Effects
o
follicular
fluid
oxidative
status
on
human
mural
granulosa
cells,
oocyte
competency
and
ICSI
parameters
Seda
Karabulut
a,b,*
,
Oya
Korkmaz
a,b,
Pelin
Kutlu
c,
Hilal
Eren
Gozel
a,b,
Ilknur
Keskin
a,ba
IstanbulMedipolUniversity,InternationalSchoolofMedicine,HistologyandEmbryologyDepartment, _Istanbul,Turkey
b
MedipolUniversity,SchoolofMedicine,Kavacık, _Istanbul,REMER(RegenerativeandRestorativeMedicineResearchCenter),KavacıkMah.EkincilerCad. No.19KavacıkKavşagı34810Beykoz, _Istanbul,Turkey
cMedicanaÇamlıcaHospital,IVFCenter, _Istanbul,Turkey
ARTICLE INFO Articlehistory: Received4June2019
Receivedinrevisedform2June2020 Accepted8June2020
Keywords: Oxidativestress Follicularfluid
Intracytoplasmicsperminjection DNA
Muralgranulosacells
ABSTRACT
Purpose:Theaimofthepresentstudywastounderstandthemolecularandgeneticalterationsinvolved
infollicularfluidoxidativeprocessbyinvestigatinghumanmuralgranulosacellsandtofindpossible
biomarkersforoocytecompetencyandICSIoutcomemeasures.
Methods:Atotalof166patientswereincludedinthestudy.Totalantioxidantandoxidantlevelsof
follicularfluidsweremeasuredonthedayofoocytepick-upand oxidativestatuswerecalculated.
Expressionprofilesofthreepotentialtargetproteinsincasesofoxidativestress(Hsp70,Tgf-β,Notch1),
DNAstatusandchromatinintegrityofmuralgranulosacellswereanalyzed.
Results:TASlevelswerepositivelycorrelatedwiththeHsp70andTgf-βexpressionpatternsofmural
granulosacells.Matureoocyterateandfertilizationrateswereaffectednegativelybythepresenceof
oxidative stressandasignificantpositivecorrelationwas foundwith theoxidativestatus andthe
fertilization rate,whereasnocorrelationwiththeremainingICSIparametersinthe overallgroup.
Conclusions:Oxidativestressdetectedinfollicularfluidadverselyaffectsfertilizationratespost-ICSI
howevernoeffectontheremainingparametersincludingembryoquality,pregnancy,andimplantation
rates.DNAdamage,chromatinintegritywereincreased,whereasHsp70andTgf-ßweredecreasedin
muralgranulosacellsincasesofoxidativestresswhichmayindirectlyreflecttheoocytecompetencyand
maybeusedasbiomarkersforICSIoutcomemeasures.
PublishedbyElsevierB.V.
Introduction
Infertilityburden millions of people all around the world, yetcurrenttreatmentstrategiesarecapableofremedyingonly 40%ofthem[1,2].Intracytoplasmicsperminjection(ICSI)isone of themost commonlyusedassistedreproduction techniques for the treatment of infertility in couples with conception failure.
Oxidativestress(OS)whichisdefinedasaconsequenceofan imbalancebetweentheamountofreactiveoxygenspecies(ROS) produced, and the capability of antioxidant mechanisms to eliminatetheseoxidizingspecies,hasbeensuggestedasoneof thecausativefactorsof femaleinfertility[3–6]accordingtothe recentdata.Importantphysiologicalrolesofoxidativestatushave
beendemonstratedtoaffectawidevarietyoffemalereproductive functionsincludingoocytematurationandquality,formation of corpus luteum, ovarian steroidogenesis, fertilization process, embryo development, and pregnancy [3–5]. ROS are knownto beside-productsofnormalmetabolicactivityandareprovedto havevariousbeneficialeffects ontheorganismatphysiological concentrations[7].However,theimbalancebetweenROS produc-tion and its elimination is reported to cause damage to the components of cellular structures suchas lipids, nucleic acids, carbohydrates,andproteins[8,9].
Studiesregardingtheimpactofoxidativestressonthefemale reproductivecapacity mostlyfocusonthemicroenvironment of thedevelopingoocyte,ovarianfolliclesandinparticular,levelsof ROSand antioxidantswithinthefollicularfluid withconflicting results[3,10–16].Althoughthereisalsosomeevidencefortherole of reactive oxygen species (ROS) in the pathophysiology of infertilityandassistedfertility[17,18]withspecialroleonoocyte qualityandimpairedoocytefertilization[12,18–20]theexisting data are conflicting and the effect of oxidative stress on the outcomeofIVFisnotclear[15,21]
* Correspondingauthorat:MedipolUniversity,InternationalSchoolofMedicine, DepartmentofHistologyandEmbryology,Kavacıkmah,Ekincilercd.No:19Beykoz, Istanbul,Turkey.
E-mailaddress:sedakarabulut@medipol.edu.tr(S.Karabulut).
https://doi.org/10.1016/j.ejogrb.2020.06.021
0301-2115/PublishedbyElsevierB.V.
