether the comparison was between endometriosispatients and healthy control patients eutopic and ectopicendometrium in endometriosis patients (Figures 1(a) and1(b)) endometriosis stages (Figures 2(a) and 2(b)) or thetwo phases of the menstrual cycle (Table 2)

Expression of EP3 was increased in ectopic endometriumof endometriosis patients (Figure 1(c)) and as shown inFigure 2(c) in both stages I-II and stages III-IV This effectwas apparent in both secretory and proliferative phases of themenstrual cycle (Table 2) These effects were all significant at119875 lt 0001 It is worth mentioning that EP3 expression wasincreased significantly in eutopic tissue during the secretoryphase even in healthy women (119875 lt 005) but at an order ofmagnitude less than in ectopic tissue

As was the case for EP3 EP4 expression was increased(119875 lt 0001) in ectopic endometrium (Figure 1(d)) althoughneither endometriosis stage (Figure 2(d)) nor phase of themenstrual cycle (Table 2) had any effect

FP expression followed distinctive patterns in eutopic(119875 lt 005) and ectopic (119875 lt 0001) endometria of endomet-riosis patients (Figure 1(e)) The increase was especiallypronounced in ectopic endometrium (119875 lt 0001) at bothdisease stages (Figure 2(e)) during both phases of the men-strual cycle (Table 2) Expression in eutopic endometriumin endometriosis patients was significantly higher during theproliferative phase than during the secretory phase while thedifference between these patients and healthy subjects duringthe proliferative phase was only marginally significant (119875 =018)

312 Prostaglandins E2 and F2120572 Transporters PGT expres-sion was significantly increased (119875 lt 0001) in ectopicendometrium (Figure 1(f)) at stages I-II (119875 lt 0001) and III-IV (119875 lt 001) of the disease (Figure 2(f)) and during bothphases of the menstrual cycle (Table 2)

Expression of total MRP4 in ectopic endometrium wasincreased similar to PGT in endometriosis stages I-II andIII-IV (Figures 1(g) and 2(g)) and during both phasesof the menstrual cycle (Table 2) However regulation ofMRP4 is more complex because of its two transcriptionalvariants namely variant 1 (coding sequence 120 4097NM 0058453) and a shorter variant 2 (coding sequence120 2699 NM 001105515) which encode distinctive func-tional proteins of different molecular mass There was nonoticeable effect of the disease on the expression of variant1 (Figure 1(h)) The ratio of variant 1 to total MRP4 wassignificantly greater (119875 lt 0001) in eutopic endometriumin subjects with or without the disease (not shown) Themeaning of this ratio is not yet known In any case it did notdiffer significantly

32 Immunohistochemical Localization of PGE2 and PGF2120572Receptors and Transporters in Eutopic and Ectopic Endome-tria in Endometriosis Patients and Healthy Subjects Cellmembranes of ectopic endometriotic tissue and eutopicendometrium from endometriosis patients and from healthysubjects were examined using an immunohistochemicaltechnique Representative immunological staining of thereceptors and transporters is shown in Figures 3 and 4


0

500

1000

1500

2000

2500

Controls Eutopic Ectopic

Endometriosis

EP1

mRN

A (

of G

APD

H)

(a)

Controls Eutopic Ectopic0

200

400

600

800

1000

Endometriosis

EP2

mRN

A (

of G

APD

H)

(b)

0

1000

2000

3000

4000

5000


Endometriosis

EP3

mRN

A (

of G

APD

H) lowastlowastlowast

lowastlowastlowast

(c)

0

1000

2000

3000

4000


Endometriosis

lowastlowastlowast

lowastlowastlowast

EP4

mRN

A (

of G

APD

H)

(d)

0

1000

2000

3000

10000200003000040000


Endometriosis

FP m

RNA

(of

GA

PDH

)

lowastlowastlowastlowastlowastlowast

lowast

(e)

0

100

200

300

400

Endometriosis


PGT

mRN

A (

of G

APD

H)

lowastlowastlowast

lowastlowastlowast

(f)

0200400600800

100040005000600070008000

Endometriosis


lowastlowastlowast

lowastlowastlowast

MRP

4 to

tal m

RNA

(of

GA

PDH

)

(g)

0

200

400

600

800

Endometriosis


MRP

4va

riant

1m

RNA

(of

GA

PDH

)

(h)

Figure 1 Expression of PGE2 and PGF2120572 receptors and prostaglandin transporters in endometrium 119899 = 30 50 and 28 respectively forhealthy (control) subjects eutopic tissue samples from endometriosis patients and ectopic tissue samples Total RNA was extracted andreverse-transcribed and mRNA was quantified by quantitative RT-PCR and normalized relative to GAPDH (internal control) (a) EP1 (b)EP2 (c) EP3 (d) EP4 (e) FP (f) PGT (g) total MRP4 and (h) MRP4 variant 1 The horizontal lines represent the mean for each set of datalowast

119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001


Controls EI-EII EIII-EIV EI-EII EIII-EIV0

500

1000

1500

2000

2500

Eutopic Ectopic

EP1

mRN

A (

of G

APD

H)

(a)


200

400

600

800

1000

Eutopic Ectopic

EP2

mRN

A (

of G

APD

H)

(b)


250

50010002000300040005000

Eutopic Ectopic

EP3

mRN

A (

of G

APD

H)


lowastlowastlowastlowastlowast

(c)


1000

2000

3000

4000

Eutopic Ectopic

lowastlowastlowast


lowastlowast

EP4

mRN

A (

of G

APD

H)

(d)


1000

2000

3000

10000200003000040000

Eutopic Ectopic

FP m

RNA

(of

GA

PDH

)



(e)


100

200

300

400

Eutopic Ectopic

PGT

mRN

A (

of G

APD

H)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(f)


200400600800

100040005000600070008000

Endometriosis

Eutopic Ectopic


lowastlowastlowastlowast

MRP

4 to

tal m

RNA

(of

GA

PDH

)

(g)


200

400

600

800

Endometriosis

Eutopic Ectopic

MRP

4va

riant

1m

RNA

(of

GA

PDH

)

(h)

Figure 2 Expression of PGE2 and PGF2120572 receptors and prostaglandin transporters in endometrium at different stages of endometriosis 119899= 30 50 and 28 respectively for healthy (control) subjects eutopic tissue samples from patients with the disease and ectopic tissue samplesTotal RNA was extracted and reverse-transcribed and mRNA was quantified by quantitative RT-PCR and normalized relative to GAPDH(internal control) (a) EP1 (b) EP2 (c) EP3 (d) EP4 (e) FP (f) PGT (g) total MRP4 and (h) MRP4 variant 1 The horizontal lines representthe mean for each set of data lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001


