Association of tissue inhibitor of metalloproteinase gene polymorphisms and unexplained recurrent spontaneous abortions in Han Chinese couples
§Guihong Song
a,b,1, Junhao Yan
a,c,d,1, Qian Zhang
a, Guangyu Li
a,c,d, Zi-Jiang Chen
a,c,d,*
aCenterforReproductiveMedicine,ProvincialHospitalAffiliatedtoShandongUniversity,Jinan,China
bShandongCollegeofTraditionalChineseMedicine,Yantai,China
cNationalResearchCenterforAssistedReproductiveTechnologyandReproductiveGenetics,Jinan,China
dTheKeyLaboratoryforReproductiveEndocrinologyofMinistryofEducation,Jinan,China
Introduction
Recurrent spontaneousabortion(RSA) is definedas threeor more consecutive pregnancy losses before the 22th week of gestationwiththesamepartnerandisaratherrarecondition,with estimatedincidencesof1%to3%ofcouples[1–3].However,RSAis associated with enormous physical and psychological harm to
patients.Basedonyearsofinvestigation,somecausesofRSA,such asabnormalchromosomesineitherpartner(particularlytranslo- cations),antiphospholipidantibodies,anduterineanomalies,have been identified. Other potential causes, such as endocrine dysfunction, thrombophilia, immune disorders, lifestyle factors andmaternalinfections,havebeensuggested[4–6].However,the etiologiesof50%ofcasesremainunexplained,andthesecasesare termedunexplainedrecurrentspontaneousabortions(URSAs)[1].
Somegeneticfactorshavebeensuggestedaspotentialetiological mechanismsofURSA.
A normal pregnancy depends on embryo implantation and placentaformation.Theseprocessesarecharacterizedbyextensive degradation and remodeling of the extracellular matrix (ECM), which depends on the regulation of the interaction of matrix metalloproteinases (MMPs) withspecific ECM components[7].
MMPs are a family of structurally related zinc-dependent endopeptidases,areabundantmacromoleculesonthecellsurface andhaverolesinmanyphysiologicalprocesses,suchasapoptosis andangiogenesis.Moreover,theactivitiesofMMPsareregulated ARTICLE INFO
Articlehistory:
Received20March2014 Receivedinrevisedform8July2014 Accepted20July2014
Keywords:
Tissueinhibitorofmetalloproteinase Singlenucleotidepolymorphisms Unexplainedrecurrentspontaneous abortion
ABSTRACT
Objective:To investigate the association of tissue inhibitor of metalloproteinase (TIMP) gene polymorphismswithunexplainedrecurrentspontaneousabortions(URSA) inawell-definedgroup ofHanChinesecouples.
Studydesign:Thisisacase-controlassociationstudy.GenomicDNAwasextractedfromperipheralblood samplesfrom84coupleswithhistoriesofthreeormorepregnancylossesand69age-matchedhealthy coupleswithatleastonelivebirthandnohistoriesofpregnancyloss.Polymerasechainreactions(PCRs) andsequencingwiththefluorescentdyedideoxy-terminationmethodwereusedtodetectthers4898in TIMP-1, rs2277698 in TIMP-2, rs2234921 and rs5749511 in TIMP-3 and rs17035945 in TIMP-4 genotypesandallelefrequencies.
Results:NeithertheallelefrequenciesnoranyofthegeneticmodelofthefiveTIMPgeneSNPs(i.e.,TIMP- 1-rs4898, TIMP-2-rs2277698, TIMP-3-rs5749511 and rs2234921, and TIMP-4-rs7035945) were significantlydifferencesbetweentheURSAcouplesandthecontrolgroup.
Conclusions: NoevidencewasfoundforanyassociationsbetweentheTIMP-1,-2,-3,or-4genesSNPs withURSAinthisHanChineseHan.
ß2014ElsevierIrelandLtd.Allrightsreserved.
§In this study,polymerase chain reaction (PCR) andsequencing with the fluorescentdyedideoxy-termination method wereusedto detect TIMPgene polymorphisms,andnostatisticallysignificantdifferencesbetweenURSAcouples andthecontrolgroupwerefound.AssociationofTissueInhibitorofMetallopro- teinaseGenePolymorphismsandUnexplainedRecurrentSpontaneousAbortionsin HanChineseCouples.
* Correspondingauthorat:CentreforReproductiveMedicine,ProvincialHospital affiliatedtoShandongUniversity,Jinan250021,China.Tel.:+008653185651190;
fax:+008656187068226.
E-mailaddress:[email protected](Z.-J.Chen).
1Contributedequallytothiswork.
ContentslistsavailableatScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology
j o urn a l hom e pa ge : ww w. e l s e v i e r. c om/ l o ca t e / e j ogr b
http://dx.doi.org/10.1016/j.ejogrb.2014.07.013
0301-2115/ß2014ElsevierIrelandLtd.Allrightsreserved.
byspecificendogenoustissueinhibitors(TIMPs),whichexerttheir effects eitherdirectly bybinding totheMMPs or indirectlyby activatingnucleartranscriptionfactorsthatcontroltheexpression ofselectMMPgenes[8–11].Somestudieshaveconfirmedthatthe balance of MMPs and TIMPs plays crucial roles in embryo implantation,trophoblastinvasion,earlyplacentation,andcervi- caldilatationandfeto-maternalmembranelysisinlatergestation.
Ifthisbalanceisimpaired,extensivedestructivedegradationofthe ECMandpregnancyfailurecanresult[12–14].
TIMPs act as signaling molecules that have cytokine-like activities and thereby influence various biological processes including cell growth, apoptosis, differentiation, angiogenesis, andoncogenesis.TherearefourTIMPs(i.e.,TIMP-1,TIMP-2,TIMP- 3,andTIMP-4),andtheyregulatenearlyallofthe26membersof the human MMP gene family [13,15]. Independently of MMP inhibition,TIMPs themselvesareable tostimulateproliferation and play a direct role in the development of the intrauterine structures[16,17]. TIMP-1is the main endogenousinhibitor of MMP-9,whichisthekeyenzymeintrophoblastinvasion.TIMP-1 has also been shown to inhibit other metalloproteinases in addition to MMPs [8,14]. Ritva Nissi [18] confirmed that the serum concentrations of TIMP-1 and TIMP-2 are significantly different(P<1010)in patients with ongoing pregnancies and those with incomplete abortions or anembryonic pregnancies.
SignificantlylowerexpressionofTIMP-1intheuterinefluidduring implantationwindowhasbeenconfirmedinwomenwithURSA [19]. Serum TIMP-1 concentrations on days 14 and 21 among pregnantwomen followingIVF-ETare significantly higherthan thoseinnon-pregnantwomen[20].DillyOCAnumba [8]found thattheserumlevelsofTIMP-2arehigherinwomenwithhistories ofURSAthaninage-matchedpregnantwomenwithnohistoryof RSA at all points of gestation. The expression of TIMP-3 is significantlydecreasedintheendometriaofwomenwithURSA, unexplained infertility or implantation failure following IVF comparedtocontrolgroups[21,22].Themaximalexpressionsof TIMP-4,whichisMMP-26’sstrongestinhibitor,occurintheearly andmid-secretoryphases,whichsuggeststhatTIMP-4playssome rolesduringimplantation[23].
Together,these resultssuggest that TIMPs areinfluential in URSAandthatTIMPgenevariationsmaybeafactorinURSA.The present study aimed to investigate the potential associations between TIMP gene polymorphisms and URSA in a Chinese population. Notably, some researchers have investigated such associations in Slovenians with URSA but did not find any associationsbetweenTIMPgenepolymorphismsandURSA[24].
Materialsandmethods Subjects
This study was performed at the Center for Reproductive MedicineofShandongUniversity,China.Eighty-fourcoupleswith
historiesofthreeormorepregnancylosseswereformedtheURSA group. All of these couples had primary RSAs without any explanatoryetiologies,includingabnormalchromosomesineither partner(particularlytranslocations),antiphospholipidantibodies, uterineanomalies,endocrinedysfunction,thrombophilia,immune disorders,lifestylefactorsandmaternalinfections.Thegestational ageofthemiscarriageswas66.1621.11days(range:35–120d).
Theageoftheearliestmiscarriagesamongthewomenwas24.94.5 yearsold,andmostrecentmiscarriageoccurreda30.85.0years.
Thecontrolgroupincluded69age-matchedhealthycoupleswithat leastonelivebirth(54(80%)couplesofourcontrolshadonlyonelive birth,and15(20%)coupleshadtwolivebirths),normalchromosomes andnohistoriesofpregnancy loss,endocrinedisorders(including diabetes and polycystic ovarian syndrome, PCOS), autoimmune diseaseorpregnancycomplications.AllofthesubjectswereChinese and were recruited at the Center for Reproductive Medicine of ShandongUniversity,Chinafrom2007to2013,andallparticipants providedwritteninformedconsent.
Thisstudywasacase-controlledassociationstudyofasample ofrecurrentspontaneousabortioncouplesandwasapprovedby theInstitutionalReviewBoardof ReproductiveMedicine,ofthe CenterforReproductiveMedicineofShandongUniversity.