ContentslistsavailableatScienceDirect
European
Journal
of
Obstetrics
&
Gynecology
and
Reproductive
Biology
Ovarian follicles are sites of developing oocytes in which complicated network of systemic and localsignaling pathways crosstalks with each other. Within these follicles, the oocyte becomes mature which is essential for its normal fertilization processandthecompetenceoftheembryo[22].Follicularfluidisa mixture that contains proteins, metabolites, polysaccharides, steroids, reactive oxygen species, and antioxidant enzymes [23,24]whichactassignalingmoleculesandcontributetoproper oocytematuration.Molecularinteractionsthatregulatethefollicle and oocyte development until ovulation, require a suitable environment provided by the local and systemic interactions between follicular cells and fluid. Mural granulosa cells are importantcomponentsofovarianfolliclesthatsurround develop-ingoocytesandwhicharethesourcesofcumulusoophorousand corona radiata cells. These cells act as a provider of different moleculessuchassugars,amino acids,signalingmoleculesand nucleotidesfortheoocyte[25]. Furthermore,thesecells supply glutathione which is an important antioxidant for the oocyte [26,27],cysteineforthesynthesisofglutathione[28]hencedefend theoocyteagainstoxidativestress[29,30]andregulatematuration of oocytes by producing regulatory signals, induce meiosis for progressionandalterthecytoskeletonoftheoocyteaswell[31]. Astheoocytesappearsasthemostcommonfactor(70–80%) thatisresponsibleforimplantationfailure[32]andqualityofthe oocytereflectstheintrinsicdevelopmentalpotentialofanoocyte, itisofgreatimportancetounderstandthenatureofthemolecular andcellularprocessesthatmayaffectthequalityandcompetence of the oocyte. Assessment of oocyte quality depends only on morphologicexaminationandnotonthemolecularcompetency duringanassistedreproductivetechnique(ART)program.Several indirectmethodsthat evaluatethecompetencyof oocyteshave beenthesubjectof severalstudiesmostlyfocusingoncumulus cellsasthereflectorsoftheoocytes[33,34].Inthelightofthedata aboutthepotentialroleofgranulosacellsasregulatorsofoocyte competence and as biomarkers for oocyte/embryo quality or pregnancyoutcome [35–41], alterations in themetabolism and protein composition of follicular cells is suggested to cause problemsinoocytematurationandcompetence[42].
Folliculogenesisisacomplexeventwhichincludethecrosstalk ofmanycellsinthefollicularenvironmentandactivationofmany pathwaysinthosecellsinastrictlycoordinatedmanner.Thisevent controltheoocyte and thus developing embryos’ fatewhich is expected to change under different follicular circumstances. Amongthose proteins,three ofwhich are importantregulators ofcellsfate(Hsp70,Tgf-β,Notch1)andpossiblytargetofoxidative damagewereanalyzedintherecentstudy.
Heatshockproteins (Hsps)areaproteinfamilythat maybe potential candidateto affectoocyte competencefunctioning as chaperonesinthecell[43].Theirexpressionincreaseasaresultof variousstressinducersincludingoxidativestress[44].Hsp70isthe most abundant,highly conserved, and sensitive protein of the superfamilyofHsps[45,46]whichinteractswithmany intracellu-lar pathwaysthat control cell proliferation,differentiation, and cellulardeath[47].Hspswereshowntobeexpressedinfollicular cells[45,48–55],andshowntohavearoleinovarianphysiology andfolliculardevelopment[56,57].
Transforming growth factor beta (Tgf-β) is a multifunctional cytokinebelongingtothetransforminggrowthfactorsuperfamily thatfunctionasagrowthfactorinfolliculardevelopment[58]. Tgf-βwasidentifiedatmRNAandproteinlevelinpreantralfolliclesof various species including human, rodents, cattle, and sheep [59–63]andlocalTgf-βmemberswereshowntoregulatepreantral folliclegrowthpositively[64].
Notch signalingis another pathway regulatingproliferation, differentiationandapoptosis,andcontrollingcell-fatedecisions between adjacent cells [57,65]. It has been reported that the
membersofNotchsignalingareexpressedinmammalianovaries, butexactfunctionsofthispathwayinfolliculardevelopmentare not clearly identified. One of the suggested functions is the regulationofgranulosacellproliferationduringfollicular devel-opment. Notch genes are reportedto be actively expressed by cumuluscellsduringfolliculogenesis[66].
Allthreeproteinsanalyzedintherecentstudydeterminethe faithof thecellbyactivatingsignalingpathwaysforsurvival or apoptosis.Becauseitisreportedthatoocytematuration, fertiliza-tion,qualityoftheresultantembryos,numberofoocytesobtained, fertilization rate, and pregnancy outcomes following in vitro fertilization are associated withthe apoptosisof cumulus cells [67–69]understandingthebehavioroftheseproteinsindifferent circumstanceswillprovidecluesforunderstandingthemolecular basisofoxidativestressinfollicularfluidandforpredictingoocyte andembryoniccompetence.
Theaimofthepresentstudywastounderstandthemolecular and genetic mechanisms involved in follicular fluid oxidative processbyinvestigatinghumanmuralgranulosacellsandtofind possible biomarkers of oocyte competency and ICSI outcome measures.Dataobtained,mayprovideuscluesabouttheeffectsof oxidativestatus onoocyte and embryo developmentcausing a successfulpregnancy.
Materialsandmethods
Studyparticipantsandstudydesign
This prospective clinical study was conducted at Medicana ÇamlıcaHospital,IVFcenterbetweenJanuary2017toJune2018on 166 cycles that were undertaken ICSI treatment because of unexplainedinfertility.Patientexclusioncriteriawereasfollows: 1) For both gender: having any anatomic and physiologic abnormality of the reproductive system as revealed by their physicaland clinicalexaminations, having chromosomal abnor-malityand a smoking history.2) For women:poor responders according to ESHRE consensus, absence of polycystic ovary syndrome as defined by the Rotterdam criteria [[70]] and endometriosis, age<25and >37years, body mass index(BMI) <19and>26kg/m2.3)Formen:anyspermabnormalityasdefined
bytheWHO2010criteria[71],varicocelehistory.ICSIcycleswith preimplantationgeneticdiagnosis,assistedhatching,total fertili-zationfailure,noembryotransferregardless ofthereason,and coasting were also excluded from the study. Patients were informed about the study and written informed consent form were taken from the couples.The study was approved by the Ethical ReviewBoard of the Istanbul Medipol University ethics committeeon27September2017.