Table 2 Effect of endometriosis andmenstrual cycle phase on expression of prostaglandin receptors and transporters in endometrium basedon real-time PCR and normalized relative to GAPDH mRNA as mean plusmn SD Also 119899 = 30 50 and 28 respectively for healthy (control)subjects eutopic tissue samples from endometriosis patients and ectopic tissue samples

GeneControls

(mean plusmn SEM)

EndometriosisEutopic

(mean plusmn SEM)Ectopic

(mean plusmn SEM)EP1

Proliferative phase 3172 plusmn 1005 3070 plusmn 4213 3073 plusmn 5411Secretory phase 2095 plusmn 3533 2002 plusmn 3757 4113 plusmn 1289

EP2Proliferative phase 5790 plusmn 1746 6660 plusmn 2224 7174 plusmn 1489Secretory phase 1982 plusmn 5172dagger 1795 plusmn 3882dagger 1126 plusmn 179

EP3Proliferative phase 2855 plusmn 6652 2976 plusmn 3444 1021 plusmn 2493lowastlowastlowast+++

Secretory phase 7394 plusmn 2006dagger 4835 plusmn 6463daggerdagger 1310 plusmn 2761lowastlowastlowast+++

EP4Proliferative phase 3438 plusmn 1267 4682 plusmn 2268 3802 plusmn 1365lowastlowastlowast+

Secretory phase 2194 plusmn 6654 1047 plusmn 1546 5834 plusmn 2052lowastlowastlowast+++

FPProliferative phase 2375 plusmn 3118 1059 plusmn 4972 9213 plusmn 1947lowastlowastlowast+++

Secretory phase 9727 plusmn 1826daggerdagger 2296 plusmn 6122daggerdagger 14268 plusmn 2742lowastlowastlowast+++

PGTProliferative phase 1604 plusmn 4058 2174 plusmn 3682 5642 plusmn 1473lowastlowast+

Secretory phase 5964 plusmn 3326daggerdagger 6760 plusmn 2736daggerdaggerdagger 8869 plusmn 2528lowastlowastlowast+++

MRP4 totalProliferative phase 1429 plusmn 6347 9705 plusmn 1551 5915 plusmn 227lowastlowast+


MRP4 variant 1Proliferative phase 1609 plusmn 1528 1578 plusmn 1882 1942 plusmn 2408Secretory phase 1962 plusmn 2435 1788 plusmn 1899 1647 plusmn 2093

Note lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus the control group +119875 lt 005 and +++119875 lt 0001 versus the eutopic group dagger119875 lt 005 and daggerdagger119875 lt 001 versus expressionin the corresponding proliferative phase daggerdaggerdagger119875 lt 0001

Staining of EP1 and EP2 in endometrial glands was weakClear staining of EP3 EP4 and FP was visible mainly inendometrial glands but the adjacent stroma also appearedpositive for these receptors However staining of FP wasmore intense in eutopic and ectopic endometrial tissuesof endometriosis patients whether in glandular cells or insurrounding stromal cells

The pattern of PGT staining was similar in endometrialtissues from endometriosis patients and healthy subjectsPositive staining forMRP4 (total) was locatedmainly in stro-mal cells from control patients but only in epithelial cells ofeutopic endometrium from endometriosis patients Howeverthis staining was more intense in ectopic tissue whether inglandular epithelial cells or in surrounding stromal cells

4 Discussion

Prostaglandins E2 and F2120572 play major roles in the regu-lation of the cyclic changes of the endometrium and arealso involved in diseases afflicting this tissue in particular

endometriosis In this study we showed that expressionof mRNA encoding prostaglandin receptors EP3 EP4 andFP and of transporters PGT and MRP4 was increased inectopic endometrium of women suffering from endometrio-sis The expression of FP receptor was increased in bothectopic and eutopic endometrium of endometriosis patientswhile expression of EP3 and EP4 was increased in ectopicendometrium only No effect on expression of EP1 or EP2receptors was observed The menstrual cycle also had asignificant effect increasing EP3 receptor expression in thesecretory phase both in the control group and in womenwith endometriosis Although the cycle did not modulateoverexpression of EP3 EP4 and FP in ectopic endometriumit did appear to affect the eutopic endometrium of thesepatients notably by causing decreased expression of FP in thesecretory phaseThis was not observed for any other receptorWhile the increase in PGF2120572 during the secretory phase inhealthy women is well documented the regulation of PGE2secretion during the menstrual cycle is less certain Studiesshow that PGE2 level may increase during the secretory


Controls Eutopic EctopicEndometriosis

EP1

EP2

EP3

EP4

FP

Negativecontrol

Figure 3 Representative immunohistochemical staining of EP1 EP2 EP3 EP4 and FP in endometrium of healthy women and in eutopicand ectopic endometrium of endometriosis patients No staining was observed in control sections incubated without the primary antibodyor with an equivalent concentration of goat IgG The original magnification was 400x


PGT

MRP4total


Negativecontrol

Figure 4 Representative immunohistochemical staining of PGT and total MRP4 in endometrium of healthy women and in eutopic andectopic endometrium of endometriosis patients No staining was observed in control sections incubated without the primary antibody orwith an equivalent concentration of goat or rat IgG The original magnification was 400x

phase or the proliferative phase or may remain the samethroughout the cycle [33ndash35] Our data corroborate a previ-ous observation of EP4 and FP expression increasing towardsthe end of the menstrual cycle and concomitant with thewithdrawal of progesterone and sloughing of the functionallayer of the endometrium peaking during the mid-lateproliferative phase and not in the secretory phase coincidentwith an elevation in the expression of PGT [36] In contrastchanges in EP3 expression across the menstrual cycle havenot been noted previously [37ndash39]

The specific roles played by PGE2 and PGF2120572 in modu-lating reproductive physiology have been demonstrated usingmice deficient in the corresponding receptors [40] The moststriking observations have been made using FP receptor andEP3 receptor knockout mice It has thus been shown that theFP receptor is indispensable in female reproduction and thatits ablation results in loss of parturition [41] Studies of micelacking individual prostaglandin receptors EP1ndash4 suggestedstrongly that EP3 was the principle receptor mediating pain[42 43] In addition several in vitro studies demonstratedthat expression of aromatase (an enzyme involved in the syn-thesis of estrogens) may be regulated through EP3 [44] Onthe other hand binding of PGE2 to the EP3 receptor regulatesvascular function-dysfunction in ocular tissues and promotesvitreal neovascular diseases such as ischemic retinopathy [45]

and also transcriptional upregulation of fibroblast growthfactor 9 [46] Although little is known about the angiogenicpotential of other prostaglandin receptors increased levelsof EP4 and FP have been reported in perivascular cells inendometrial adenocarcinomas [27 47] More recent studieshave demonstrated that selective blockade of EP4 signalinginhibits proliferation and adhesion of human endometrioticepithelial and stromal cells through suppression of integrin-mediated mechanisms [48ndash50] It has also been shown thatanomalies in cell adhesion morphology and proliferationcan occur after binding of ligands to EP4 and FP or activationof downstream signaling pathways such as MAPK and PI3K[47 51ndash53] Our results suggest that the specific increase inexpression of these receptors is not insignificant and that itmay contribute to the principal symptoms associated withendometriosis Very few authors have studied the role ofMRP4 in endometriosis [54] Increased MRP4 expressionhas been shown in malignant prostate tissue [55] in acutemyeloid leukemia [56] and in colorectal neoplasia [57]while increased PGT expression has been associated withepithelialmalignancy [58]Our results show for the first timeincreased expression of both prostaglandin transporters inendometriotic tissues We observed significant increases ofPGT expression in eutopic endometrium during the prolif-erative phase of the menstrual cycle both in healthy women


and in patients with endometriosis as well as in ectopicendometrium As reported previously [54 59] we alsoobserved significantly increased expression of total MRP4 inectopic tissue while MRP4 variant 1 showed no noticeablechange Recently described as a prostaglandin efflux trans-porter MRP4 was expressed at much the same level through-out the menstrual cycle Based on these results we suggestthat the observed overexpression of MRP4 is most likely dueto variant 2 and not to the combination of the two variants asis often reportedThe increases in both transportermoleculesappear concomitant specifically in ectopic lesions Althoughtheir actions seem to be different these transporters mightwork in concert to improve prostaglandin dispatching

Immunohistochemistry experiments largely support themRNA expression results In eutopic endometrium PGE2and PGF2120572 receptors and transporters were found at greaterabundance in glandular epithelial cells than in stromalcells consistent with increased epithelial MRP4 expressiondemonstrated in endometriosis [54] and cancer [55] Further-more staining of PGT FP EP3 and EP4 in ectopic endome-trial tissue was intense as reported previously [60 61] Lowlevels of EP1 and EP2 were also found in the glandularepithelium and in ectopic tissue Our results corroboratethose of Arosh et al [62] who reported that EP2 staining inthe endometrium of cattle was expressed mainly in glandularepithelial cells

Elevated MRP4 and PGT expression particularly inepithelial glandular cells may result in increased availabilityof PGE2 and PGF2120572 which through engagement of theirEP3 EP4 or FP receptors (also elevated in endometriosis)may activate intracellular signals such as the diacylglycerolor cyclic AMP pathways Aberrant transport and signalingby prostaglandin receptors in the endometrium [47 63]as observed in the present study therefore might promoteuterine pathologies such as endometriosis Although themodifications observed in the eutopic endometrium ofendometriosis patients appear slight they nevertheless affectsensitivity to PGE2 andPGF2120572 and thus disrupt normal func-tion The proinflammatory environment of the peritonealcavity of women with endometriosis induces significantoverexpression of the majority transporters and receptorsrequired to regulate PGE2 and PGF2120572 In addition to having arole in the pathogenesis of the disease this could also disruptthe entire female reproductive tract Since the female genitaliabathe in peritoneal liquid [64] an increased level of PGE2 andPGF2120572 could act on the whole system and thereby influencethe reproductive process

Although qualitative immunohistochemistry has limita-tions that should not be overlooked Immunohistochemicalconfirmation of mRNA expression reinforces the importanceof our observations especially in view of the inevitablevariability associated with categorizing of clinical symptomsandor staging of patients In conclusion this study revealsfor the first time that endometriosis can affect the regulationof PGE2 and PGF2120572 activity at the points of reception on thecell surface and transport into the target cells

Capsule Eutopic and ectopic endometria of endometriosispatients display distinct anomalies in levels of expression ofprostaglandins receptivity and uptake factors

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

A special acknowledgement is due to the contribution ofDr Ali Akoum who passed away in July 2014 Dr Akoumwas a principal investigator in the endometriosis screeningstudy without which the present study would not have beenpossible This paper was supported by Grants MOP-120769andMOP-123259 to Ali Akoum from the Canadian Institutesfor Health Research Ali Akoum is also FRQ-S (Fonds de laRecherche du Quebec-Sante) ldquoNational Researcherrdquo HalimaRakhila is the recipient of a doctoral studentship from theldquoReseau Quebecois en Reproductionrdquo

References

[1] L C Giudice ldquoClinical practice Endometriosisrdquo The NewEngland Journal of Medicine vol 362 no 25 pp 2389ndash23982010

[2] A Akoum A Lemay C Brunet and J Hebert ldquoSecretion ofmonocyte chemotactic protein-1 by cytokine-stimulated endo-metrial cells of women with endometriosisrdquo Fertility andSterility vol 63 no 2 pp 322ndash328 1995

[3] Y Yang P Degranpre A Kharfi and A Akoum ldquoIdentifi-cation of macrophage migration inhibitory factor as a potentendothelial cell growth-promoting agent released by ectopichuman endometrial cellsrdquoThe Journal of Clinical Endocrinologyamp Metabolism vol 85 no 12 pp 4721ndash4727 2000

[4] K Khoufache P K Bondza N Harir et al ldquoSoluble human IL-1 receptor type 2 inhibits ectopic endometrial tissue implanta-tion and growth identification of a novel potential target forendometriosis treatmentrdquo The American Journal of Pathologyvol 181 no 4 pp 1197ndash1205 2012

[5] M Y Dawood F S Khan-Dawood and L Wilson Jr ldquoPeri-toneal fluid prostaglandins and prostanoids in women withendometriosis chronic pelvic inflammatory disease and pelvicpainrdquoAmerican Journal of Obstetrics amp Gynecology vol 148 no4 pp 391ndash395 1984

[6] T-C Lee and H-C Ho ldquoEffects of prostaglandin E2

andvascular endothelial growth factor on sperm might lead toendometriosis-associated infertilityrdquo Fertility and Sterility vol95 no 1 pp 360ndash362 2011