Follicularfluidsof166patientswerecollectedandtotaloxidant andtotalantioxidantlevelsweremeasuredonthedayofoocyte pick-up(OPU).Toanalyzetheeffectsofoxidativestressonmural granulosa cells, three potential target proteins’ (Hsp70, Tgf-β, Notch1) expression levels, their intracellular localization, DNA fragmentationlevels,andchromatinintegritiesweredetermined. Oocytequality,numberofoocytescollected,normalfertilization (2 P N) rate, embryo development rate, embryo qualities, pregnancyrate,implantationrate,clinicalpregnancy,and sponta-neousmiscarriagerateswereassessedforeachpatientinorderto definetheeffectsofoxidativestressonICSIoutcomemeasures. Granulosacellscollection
Follicularaspiratesofpatientswerecollectedseparatelyfrom eachfolliclebyasinglelumenfolliclepunctureneedle(Swemed, Sweden)onthedayofoocytepick-upwithoutperformingflushing. Follicular samples with blood contamination were discarded.
Cumulus-oocyte complexes (COC) within the aspirates were collectedunderstereo-microscopeand placed intoapreviously equilibratedculturedish.Allofthefollicularfluidswerecollected individually, numbered and the fluids of mature oocytes were pooled in a separate sterile containerafter oocyte denudation because only the mature oocytes which are checked for the presence of 1 st polar body (metaphase II), were used for microinjection.Pooledaspirateswerecentrifugedat600gfor10 min and supernatants and sedimented pellets were seperated. Aspirates were used for oxidative status analysis and the sedimented pellet were used as the source of granulosa cells. Granulosacellsweredividedintotwoportions,firstportionfor preparingthesmearsforimmunocytochemistry,immuno fluores-cence, toluidine blue, and TUNEL analysis, and the remaining portionwasstoredat 20Cforproteinextraction.
OvarianstimulationandICSIprocedures
Allwomenwereundertakenovulationinductionusingashort or long gonadotrophin-releasing hormone (GnRH) analog sup-pression protocol or a GnRH antagonist protocol and human menopausalgonadotrophins orrecombinant follicle-stimulating hormone(FSH).Oocyte-cumuluscomplexes(OCC)wererecovered 36 h after the administration of 5000 IU of human chorionic gonadotrophin(Ovitrelle,MerckSerono,Italy).
Cumulus and corona cells surrounding the oocytes were removedenzymatically(Hyase-10XTM,Vitrolife,Sweden)byusing
denudation pipettes. Nuclear maturation of the oocytes was assessed under inverted microscope with X40 magnification. Oocytes with a polar body (PB) in their first microscopic examination after denudation and oocytes that extrude their polarbodywithin4hafterdenudationweredefinedasamature oocyteinMetaphaseIIstage.Theoocyteswereclassifiedintotwo categories as good and poor morphology according to their cytoplasmicstatus,PBmorphology,zonamorphology,shape,and size.Oocyteswithadiameterof120 130
m
m,anintactshape,a homogeneous cytoplasm, no vacuolization, granulation and refractilebodyformation,non-fragmentedpolarbody,andazona pellucidabelow10m
mwereclassifiedas‘goodmorphology’,while theremainingwereclassifiedas‘poor-morphology’(Fig.1).Oocyte qualitywas denotedasthepercentage(%) ofgoodmorphology oocytespertotalnumberofmatureoocytes.Matureoocytes weremicroinjected with a motile sperm as describedpreviouslybyVanSteirteghemetal.,1993[72].Further cultureofinjectedoocyteswasperformedin25
m
Lmicrodropsof culturemedium (IVF,Vitrolife,Sweden) underlight paraffin oil (OVOIL,Vitrolife,Sweden).Fertilizationwasconfirmedafter16–18 hbytheobservationoftwodistinctpronuclei(2PN)andtwopolar bodies.Fertilizationratewascalculatedasthenumberoffertilizedoocytedivided bythetotalnumber ofmatureoocytesforeach couple. Oocytes with 2 P N were observed for embryonic development onday 2and 3 following microinjection.Embryo development rates were calculated as the number of embryos developeddividedbythetotalnumberofmatureoocytes,foreach couple. Quality of the embryosat cleavage stagewas assessed underaninvertedmicroscopewithX40magnificationaccordingto thecriteriaofStaessenetal.,1992[73].GradeAandBembryos weregroupedasgoodqualityembryosandgradeCandDembryos were grouped as poor quality embryos. All ICSI procedures including microinjection, embryo, and oocyte grading was performedbythesameembryologist.Goodqualityembryorate wascalculatedasthenumberofgoodqualityembryosdividedby thetotalnumberofembryos,foreachcouple.Embryoswith4-cell inday2weregroupedasnormallycleavedembryosandembryos withlessormorethan4-cellinday2weregroupedasabnormally cleavedembryos.Normally/abnormallycleavedembryorateswere calculatedasthenumberofnormally/abnormallycleavedembryos developeddividedbythetotalnumberofembryos,foreachcouple. Embryotransferswereperformedbythesameclinicianonday 3or5accordingtothenumberandqualityofdevelopingembryos. Wallace catheter (1816 N, SmithsMedical, USA) were used for embryotransfer.β-hCGlevelinthebloodwasmeasuredtwelve daysafterembryotransferanda50mIU/mLß-hCGvalueonday 12, indicated a positive pregnancy. Biochemical pregnancy (positive hCG without a gestational sac), implantation rate (number of gestational sacs/number of embryos transferred), clinicalpregnancy rate (intrauterinepregnancy withfetal heart activity),andfirst-trimesterspontaneousmiscarriage(pregnancy failureaftervisualizationofintrauterinegestationsac),werealso assessed.