[7] A I Koskimies A Tenhunen and O Ylikorkala ldquoPeritonealfluid 6-ketoprostaglandin F1120572 thromboxane B2 in endometrio-sis and unexplained infertilityrdquo Acta Obstetricia et GynecologicaScandinavica vol 63 no 123 pp 19ndash21 1984

[8] S H Ferreira ldquoProstaglandins pain and inflammationrdquoAgentsand Actions Supplements vol 19 pp 91ndash98 1986

[9] M S Arayne and S S Ul Hasan ldquoProstaglandins in pain andinflammatiinrdquo Journal of the Pakistan Medical Association vol27 no 5 pp 326ndash330 1977

[10] O Ylikorkala A Koskimies T Laatkainen A Tenhunen andL Viinikka ldquoPeritoneal fluid prostaglandins in endometriosistubal disorders and unexplained infertilityrdquo Obstetrics andGynecology vol 63 no 5 pp 616ndash620 1984

[11] N M Pobedinskiı M A Khachikian V P Zykova and N DFanchenko ldquoBlood levels of prostaglandins in fertile women


and in women with different variants of endocrine infertilityrdquoAkusherstvo i Ginekologiia no 8 pp 14ndash18 1982

[12] R S Schenken R H Asch R F Williams and G D HodgenldquoEtiology of infertility in monkeys with endometriosis mea-surement of peritoneal fluid prostaglandinsrdquo American Journalof Obstetrics and Gynecology vol 150 no 4 pp 349ndash353 1984

[13] K Shirasuna K Sasahara M Matsui T Shimizu and AMiyamoto ldquoProstaglandin F2120572 differentially affects mRNAexpression relating to angiogenesis vasoactivation and pros-taglandins in the early and mid corpus luteum in the cowrdquoJournal of Reproduction and Development vol 56 no 4 pp428ndash436 2010

[14] M Majima H Amano and I Hayashi ldquoEndogenous prostagl-andins and angiogenesisrdquo Nihon Yakurigaku Zasshi vol 117 no4 pp 283ndash292 2001

[15] U Ulug S Goldman I Ben-Shlomo and E Shalev ldquoMatrixmetalloproteinase (MMP)-2 and MMP-9 and their inhibitorTIMP-1 in human termdecidua and fetalmembranes the effectof prostaglandin F2120572 and indomethacinrdquo Molecular HumanReproduction vol 7 no 12 pp 1187ndash1193 2001

[16] H-H Hsu W-S Hu Y-M Lin et al ldquoJNK suppression isessential for 17120573-Estradiol inhibits prostaglandin E2-InduceduPAandMMP-9 expressions and cellmigration in humanLoVocolon cancer cellsrdquo Journal of Biomedical Science vol 18 no 1article 61 2011

[17] H Ota S Igarashi M Sasaki and T Tanaka ldquoDistribution ofcyclooxygenase-2 in eutopic and ectopic endometrium in endo-metriosis and adenomyosisrdquo Human Reproduction vol 16 no3 pp 561ndash566 2001

[18] C Carli C N Metz Y Al-Abed P H Naccache and AAkoum ldquoUp-regulation of cyclooxygenase-2 expression andprostaglandin E

2

production in human endometriotic cells bymacrophage migration inhibitory factor involvement of novelkinase signaling pathwaysrdquo Endocrinology vol 150 no 7 pp3128ndash3137 2009

[19] H Fan and X L Fang ldquoExpression of cylooxygenase-2 in endo-metriosisrdquo Zhong Nan Da Xue Xue Bao Yi Xue Ban vol 30 no1 pp 92ndash95 2005

[20] S K Banu J Lee V O Speights Jr A Starzinski-Powitz andJ A Arosh ldquoCyclooxygenase-2 regulates survival migrationand invasion of human endometriotic cells through multiplemechanismsrdquo Endocrinology vol 149 no 3 pp 1180ndash1189 2008

[21] E A Willman W P Collins and S G Clayton ldquoStudies in theinvolvement of prostaglandins in uterine symptomatology andpathologyrdquo BJOG vol 83 no 5 pp 337ndash341 1976

[22] V Lundstrom K Green and K Svanborg ldquoEndogenous pros-taglandins in dysmenorrhea and the effect of prostaglandinsynthetase inhibitors (PGSI) on uterine contractilityrdquo ActaObstetricia et Gynecologica Scandinavica vol 58 no 87 pp 51ndash56 1979

[23] P Stromberg M Akerlund M L Forsling and H KindahlldquoInvolvement of prostaglandins in vasopressin stimulation ofthe human uterusrdquo British Journal of Obstetrics amp Gynaecologyvol 90 no 4 pp 332ndash337 1983

[24] S Z A Badawy V Cuenca and L Marshall ldquoPeritoneal fluidprostaglandins in patients with endometriosisrdquoContributions toGynecology and Obstetrics vol 16 pp 60ndash65 1987

[25] S Z A Badawy L Marshall and V Cuenca ldquoPeritoneal fluidprostaglandins in various stages of the menstrual cycle role ininfertile patients with endometriosisrdquo International Journal ofFertility vol 30 no 2 pp 48ndash52 1985

[26] H Rakhila C Carli M Daris M Lemyre M Leboeuf and AAkoum ldquoIdentification of multiple and distinct defects in pros-taglandin biosynthetic pathways in eutopic and ectopic endo-metrium of women with endometriosisrdquo Fertility and Sterilityvol 100 no 6 pp 1650e2ndash1659e2 2013

[27] H N Jabbour and K J Sales ldquoProstaglandin receptor signallingand function in human endometrial pathologyrdquo Trends inEndocrinology andMetabolism vol 15 no 8 pp 398ndash404 2004

[28] H N Jabbour K J Sales O P M Smith S Battersby and SC Boddy ldquoProstaglandin receptors are mediators of vascularfunction in endometrial pathologiesrdquo Molecular and CellularEndocrinology vol 252 no 1-2 pp 191ndash200 2006

[29] J Lee J AMcCracken S K Banu and J AArosh ldquoIntrauterineinhibition of prostaglandin transporter protein blocks release ofluteolytic PGF2alpha pulses without suppressing endometrialexpression of estradiol or oxytocin receptor in ruminantsrdquoBiology of Reproduction vol 89 no 2 article 27 2013

[30] Y Chi and V L Schuster ldquoThe prostaglandin transporter PGTtransports PGH

2

rdquo Biochemical and Biophysical Research Com-munications vol 395 no 2 pp 168ndash172 2010