Follicularfluidoxidativestatusdetermination
TotalAntioxidantStatus(TAS)andTotalOxidantStatus(TOS) levelsinfollicularfluidsweredeterminedbyusingRelassaykits (RelAssayDiagnostics,Turkey).ForTASlevels,TotalAntioxidant Status AssayKit was usedand theabsorbancesweremeasured using a spectrophotometer (Molecular Devices SpectraMax i3 Multi-ModeMicroplatereader).
TOS levelsweremeasuredbyTotalOxidantStatusAssayKit, whose method is as follows: oxidants present in the sample oxidizeferrous-o-dianisidinecomplexintotheferricions[74].The absorbancesweremeasuredat530nm.Theresultsareexpressed in micromoles hydrogenperoxide equivalent per liter(H2O2eq
m
mol/m
L).TASlevelsweremeasuredbyTotalAntioxidantStatusAssayKit whosemethodisasfollows:thehydroxylradicalastheproductof Fenton reaction reacts with colorless o-dianisidine to form a
Fig.1.(a)Agoodqualityoocytewithahomogeneous,brightooplasmandanintactpolarbody(b)Apoorqualityoocytewithmanyvacuoles,granulationandalarge perivitellinespace(X40).
radicalbrightyellowishbrowndianisylsubstrate.Theabsorbances were measured at 660 nm. The measurement results were expressed in Trolox equivalent of millimoles per liter (TroloxEq
m
mol/m
L)[75].Theintraassay%CVvaluesfortheTASmeasurementwere2.36% forthe0.50(0.35 0.65)mmolTroloxequiv/Land2.24%forthe2.0 (1.7–2.3)mmolTroloxequiv/L.TheintraassayCV%valuesforTOS measurementswere3.57%for5.5(3.0–8.0)
m
mol/Land5.17%for 19.5(16–23)m
mol/L.OxidativeStressIndex(OSI)wascalculated withtheformula:TotalOxidantStatus(TOS)/TotalAntioxidant Level(TAS).ProteinanalysisbyImmunocytochemistryandimmunofluorescence Immunocytochemistry analysiswas performedto determine theexpressionandimmunolocalizationofHsp70,Tgf-βandNotch1 proteins(Fig.2).Immunofluorescenceanalysiswasperformedto determinetheexpression levelsby measuringrelative staining intensity,andwesternblotwasperformedinordertoconfirmthe results(Fig.3).
For immunocytochemistry, granulosa cells were fixed with freshlyprepared4%paraformaldehyde(PFA)atroomtemperature for 20 min.Cells were washedwith phosphatebuffered saline
(PBS)duringthewholeprocedure.Toinhibitendogenoushydrogen peroxidase activity, slides were incubated with 3% hydrogen peroxidesolutionfor20min.Cellswereincubatedwithboiling citrate buffer for 5 min for antigen retrieval. Protein blocking solution was applied for 10 min at room temperature in a humidified chamber. The cells werethen incubatedwith anti-bodies(anti-Hsp70antibody(1:200,sc-24,SantaCruz,CA,USA), anti-Tgf-β antibody (1:200, sc-146, Santa Cruz), anti-Notch1 antibody(1:200,sc-6014-R,SantaCruz)),overnightat+4C.After
washingwithPBS,biotinylatedgoatanti-polyvalentsolutionwas performedfor10minatroomtemperature,andthesampleswere incubatedwithstreptavidin-peroxidaseforanother10min.DAB (3,3-diaminobenzidine)was appliedforcounterstainingfor10 min,and hematoxylinwas appliedfor 5 min.Slides werethen rinsedwithtapwateranddehydratedbyusingseriesofalcohol withincreasing concentrationand then mounted. Imageswere obtainedusingNikonEclipseNi(Nikon,Japan)microscopewith 100Xoil-immersionobjective.
For immunofluorescence,granulosa cellswerefixed with4% paraformaldehydefor15minatroomtemperature.Granulosacells werewashedwithPBSduringthewholeprocedure.Slideswere permeabilized with 0.1 % Triton X-100 for 10 min at room temperatureandwereblockedwith10%normalgoatserumand
Fig.2. ImmunocytochemistrystainingresultsforNotch1,Hsp70,andTgf-βproteinexpressionsinhumanmuralgranulosacellsofLOSandHOSgroups(40X).Arrowsindicate Notch1(+)cells(a)inLOSgroupand(b)inHOSgroup.ArrowsindicateHsp70(+)cells(c)inLOSgroupand(d)inHOSgroup.ArrowsindicateTgf-β(+)cells(e)inLOSgroupand (f)inHOSgroup.Arrowheadsindicate(-)staining.
0.3%TritonX-PBSfor30min.Thecellswerethenincubatedwith anti-Hsp70 antibody (1:200, sc-24, Santa Cruz Biotechnology, SantaCruz, CA, USA), anti-Tgf-β antibody (1:200, sc-146, Santa Cruz),anti-Notch1antibody(1:200,sc-6014-R,SantaCruz).Bound antibodieswerevisualizedwithAlexaFluor5681-conjugatedgoat anti-mouse IgG (1:200, ab175473, Abcam, UK) for Hsp70, and Alexa Fluor 4881-conjugated goat anti-rabbit IgG (1:200, ab 150077,Abcam,UK)foranti-Notch1andanti-Tgf-β.Fluorescence imageswereobtainedwithZeissCellObserverSDSpinningDisk Time-Lapse Microscope (Carl Zeiss, Jena, Germany) with a magnification of 400.In average,100 cells were counted for eachpatientineverygroup.Negativecontrolsweretestedineach slideforqualitycontrol.Relative intensitywascalculatedasthe ratioofintensitytoareaofeachcellviatoolsthatareprovidedby ZENprogram(CarlZeiss,Jena,Germany)ineachslide.