[31] N Lacroix-Pepin G Danyod N Krishnaswamy et al ldquoThemultidrug resistance-associated protein 4 (MRP4) appears as afunctional carrier of prostaglandins regulated by oxytocin in thebovine endometriumrdquo Endocrinology vol 152 no 12 pp 4993ndash5004 2011

[32] M-H Wu Y Shoji P-C Chuang and S-J Tsai ldquoEndometrio-sis disease pathophysiology and the role of prostaglandinsrdquoExpert Reviews in Molecular Medicine vol 9 no 2 pp 1ndash202007

[33] M A Lumsden R W Kelly M H Abel and D T BairdldquoThe concentrations of prostaglandins in endometrium duringthe menstrual cycle in women with measured menstrual bloodlossrdquo Prostaglandins Leukotrienes and Medicine vol 23 no 2-3pp 217ndash227 1986

[34] S A Milne G B Perchick S C Boddy and H N JabbourldquoExpression localization and signaling of PGE2 and EP2EP4receptors in humannonpregnant endometrium across themen-strual cyclerdquo Journal of Clinical Endocrinology and Metabolismvol 86 no 9 pp 4453ndash4459 2001

[35] M C P Rees A B M Anderson L M Demers and A CTurnbull ldquoEndometrial and myometrial prostaglandin releaseduring the menstrual cycle in relation to menstrual blood lossrdquoThe Journal of Clinical Endocrinology ampMetabolism vol 58 no5 pp 813ndash818 1984

[36] J Kang P Chapdelaine J Parent E Madore P Y Laberge andM A Fortier ldquoExpression of human prostaglandin transporterin the human endometrium across themenstrual cyclerdquo Journalof Clinical Endocrinology and Metabolism vol 90 no 4 pp2308ndash2313 2005

[37] T Namba Y Sugimoto M Negishi et al ldquoAlternative splicingof C-terminal tail of prostaglandin E receptor subtype EP3determines G-protein specificityrdquoNature vol 365 no 6442 pp166ndash170 1993

[38] K Sakamoto T Ezashi K Miwa et al ldquoMolecular cloning andexpression of a cDNAof the bovine prostaglandin F

2

120572 receptorrdquoThe Journal of Biological Chemistry vol 269 no 5 pp 3881ndash3886 1994

[39] J A Arosh S K Banu P Chapdelaine et al ldquoMolecular cloningand characterization of bovine prostaglandin E

2

receptorsEP2 and EP4 expression and regulation in endometrium andmyometrium during the estrous cycle and early pregnancyrdquoEndocrinology vol 144 no 7 pp 3076ndash3091 2003


[40] S Narumiya Y Sugimoto and F Ushikubi ldquoProstanoidreceptors structures properties and functionsrdquo PhysiologicalReviews vol 79 no 4 pp 1193ndash1226 1999

[41] S Narumiya and G A FitzGerald ldquoGenetic and pharmaco-logical analysis of prostanoid receptor functionrdquoThe Journal ofClinical Investigation vol 108 no 1 pp 25ndash30 2001

[42] A Ueno H Matsumoto H Naraba et al ldquoMajor roles of pros-tanoid receptors IP and EP

3

in endotoxin-induced enhance-ment of pain perceptionrdquo Biochemical Pharmacology vol 62no 2 pp 157ndash160 2001

[43] T Minami H Nakano T Kobayashi et al ldquoCharacterization ofEP receptor subtypes responsible for prostaglandin E

2

-inducedpain responses by use of EP

1

and EP3

receptor knockout micerdquoBritish Journal of Pharmacology vol 133 no 3 pp 438ndash4442001

[44] J A Richards and R W Brueggemeier ldquoProstaglandin E2

regulates aromatase activity and expression in human adiposestromal cells via two distinct receptor subtypesrdquo The Journal ofClinical Endocrinology amp Metabolism vol 88 no 6 pp 2810ndash2816 2003

[45] F Sennlaub F Valamanesh A Vazquez-Tello et al ldquoCyclooxy-genase-2 in human and experimental ischemic proliferativeretinopathyrdquo Circulation vol 108 no 2 pp 198ndash204 2003

[46] P-C Chuang H S Sun T-M Chen and S-J Tsai ldquoPros-taglandin E

2

induces fibroblast growth factor 9 via EPS-dependent protein kinase C120575 and Elk-1 signalingrdquo Molecularand Cellular Biology vol 26 no 22 pp 8281ndash8292 2006

[47] K J Sales S A Milne A R W Williams R A Andersonand H N Jabbour ldquoExpression localization and signaling ofprostaglandin F

2120572

receptor in human endometrial adenocarci-noma regulation of proliferation by activation of the epidermalgrowth factor receptor and mitogen-activated protein kinasesignaling pathwaysrdquo Journal of Clinical Endocrinology andMetabolism vol 89 no 2 pp 986ndash993 2004

[48] J Lee S K Banu R C Burghardt A Starzinski-Powitz andJ A Arosh ldquoSelective inhibition of prostaglandin E2 receptorsEP2 and EP4 inhibits adhesion of human endometriotic epithe-lial and stromal cells through suppression of integrin-mediatedmechanismsrdquo Biology of Reproduction vol 88 no 3 p 77 2013

[49] J Lee S K Banu R Rodriguez A Starzinski-Powitz and J AArosh ldquoSelective blockade of prostaglandin E

2

receptors EP2and EP4 signaling inhibits proliferation of human endometri-otic epithelial cells and stromal cells through distinct cell cyclearrestrdquo Fertility and Sterility vol 93 no 8 pp 2498ndash2506 2010

[50] J Lee S K Banu T Subbarao A Starzinski-Powitz andJ A Arosh ldquoSelective inhibition of prostaglandin E

2

recep-tors EP2 and EP4 inhibits invasion of human immortalizedendometriotic epithelial and stromal cells through suppressionof metalloproteinasesrdquo Molecular and Cellular Endocrinologyvol 332 no 1-2 pp 306ndash313 2011

[51] K L Pierce H Fujino D Srinivasan and J W Regan ldquoActiva-tion of FP prostanoid receptor isoforms leads to rho-mediatedchanges in cellmorphology and in the cell cytoskeletonrdquo Journalof Biological Chemistry vol 274 no 50 pp 35944ndash35949 1999