Electrophoresis(SDS-PAGE)andWesternblotting
Celllysisprocedurewasperformedsubsequenttothethawing ofthesamplesatroomtemperature.Sampleswerewashedtwice withcooledPBSbycentrifugingat2500Gfor10min.A400
m
L lysis buffer solution (M-PER Mammalian Protein Extraction Reagent; Thermo Scientific, Wien, Austria) supplemented with 10m
L/mLprotease-phosphataseinhibitorcocktail(HaltProtease InhibitorCocktail;ThermoScientific)at1:100wasaddedtothe pelletonice.Thesuspensionsweremixedandleftonicefor20min whileshakinggently.Suspensionswerethencentrifugedfor15 minat14,000g,andthesupernatantwas transferredtoa new tube.BCAassay(PierceBCAProteinAssayKit;ThermoScientific) wasusedforproteinassayaccordingtotheinstructionmanual.A mathematicalcurvewasplottedbymeasuringserialBSAstandards ofpre-knowndilutions.Protein concentrationsof theunknown sampleswerecalculatedaccordingtothestandardcurveobtained. Extractedproteins(20m
g)weremixedwitha4Xloadingdye andrunonagelwitha4%concentrationfor15minat90V,andsubsequentlyona10%separatinggelfor1.5hat100Vatroom temperature.Marker(PageRulerTMPlus;ThermoScientific)was loadedoneachgelformolecularweightanalysis.Thegelswere thentransferredelectrophoretically(100V)onaPVDFmembrane (Hybond-P;AmershamBiosciences,Buckinghamshire,UK)for1h. Membraneswereblockedfor1hin5%(W/v)skimmilkpowderin Tris-buffered(20mM)salinecontaining1%Tween20(TBS-T)and wereincubatedovernightwithprimaryantibodies:Hsp70/Hsc70 (sc24,SantaCruzBiotechnologies,USA),Tgf-β1(sc-146,SantaCruz Biotechnologies, USA), Notch1(C-20)-R (sc-6014-R, Santa Cruz Biotechnologies,USA),ß-Actin(4967,Cellsignaling,Netherlands). Membraneswerewashedthreetimesandincubatedfor1hwith thesuitablesecondaryantibody(goatanti-mouse,ab175473,goat anti-rabbit, ab150077, Abcam, England), then exposed to HRP conjugated antibody (goat anti-mouse IgGHRP; Santa Cruz Biotechnologies, USA and anti-rabbit IgG HRP-linked antibody, Cell Signalling Technologies, Netherlands). After incubation, membranes were washed with TBS-T and the immunoreactive bandintensitieswereanalyzedwithChemiDocMolecularImager (Bio-Rad, Philadelphia, PA, USA). The integrated density values werecalculatedbycomparingthesignalsoftargetproteinstothat ofthehousekeepingActin.
DNAfragmentationandchromatinintegrityassessment
TUNELtestwasusedtoevaluateDNAfragmentationlevelsand Toluidineblue(TB)stainingwasperformedinordertodetermine thechromatinintegrityofcumuluscells(Fig.4).TBstainingwas evaluated under light microscope as positive (dark-stained) or negative(pale-stained)bycomparingdyeintake.
ForTUNELprocedure,slideswerefixedinfreshlyprepared4% PFAatroomtemperaturefor20min.Forpermeabilization,0.1% Triton-XinPBSwasusedonicefor2min.DNAfragmentationwas determinedbyTUNELassayusingacommerciallyavailablekit(In SituCellDeathDetectionKit,fluorescein,Roche,Indianapolis,IN,
Fig.3. ImmunofluorescencestainingresultsforNotch1,Hsp70,andTgf-βproteinexpressionsinhumanmuralgranulosacellsinLOSandHOSgroup(40X).Arrowsindicate Notch1(+)cells(a)inLOSgroupand,(b)inHOSgroup.Hsp70(+)cells(c)inLOSgroupand(d)inHOSgroup.Tgf-β(+)cells(e)inLOSgroupand(f)inHOSgroup. (g)RepresentativeproteinbandsforHsp70,Notch1andß-actin(42kDa)expressionlevelsof5patientsofLOS(L)and5patientsofHOS(H).(h)Relativeexpressionlevels (meandensitometry)ofHsp70,andNotch1proteinobtainedfromwesternblottinganalyses.Barsrepresentthemeansanderrorbarsrepresentstandarddeviationofthe quantifiedproteins.*p<0.05(statisticallysignificant).
USA).Cellswereincubatedat37CwithTUNELreactionsolution (whichisobtainedfromkit)for1h.Slideswererinsedthreetimes withPBS.DAPIwasusedtolabelnuclei.Cellswereexaminedunder Zeiss Cell Observer SD Spinning Disk Time-Lapse Microscope (CarlZeiss,Jena,Germany)with400magnification.Aminimum of 100 granulosa cells were chosen randomly in at least 10 microscopicfieldswithamagnificationof10Xandtheresultswere givenasmeanpercentage(%).TUNEL-positivecellswerecounted tocalculatetheratioofapoptoticcellsineachgroup.Cellsstained in green werethose with fragmented DNA, and not-stained in greenwerewithoutfragmentedDNA.
ForToluidinebluestaining,cellswerefixedinfreshlyprepared 96%ethanol-acetone(1:1)at4Cfor1handhydrolyzedin0.1N HClat4Cfor5min.Slideswererinsedthreetimesindistilled waterfor2minandwerestainedwith0.05%Toluidinebluefor5 minatroomtemperature.Theslideswerethenrinsedthoroughly indistilled water beforemounting.Imageswereobtained with NikonEclipse(Nikon,Japan)microscopeby100xoil-immersion objective.