[52] H Sheng J Shao M K Washington and R N DuBoisldquoProstaglandin E2 increases growth and motility of colorectalcarcinoma cellsrdquo Journal of Biological Chemistry vol 276 no21 pp 18075ndash18081 2001

[53] V Capra A Habib M R Accomazzo et al ldquoThromboxaneprostanoid receptor in human airway smoothmuscle cells a rel-evant role in proliferationrdquo European Journal of Pharmacologyvol 474 no 2-3 pp 149ndash159 2003

[54] I Gori Y Rodriguez C Pellegrini et al ldquoAugmented epithelialmultidrug resistance-associated protein 4 expression in peri-toneal endometriosis regulation by lipoxin A4rdquo Fertility andSterility vol 99 no 7 pp 1965e2ndash1973e2 2013

[55] L H Lye J G Kench D J Handelsman et al ldquoAndrogenregulation ofmultidrug resistance-associated protein 4 (MRP4ABCC4) in prostate cancerrdquo The Prostate vol 68 no 13 pp1421ndash1429 2008

[56] S Copsel C Garcia F Diez et al ldquoMultidrug resistance protein4 (MRP4ABCC4) regulates cAMP cellular levels and controlshuman leukemia cell proliferation and differentiationrdquo TheJournal of Biological Chemistry vol 286 no 9 pp 6979ndash69882011

[57] V R Holla M G Backlund P Yang R A Newman and R NDuBois ldquoRegulation of prostaglandin transporters in colorectalneoplasiardquo Cancer Prevention Research vol 1 no 2 pp 93ndash992008

[58] K Guda S P Fink G L Milne et al ldquoInactivating mutationin the prostaglandin transporter gene SLCO2A1 associatedwith familial digital clubbing colon neoplasia and NSAIDresistancerdquo Cancer Prevention Research vol 7 no 8 pp 805ndash812 2014

[59] R Kumar A-C Clerc I Gori et al ldquoLipoxin A4

preventsthe progression of de novo and established endometriosis in amouse model by attenuating prostaglandin E

2

production andestrogen signalingrdquo PLoS ONE vol 9 no 2 Article ID e897422014

[60] M C Keightley K J Sales and H N Jabbour ldquoPGF2120572-F-pros-tanoid receptor signalling via ADAMTS1 modulates epithelialcell invasion and endothelial cell function in endometrialcancerrdquo BMC Cancer vol 10 article 488 2010

[61] S K Banu J A Arosh P Chapdelaine and M A FortierldquoExpression of prostaglandin transporter in the bovine uterusand fetal membranes during pregnancyrdquo Biology of Reproduc-tion vol 73 no 2 pp 230ndash236 2005

[62] J A Arosh S K Banu S Kimmins P Chapdelaine L AMacLaren and M A Fortier ldquoEffect of interferon-120591 on pros-taglandin biosynthesis transport and signaling at the timeof maternal recognition of pregnancy in cattle evidence ofpolycrine actions of prostaglandin E

2

rdquo Endocrinology vol 145no 11 pp 5280ndash5293 2004

[63] H N Jabbour S A Milne A R W Williams R A Andersonand S C Boddy ldquoExpression of COX-2 and PGE synthase andsynthesis of PGE

2

in endometrial adenocarcinoma a possibleautocrineparacrine regulation of neoplastic cell function viaEP2EP4 receptorsrdquo British Journal of Cancer vol 85 no 7 pp1023ndash1031 2001

[64] E Oral D L Olive and A Arici ldquoThe peritoneal environmentin endometriosisrdquo Human Reproduction Update vol 2 no 5pp 385ndash398 1996


other specific prostaglandins are present at abnormally highlevels in uterine tissues of women suffering from menorrha-gia dysmenorrhoea or endometriosis [21ndash23] It is also wellknown that PGE2 and PGF2120572 are more concentrated in theperitoneal fluid of endometriosis patients [5 10 12 24 25]Such evidence has led us to investigate a possible role of theseprostaglandins in the pathogenesis of endometriosis Ourrecent comparison of endometrium (eutopic and ectopic)from endometriosis patients to healthy eutopic endometriumshowed overproduction of PGE2 and PGF2120572 apparentlypromoted by increased expression of enzymes such as COX-2with cPGES or AKR1C3 [26]

Regulation of production is not the only means bywhich the body modulates the action of PGE2 and PGF2120572Expression of prostaglandin receptors and transporters mayalso be regulated Once released as messenger moleculesprostaglandins act locally on receptors in an autocrine orparacrine manner The four main subtypes of PGE2 receptorare designated as EP1 EP2 EP3 and EP4 while the PGF2120572receptor is called FP [27 28] The receptor subtype deter-mines the nature of the physiological response Receptioneither elicits the intracellular calcium-inositol triphosphatepathway or increasesdecreases cyclic adenosine monophos-phate (cAMP) activity Engagement of some receptors mayelicit both pathways depending on cell type and receptorsplice variety

Prostaglandins were originally believed to exit fromproducer cells via passive diffusion because of their stronglylipophilic characterThediscovery of the prostaglandin trans-porter protein PGT (SLCO2A1) which mediates prostagl-andin uptake and release [29 30] demonstrated that diffu-sion alone did not explain the penetration of prostaglandinsthrough the cell membrane Furthermore a specific trans-porter namely multidrug resistance protein 4 (MRP4ABCC4) of the ATP-binding cassette transporter superfam-ily has been shown to mediate prostaglandin release [31]Whether or not MRP4 is the only transporter that does thisis still unclear

Although it is clear that PGE2 and PGF2120572 play importantroles in a number of female reproductive physiological proc-esses as well as in endometriosis-associated infertility andpain [5 10 12 32] current understanding of these rolesremains incomplete In the present study we analyzed theexpression of EP1 EP2 EP3 EP4 FP PGT and MRP4 inendometriosis patients in comparison to their expression innormal eutopic endometrium We observed marked differ-ences between eutopic and ectopic endometria in terms ofprostaglandins receptivity and transport readiness

2 Materials and Methods

21 Patients and Tissue Collection The study received ap-proval from the Human Research Ethics Committee atSaint-Francois drsquoAssise Hospital and informed consent wasobtained from all participants who were recruited betweenFebruary 2002 and March 2007 Endometriosis patients wereaged 342 plusmn 36 years (119899 = 78) and were consulting forpelvic pain andor infertility They were diagnosed usinglaparoscopy and the disease stage (IndashIV) was determined

according to the Revised American Fertility Society classifi-cation system Endometriotic tissue samples were collectedfrom 28 of these patients We also recruited healthy womenaged 353 plusmn 38 (119899 = 30) scheduled for tubal ligation Theseparticipants had no pelvic pathology or sign of endometrialhyperplasia or neoplasia and had not received any anti-inflammatory or hormonal medication for at least 3 monthsMenstrual cycle dating was determined using the cyclehistory

Endometrial and endometriotic biopsies were obtainedduring laparoscopy Tissue was placed immediately at 4∘C insterile Hankrsquos balanced salts solution (HBSS) (GIBCO Invit-rogen Corp Burlington ON Canada) containing 100 IUmLpenicillin 100 120583gmL streptomycin and 025 120583gmL ampho-tericin and transported to the laboratory After washingin HBSS at 4∘C samples were frozen at minus80∘C in Eppen-dorf tubes for quantitative real-time PCR (qRT-PCR) orembedded in paraffin and stored at room temperature forimmunohistochemical analysis

22 Quantitative Real-Time PCR Total RNA was extractedfrom endometrial tissue using the TRIzol reagent accordingto the manufacturerrsquos instructions (Invitrogen Life Technolo-gies Inc Grand Island NY USA) and reverse-transcribedin the presence of random hexamers The qRT-PCR reactionwas carried out in an ABI 7000 Thermal Cycler (AppliedBiosystems Foster City CA USA) The standard reactionmixture contained 2120583L of RT product 05 120583L of each primer(final concentration 01mM) 125 120583L SYBR Green PCRMaster Mix (Invitrogen Life technologies Inc Grand IslandNY USA) consisting of Taq DNA polymerase reaction bufferdNTP mix SYBR green I MgCl

2 and Taq DNA polymerase

Following denaturing for 2min at 95∘C the reactions werecycled 45 times with denaturing for 15 sec at 95∘C and anneal-ing for 60 sec at 60∘C The primer sequences are listed inTable 1 The primers were designed using Primer Express 20(Applied Biosystems Foster City CA USA) to span intron-exon boundaries to avoid amplification of genomic DNA andselected to have compatible Tm values (59ndash61∘C) A relativequantification method was used Expression of mRNA ofEP1 EP2 EP3 EP4 FP PGT total MRP4 and MRP4 variant1 was normalized to that of the gene GAPDH After eachrun melting curve analysis (55ndash95∘C) was performed toverify the specificity of the PCR reaction All samples weretested in duplicate and each run included a template controlBaseline curves melting curves melting points crossingpoints slopes and errors were monitored for each gene

23 Immunohistochemical Probe Endometrium was fixedin 10 formalin (Fisher Scientific New Jersey USA) andthen embedded in paraffin Serial tissue sections 4120583m thickwere rinsed in phosphate buffered saline (PBS) and treatedwith 3 hydrogen peroxide to block endogenous peroxidaseactivity All antibodies were diluted in PBS containing 02bovine serum albumin and 01 Tween 20 Sections wereincubated for twohours at room temperaturewith the specificantibody (Cayman Ann Arbor USA) Rabbit polyclonalanti-human EP2 was diluted 1 800 and rabbit polyclonalantibodies directed against human EP1 EP3 EP4 FP and













3 Results










0

500

1000

1500

2000

2500


Endometriosis

EP1

mRN

A (

of G

APD

H)

(a)


200

400

600

800

1000

Endometriosis

EP2

mRN

A (

of G

APD

H)

(b)

0

1000

2000

3000

4000

5000


Endometriosis

EP3

mRN

A (

of G

APD


lowastlowastlowast

(c)

0

1000

2000

3000

4000


Endometriosis

lowastlowastlowast

lowastlowastlowast

EP4

mRN

A (

of G

APD

H)

(d)

0

1000

2000

3000

10000200003000040000


Endometriosis

FP m

RNA

(of

GA

PDH

)


lowast

(e)

0

100

200

300

400

Endometriosis


PGT

mRN

A (

of G

APD

H)

lowastlowastlowast

lowastlowastlowast

(f)

0200400600800

100040005000600070008000

Endometriosis


lowastlowastlowast

lowastlowastlowast

MRP

4 to

tal m

RNA

(of

GA

PDH

)

(g)

0

200

400

600

800

Endometriosis


MRP

4va

riant

1m

RNA

(of

GA

PDH

)

(h)





500

1000

1500

2000

2500

Eutopic Ectopic

EP1

mRN

A (

of G

APD

H)

(a)


200

400

600

800

1000

Eutopic Ectopic

EP2

mRN

A (

of G

APD

H)

(b)


250

50010002000300040005000

Eutopic Ectopic

EP3

mRN

A (

of G

APD

H)



(c)


1000

2000

3000

4000

Eutopic Ectopic

lowastlowastlowast


lowastlowast

EP4

mRN

A (

of G

APD

H)

(d)


1000

2000

3000

10000200003000040000

Eutopic Ectopic

FP m

RNA

(of

GA

PDH

)



(e)


100

200

300

400

Eutopic Ectopic

PGT

mRN

A (

of G

APD

H)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(f)


200400600800

100040005000600070008000

Endometriosis

Eutopic Ectopic



MRP

4 to

tal m

RNA

(of

GA

PDH

)

(g)


200

400

600

800

Endometriosis

Eutopic Ectopic

MRP

4va

riant

1m

RNA

(of

GA

PDH

)

(h)




GeneControls

(mean plusmn SEM)




















4 Discussion





EP1

EP2

EP3

EP4

FP

Negativecontrol



PGT

MRP4total


Negativecontrol













Acknowledgments


References






















2














2










2



2






3



2


1

and EP3






2



2120572




2



2












2





2



2















3 Results










0

500

1000

1500

2000

2500


Endometriosis

EP1

mRN

A (

of G

APD

H)

(a)


200

400

600

800

1000

Endometriosis

EP2

mRN

A (

of G

APD

H)

(b)

0

1000

2000

3000

4000

5000


Endometriosis

EP3

mRN

A (

of G

APD


lowastlowastlowast

(c)

0

1000

2000

3000

4000


Endometriosis

lowastlowastlowast

lowastlowastlowast

EP4

mRN

A (

of G

APD

H)

(d)

0

1000

2000

3000

10000200003000040000


Endometriosis

FP m

RNA

(of

GA

PDH

)


lowast

(e)

0

100

200

300

400

Endometriosis


PGT

mRN

A (

of G

APD

H)

lowastlowastlowast

lowastlowastlowast

(f)