Statistics
AllstatisticalanalyseswereperformedusingStatisticalPackage for Social Sciences (SPSS, Version 21 for Windows; SPSS, Inc., Chicago,IL,USA).Samplesizewascalculatedforasignificancelevel of5%andapowerhigherthan80%,inordertodetectadifference betweenmeanshigherthan15%.StatMateforWindows (Graph-PadSoftware, USA)program package was usedfor sample size calculations.Univariate(mean,standarddeviation,andfrequency) and bivariate (Student’s t-test, Mann–Whitney-Uand Wilcoxon tests) descriptive statistics wereperformed. Statistical compar-isons for categorical variables were carried out using the chi-square test. The Kolmogorov–Smirnov test was used toassess complianceofthevariablestonormaldistribution.Associations amongquantitativevariableswereanalyzedusingthePearson’s correlationcoefficient.
All tests were conducted using a p-value 0.05 defining statistical significance. The data were expressed as mean standard deviation for continuous variables and number of
Fig.4. Tunel(DNAfragmentation)andToluidineBlue(chromatineintegrity)stainingresultsforNotch1,Hsp70,andTgf-βproteinexpressionsinhumanmuralgranulosacells inLOSandHOSgroups(40X).Tunelstaining:ArrowheadrepresentsTunel(-)cells,arrowsindicateTunel(+)cells(a)inLOSand(b)inHOSgroup.Toluidinebluestaining (40X).ArrowsindicateToluidineBlue(+)cells,arrowheadsindicateToluidineBlue(-)cells(c)inLOSand(d)inHOSgroupe.DNAfragmentationandchromatinintegrityrates ofLOSandHOSgroupswereshowninbars.Resultsweregivenaspercentage(%).*p<0.005(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderis referredtothewebversionofthisarticle).
Table1
Oxidativeparameters(TAClevels,TOClevels)accordingtoLOS(<10mmol/L)andHOS(>10mmol/L)subgroups.
Oxidativeparameters LOS HOS pvalue
TASlevel(meanO.D.) 309095 036026 0000* TOSlevel(meanO.D.) 1197373 1305531 054 Oxidativestatus(TOS/TASratio)(meanO.D.) 422197 685677, 0000* ResultsweregivenasmeanO.D.SD.
cases(n)andpercentageofoccurrence(%)forqualitativevariables. Receiver operating characteristic (ROC) curves were used to determinethesensitivityand specificity values andareaunder curve.
Results
Meanantioxidantandoxidantlevelswithinthefollicularfluid intheoverallgroupundergoingICSItreatmentweremeasuredto be1.64and12.32OD(opticaldensity)resp.,andthemeanlevelof oxidative statuswhichindicatesthe balancebetween oxidants and antioxidantswas calculatedtobe41.49 OD.Patients were dividedintotwogroupaccordingtotheirfollicularfluidoxidative statusaslowoxidativestress(LOS,10OD)andhighoxidative stress(HOS,>10OD).Follicularfluidoxidativestatuswithineach grouparepresentedinTable1.Demographicdataofthegroups were similar between two groups in terms of maternal age, numberofpreviouscycle,E2levelonthedayofoocytepick-up, controlledoverstimulationperiod,numberofoocytescollected, numberofembryostransferredandthedayofembryotransfer (Table2,p>005).
TheoxidativelevelwascalculatedasTOS/TASratio.TASlevels weresignificantlyincreasedinHOSpatientsalthoughTOSlevels weresimilar(p=0.000;p=0.545resp.).Asaresult,TASlevelswere foundtobethedeterminativeparameterofoxidativestresslevelin bothgroups.
Expressionprofilesof three possible targetproteins (Hsp70, Tgf-β,Notch1)ofoxidativestresswereanalyzedinmuralgranulosa cells.Accordingtotheresultsofimmunocytochemistry,Hsp70and Notch1werefoundtobeexpressedstatisticallylowerintheHOS groupwhereastherewasnodifferenceinTgf-βexpressionrates betweenthegroups(Table3,Fig.2,p=0.0026,p=0.0047resp.). Theresultsobtainedwerefurtherconfirmedbyimmuno fluores-cenceandwesternblotanalysis(Fig.3).
Chromatinintegrityrateswhichisassessedbytoluidineblue paintingweresignificantlydecreasedinHOSgroupthanLOSgroup (17.2%; 81 %resp,p= 0.0023)althoughthe differenceinDNA fragmentation rates did not reach statistical significance level (75%;66.15%resp.p=012).(Fig.4).
ICSIoutcomeparametersincludingoocytequality,fertilization, embryodevelopment,embryoquality,clinicalpregnancy, implan-tationandmiscarriagerateswerealsoassessedtoseetheeffectof oxidativestatusonICSIoutcome(Table2).Matureoocyteratesand fertilizationratesweretheonlytwoparameterthatwereaffected negativelyfromtheoxidativestresswhich wereobservedtobe decreasedsignificantlyinHOSgroup(p=002,p =0011,resp.). OtherICSIoutcomemeasureswerenotaffectedsignificantlybythe presenceofoxidativestress(p>005,Table2).
We then performed correlation analysis to analyze the correlation with the expression pattern of the three possible targetproteinswithTAS,TOSlevelsandICSIoutcomemeasures. Wefounda positivecorrelationbetweentheTASlevelsandthe expressionsofHsp70(p=0.001,r=0.488)althoughtherewereno correlation between TOS levels and expression profiles of investigatedproteins(p>005).