0200400600800

100040005000600070008000

Endometriosis


lowastlowastlowast

lowastlowastlowast

MRP

4 to

tal m

RNA

(of

GA

PDH

)

(g)

0

200

400

600

800

Endometriosis


MRP

4va

riant

1m

RNA

(of

GA

PDH

)

(h)





500

1000

1500

2000

2500

Eutopic Ectopic

EP1

mRN

A (

of G

APD

H)

(a)


200

400

600

800

1000

Eutopic Ectopic

EP2

mRN

A (

of G

APD

H)

(b)


250

50010002000300040005000

Eutopic Ectopic

EP3

mRN

A (

of G

APD

H)



(c)


1000

2000

3000

4000

Eutopic Ectopic

lowastlowastlowast


lowastlowast

EP4

mRN

A (

of G

APD

H)

(d)


1000

2000

3000

10000200003000040000

Eutopic Ectopic

FP m

RNA

(of

GA

PDH

)



(e)


100

200

300

400

Eutopic Ectopic

PGT

mRN

A (

of G

APD

H)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(f)


200400600800

100040005000600070008000

Endometriosis

Eutopic Ectopic



MRP

4 to

tal m

RNA

(of

GA

PDH

)

(g)


200

400

600

800

Endometriosis

Eutopic Ectopic

MRP

4va

riant

1m

RNA

(of

GA

PDH

)

(h)




GeneControls

(mean plusmn SEM)




















4 Discussion





EP1

EP2

EP3

EP4

FP

Negativecontrol



PGT

MRP4total


Negativecontrol













Acknowledgments


References






















2














2










2



2






3



2


1

and EP3






2



2120572




2



2












2





2



2




0

500

1000

1500

2000

2500


Endometriosis

EP1

mRN

A (

of G

APD

H)

(a)


200

400

600

800

1000

Endometriosis

EP2

mRN

A (

of G

APD

H)

(b)

0

1000

2000

3000

4000

5000


Endometriosis

EP3

mRN

A (

of G

APD


lowastlowastlowast

(c)

0

1000

2000

3000

4000


Endometriosis

lowastlowastlowast

lowastlowastlowast

EP4

mRN

A (

of G

APD

H)

(d)

0

1000

2000

3000

10000200003000040000


Endometriosis

FP m

RNA

(of

GA

PDH

)


lowast

(e)

0

100

200

300

400

Endometriosis


PGT

mRN

A (

of G

APD

H)

lowastlowastlowast

lowastlowastlowast

(f)

0200400600800

100040005000600070008000

Endometriosis


lowastlowastlowast

lowastlowastlowast

MRP

4 to

tal m

RNA

(of

GA

PDH

)

(g)

0

200

400

600

800

Endometriosis


MRP

4va

riant

1m

RNA

(of

GA

PDH

)

(h)





500

1000

1500

2000

2500

Eutopic Ectopic

EP1

mRN

A (

of G

APD

H)

(a)


200

400

600

800

1000

Eutopic Ectopic

EP2

mRN

A (

of G

APD

H)

(b)


250

50010002000300040005000

Eutopic Ectopic

EP3

mRN

A (

of G

APD

H)



(c)


1000

2000

3000

4000

Eutopic Ectopic

lowastlowastlowast


lowastlowast

EP4

mRN

A (

of G

APD

H)

(d)


1000

2000

3000

10000200003000040000

Eutopic Ectopic

FP m

RNA

(of

GA

PDH

)



(e)


100

200

300

400

Eutopic Ectopic

PGT

mRN

A (

of G

APD

H)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(f)


200400600800

100040005000600070008000

Endometriosis

Eutopic Ectopic



MRP

4 to

tal m

RNA

(of

GA

PDH

)

(g)


200

400

600

800

Endometriosis

Eutopic Ectopic

MRP

4va

riant

1m

RNA

(of

GA

PDH

)

(h)




GeneControls

(mean plusmn SEM)




















4 Discussion





EP1

EP2

EP3

EP4

FP

Negativecontrol



PGT

MRP4total


Negativecontrol













Acknowledgments


References






















2














2










2



2






3



2


1

and EP3






2



2120572




2



2












2





2



2





500

1000

1500

2000

2500

Eutopic Ectopic

EP1

mRN

A (

of G

APD

H)

(a)


200

400

600

800

1000

Eutopic Ectopic

EP2

mRN

A (

of G

APD

H)

(b)


250

50010002000300040005000

Eutopic Ectopic

EP3

mRN

A (

of G

APD

H)



(c)


1000

2000

3000

4000

Eutopic Ectopic

lowastlowastlowast


lowastlowast

EP4

mRN

A (

of G

APD

H)

(d)


1000

2000

3000

10000200003000040000

Eutopic Ectopic

FP m

RNA

(of

GA

PDH

)



(e)


100

200

300

400

Eutopic Ectopic

PGT

mRN

A (

of G

APD

H)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(f)


200400600800

100040005000600070008000

Endometriosis

Eutopic Ectopic



MRP

4 to

tal m

RNA

(of

GA

PDH

)

(g)


200

400

600

800

Endometriosis

Eutopic Ectopic

MRP

4va

riant

1m

RNA

(of

GA

PDH

)

(h)




GeneControls

(mean plusmn SEM)




















4 Discussion





EP1

EP2

EP3

EP4

FP

Negativecontrol



PGT

MRP4total


Negativecontrol













Acknowledgments


References






















2














2










2



2






3



2


1

and EP3






2



2120572




2



2












2





2



2





GeneControls

(mean plusmn SEM)




















4 Discussion





EP1

EP2

EP3

EP4

FP

Negativecontrol



PGT

MRP4total


Negativecontrol













Acknowledgments


References






















2














2










2



2






3



2


1

and EP3






2



2120572




2



2












2





2



2





EP1

EP2

EP3

EP4

FP

Negativecontrol



PGT

MRP4total


Negativecontrol













Acknowledgments


References






















2














2










2



2






3



2


1

and EP3






2



2120572




2



2












2





2



2




PGT

MRP4total


Negativecontrol













Acknowledgments


References






















2














2










2



2






3



2


1

and EP3






2



2120572




2



2












2





2



2











Acknowledgments


References






















2














2










2



2






3



2


1

and EP3






2



2120572




2



2












2





2



2












2














2










2



2






3



2


1

and EP3






2



2120572




2



2












2





2



2







3



2


1

and EP3






2



2120572




2



2












2





2



2



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Abnormal Expression of Prostaglandins E2 and F2α Receptors ... · ClinicalStudy Abnormal...

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