Among all ICSI parameters, fertilization rate is the only parameterthatisfoundtobecorrelatedwithoxidativestatusof follicularfluid(p=0.001,r=0.518).Therewerenocorrelationwith theremainingparametersincludingembryodevelopment,embryo quality,clinicalpregnancy,implantationandspontaneous miscar-riageratesintheoverallgroup(p>005).
Discussion
Oxidativestressisknowntoplayamajorrolein pathophysi-ology of infertility, yetthe causes, effects, andthe molecular mechanisms underlying this phenomenon is not well under-stood.Thereareseveralstudiesreportingthenegativeeffectsof follicular fluid oxidative stress on fertilization capacity [12,13,20,76] while the others had foundnocorrelation [77]. Underphysiologicalconditionstheantioxidantdefense mecha-nism of oocytes and ovaries, consisting of enzymatic antiox-idantssuchassuperoxidedismutase(SOD),andnon-enzymatic antioxidants (GSH), detoxifies excess ROS maintaining the oxidant/antioxidantbalance.However, increasedlevelsof ROS beyondthephysiologicalrangemayleadtoOSandcauseawide range ofmolecular damages,including lipidperoxidationand protein and DNAdamage resultingin deterioration of oocyte quality[78–81].
Granulosa cells are the common cell population that are exposedand thusareaffectedfromthefollicular microenviron-ment.Hencetheyactbothasabridgeandabarrierbetweenthe oocyte and the follicular microenvironment. As their activity directlyaffectsparacrinesignalingwiththeoocytestheysurround, understandingtheirbehaviorunderdifferentoxidativeconditions maygivevaluable clues abouttheeffect ofoxidativestatus on oocyte’sdevelopmentalcompetenceandthesuccessofICSIcycle usingtheseoocytes.
Inthisaspect,theexpressionpatternsofthreepossibletarget proteins (Hsp70, Tgf-β, Notch1) of oxidative stress, which are involvedinimportantregulatorysignalingpathwaysinthecells wereinvestigated.Allthreeproteinswereactivelyexpressed in granulosa cells of patientsbut with differentrates. Hsp70 and Notch1werefoundtobeexpressedstatisticallylowerintheHOS groupwhereastherewasnodifferenceinTgf-βexpressionrates betweenthegroups.
Table2
DemographicdataandICSIoutcomemeasuresaccordingtoLOS(<10mmol/L)andHOS(>10mmol/L)subgroups.
ICSIoutcomemeasures LOS HOS pvalue
Maternalage 336615 344616 036
NumberofpreviousIVFcycle 170098 176125 088 EstradiolLevel 31,92819,038 51,67813,135 029 Controlledovarianstimulationperiod(day) 652172 656131 038 Meannumberofoocytesretrieved 753610 706508 062 Meannumberofmatureoocytes 576357 3,12,5 002* Normalfertilizationrate(%)(#of2PN/#ofmatureoocytes) 72762102 62541416 0011* Embryodevelopmentrate(%)(#of6 8cellonday3/#of2PN) 811112 731167 021 Topqualityembryorate(%) 451343 371446 025 Pregnancyrate(positiveß-hcG)(%) 502 396 030
Clinicalpregnancyrate(%) 413 322 0,4
Implantationrate(%) 245 206 032
Spontaneousmiscarriagerate(%) 532 912 018
ResultsweregivenasmeanO.D.SD.
Hsp70isoneoftheimportantmembersofHeatshockproteins (Hsp) that protect the cells against damages caused by stress conditions,andtomediatethetransportofdamagedproteinsto targetorganellesfortheirrepairordegradation.Wefoundalower Hsp70expressionrateinHOSgroup.Itmaybesuggestedthatthe oxidativefollicularenvironmentsuppressHsp70expressionwhich may,inturn,decreasethetoleranceofcellstooxidativestressand causethemtobecomemorevulnerable.Thereisnodatainthe literatureanalyzingtheHsp70expressionrate ofgranulosacells underdifferentoxidativestressconditions.
Tgf-β superfamily members have found to be selectively expressedbyoocytesfromprimaryfolliclesinrodents,andfrom primordial follicles in cows and sheep [59–63,78,79,82–84]. Varioustypeswereshowntobeexpressedbygranulosacellsof theearly folliclestages,makingthesecells potentialtargetsfor paracrinesignalingandlocalTgf-βfromthecaandgranulosacells implicatedaspositiveregulatorsofpreantralfolliclegrowth[85]. Binelli and Murphy indicated an inhibitory effect of Tgf-β on primaryfolliclesurvivaland/orprogressiontothelatepreantral/ earlyantralstagewhileothersindicatedlackofanyeffects[86,87]. Tgf-βisreportedtostimulateFollicle-stimulatinghormone(FSH) receptor expression, amplify FSH-induced aromatase activity, inhibin production, progesterone production and luteinizing hormone(LH)receptor induction[69,88–91] andits expression werereportedtochangeunderdifferentcircumstancesincluding female age [92]. We found no significant difference in Tgf-β expressionratesbetweenthegroupswhichprovidesevidencethat follicularfluid oxidative status do not haveany effect onTgf-β expressionsingranulosacells.
TheotherproteinevaluatedwasNotch1,whichisa transmem-branereceptor and controls cell-fatedecisions betweentightly adjacentcellsbymodulatingcellproliferation,differentiation,and survival[65].Notchgenesareshowntobeactivelyexpressedby cumulus cells during folliculogenesis [66]. Notch signaling is showntobeinvolvedinovarianfollicledevelopmentbyregulating granulosacellproliferation[68].Wefoundasignificantdecreasein Notch1expressionratesinHOSpatientswhichmayinfluencethe fateofgranulosacellsandpossiblytheoocyteneighboringthem. There is also no data about the effects of oxidative stress on granulosaTgf-βexpressionrates.
ICSIoutcomemeasureswereanalyzedtoanalyzetheeffectsof oxidative status on ICSI outcome. Beyond all ICSI parameters includingoocytequality, embryo development,embryo quality, clinical pregnancy, implantation, and spontaneous miscarriage ratesthetwoparametersincludingtheoocytematurationrateand thefertilizationratewerenegativelyaffectedfromthefollicular fluid oxidative environment. Mature oocyte number were also
found to be significantly lower in HOS group indicating the possiblenegativeroleofoxidativestressonoogenesis.
Hsp70andNotch1activitywassignificantlyloweringranulosa cells of HOS patients according toour findings which may be correlated with the oocyte maturation. One of the possible suspected protein is Hsp70 which is shown to have role in regulatingestradiolbiosynthesisunderstressconditions[93].
Resultsofthisstudyprovideevidencetoexplaintheretarded oocytematurationratedetectedinHOSgroupofourstudy.Second suspectedproteinforretardedoocytematurationisNotch1,which isknowntoregulatefolliculogenesisthusoogenesis.As matura-tionandcompetencyofanoocyteiscloselyrelatedtothesurvival signalsitreceives,Notch1mayhaveeffectonthedevelopmental capacityofoocytesbyparacrinesignalingfromgranulosacells.
This studyshows a decrease in the activities of Hsp70and Notch1 of granulosa cells with mature oocyte number under oxidativecircumstance,thusprovidingindirectevidenceforthe roleoftheseproteinsinoocytematuration.Theotherparameter thatisinfluencedbyoxidativestresswasthefertilizationrate.We observedasignificantlydecreasedfertilizationrateinHOSgroup indicating the possible role of oxidative status on pronucleus formationorgametesactivation.Oneofthereasonsmaybethe lowerHsp70expressionrates in HOSgroupwhich maynot be enough to overwhelm the damage caused by oxidative stress. Increased oxidative attack and decreased cellular protection mechanismsmayresultindamagedormisfoldedproteinswhich contribute the fertilization process. We found no significant differencefortheotherICSIoutcomemeasuresanalyzedincluding oocyte quality, embryo development, embryo quality, clinical pregnancy,implantation,andspontaneousmiscarriagerates.The correlation analysis results also confirmed these findings by observingasignificantpositivecorrelationwiththefertilization rate which, in turn, showed that higher Hsp70 and Notch1 expressionsmaybeusedasbiomarkersofmatureoocytes,leading tosuccessfulfertilization.Ourresultsdonotseeminaccordance with several other studies which reported negative effects of oxidativestatusonembryoformation,quality,andpregnancyrate after ICSI[2,13,18]. The difference obtainedmaybe becauseof differences in oxidative status determination methods and different patient population. Oxidative status of the follicular fluidisnotfoundtobepredictiveforpregnancyassessmentinan ICSIcycleintherecentstudy.
Thegeneticstabilityofthesecellswerealsoanalyzedwhichis showntoaffectoocyteprogression[94–96].Wefoundanincreased DNAfragmentationratesinHOSgroupalthoughthedifferencedid not reach statistical significance. But the chromatin integrity which is assessed by toluidineblue paintingwere significantly
Table3
ExpressionrateandlevelofHsp70,Tgf-ß,Notch1accordingtoaccordingtoLOS(<10mmol/L)andHOS(>10mmol/L)subgroups.Resultsweregivenaspercentage(%)unless otherwisestated.Abbreviations:IF,Immunofluorescence*p<0.05.
Proteinsofinterest LOS HOS pvalue
Hsp70
Immunostainedcell(%) Expressionlevel
IF(Meanfluorescenceintensity) Westernblot(Meandensitometry)
7591461 180.13 0,20,1 566828951210520.12013 0026* 0.64 0.24 Tgf-β Immunostainedcell(%) Expressionlevel
IF(Meanfluorescenceintensity) Westernblot(Meandensitometry)
820413091850830.24033 731918382051090.320,3 0067034042
Notch1
Immunostainedcell(%) Expressionlevel
IF(Meanfluorescenceintensity) Westernblot(Meandensitometry)
78,8818174121,10.4017 721714594251,70.26023 0047* 0.43 0.3
decreasedinHOSgroupprobablyaddressingsomeproblemson DNArepackaging.DNAmethylationplaysacentralrole ingene expressionviaepigeneticwhichmodifiesthegeneticmaterialand DNA packaging. The decreased Hsp70 may be one of the responsibleproteinsofDNAmethylationwhichisreportedtobe negativelyassociatedwiththeexpressionofHsp70understress conditionsindifferenttissues[97]. Thedecreaseinfertilization ratesinourstudymaybearesultofthisconsequencewhichmay affectfertilizationprocess byalteringchromatinrearrangement during pronucleus formation. However, this study was not designed to investigate the association of individual follicular fluid status with the quality of the corresponding oocyte and embryo.Furtherinvestigationsareneededtoevaluateindividual follicles,otheroxidativestressmarkersortheclinical character-istics ofpatients. Strategiesaimed atreducing oxidative stress, includingtheaddition of antioxidantsin the oocyteincubation mediumorembryoculturemedium,maybetestedtoseewhether theseeffectsaredisappearedornot.Furtherstudiesshouldfocus on possible other pathways and crosstalks between them to understandthemolecularmechanismsunderlying thesituation and thepossible potentialof Hsp70 and Notch1 tobe usedas biomarkersforICSIoutcomeshouldbefurtherinvestigatedinthe lightofthedataobtainedinthisstudy.
Funding
Thisresearchdidnotreceiveanyspecificgrantfromfunding agenciesinthepublic,commercial,ornot-for-profitsectors. DeclarationofComeptingInterest
Theauthorsdeclarenoconflictofinterest References
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