DNAextraction
Genomic DNA wasextracted from all participantsfrom the peripheralbloodleukocytesviaastandardprocedureinvolvingthe useoftheQIAampDNAminikit(QIAGEN,Hilden,Germany).The sampleswerestoredat208C,andtheDNAconcentrationswere 25–60ng/
m
l.TIMPgeneSNPs
FiveTIMPgeneSNPs(rs4898inTIMP-1,rs2277698inTIMP-2, rs2234921andrs5749511inTIMP-3andrs17035945inTIMP-4) were selected for the present study; these SNPs have been extensivelystudiedandarewelldescribed.Theallelefrequencies offouroftheseSNPS,withtheexceptionofrs5749511inTIMP-3 (MAF<5%)have been observedto be minorallele frequencies (MAF)>5%.OurreasonforchoosingtheseSNPswastoconfirmthe resultsfromastudyofaSlovenianpopulation[24].Someevidence hasshownthatthesefiveSNPs,twoofwhich(rs4898inTIMP-1, rs2277698inTIMP-2)arelocatedinexons,mightinfluenceprotein expression,regulatemRNAstabilityandtranslationandbelinked tootherfunctionalpolymorphisms[25–28].
Genotypeanalyses
Genotyping for all of the SNPs was performed utilizing polymerase chain reactions (PCRs) and the Sanger fluorescent dyedideoxy-terminationmethod(alsocalledthechaintermina- tion method). All primerswere designed by MolecularBiology
Table1
PrimersandPCRamplificationconditionsforTIMPgeneSNPs.
SNPID Primes(50–30) Annealing,cycle PCRproduct
TIMP-1(Xp11.23)rs4898 F-CGCAGCGAGGAGTTTCTCAT
R-TCAGGGTCCAGGCACTCACT
588C,35 380bp
TIMP-2(17q25.3)rs2277698 F-GAATTCACCAACTGTGTGGC R-CCAGGAAATTGGCAGGTAGT
588C,35 365bp
TIMP-3(22q12.3)rs2234921 F-CCTCTTGCCTTTCTACTTTCTGTCT R-GAGACCTTGACTGTGCTTGGTG
608C,35 559bp
rs5749511 F-CTTTCCTATGGCTAAAGTGCTCA
R-AGCAGGATTCTGTATCTCACCGT
588C,35 494bp
TIMP-4(3p25.2)rs17035945 F-GTCCATTCTGGCTCTCACATTG R-CTCCCAAACCCCATTAGTCT
608C,35 378bp
Insights Oligo. The primers and PCR reaction conditions are presentedinTable1.
Statisticalanalyses
Thestatisticalanalyseswereperformed withtheSPSSv.19.0 software package. Differences in the genotypes and allele frequenciesbetween thepatientsandcontrolsweredetermined with Pearson’s chi-square (
x
2) tests. The power analysis was calculatedusingtheGeneticPowerCalculator(http://pngu.mgh.- harvard.edu/purcell/gpc/).TheHardy–Weinbergequilibriumtest wasadoptedtoexaminethedemographicrepresentationsofthe URSAandthecontrolgroups.TheassociationsoftheTIMPgene polymorphismswithURSAwereestimatedbycalculatingtheodds ratios (OR) and 95% confidence intervals. P-values<0.05 were consideredtobesignificant.Results
In this study, the power tests produced results of 80.6%
(rs4898),98.4%(rs2277698),81.1%(rs2234921),77%(rs574951), and94% (rs17035945). Thegenotype distributions ofall of the investigatedpolymorphismsintheURSAandcontrolgroupswere inHardy–Weinbergequilibrium(P>0.05,Table4).Thegenotypes andallelefrequenciesofTIMP-1,-2,-3,and-4arepresentedin Tables2and3.Thegenotypeandallelefrequenciesofourcontrol grouparesimilartothosethathavepreviouslybeenpublished[29]
(http://www.ncbi.nlm.nih.gov/projects/SNP) for a Han Chinese Hanpopulation;however,theallelefrequencyofrs2277698(A/G) inTIMP-2wasdifferentfromthatofaSlovenianpopulation(C/T) [24]. Nosignificant differencesbetween theURSA patients and controlswereobservedinthedistributionsofgenotypes,theallele frequenciesoranyofthegeneticmodels.
Comment
Totheextentofourknowledge,thisis thefirstreportfrom China to explore the potential associations of TIMP gene polymorphisms with URSA. This study was conducted in 84 well-characterized homogeneous RSA couples who had had at least three consecutive spontaneous abortions of unknown etiologies and 69 control couples with at least one live birth andnoprevioushistoriesofmiscarriage,pre-eclampsia,pre-term delivery, ectopic pregnancy or any other pregnancy-related complications. In this study,no significant differencesbetween thepatientsandthecontrolswereobservedinanyofthealleleor genotype frequencies, which suggests that suggested that the polymorphisms of rs4898 in TIMP-1, rs2277698 in TIMP-2, rs2234921andrs5749511inTIMP-3,andrs17035945inTIMP-4 mightnotbeassociatedwithURSAamongHanChinesecouples.
Thedevelopment ofa histologically andfunctionallynormal endometriumiscriticalforsubsequentendometrialdecidualiza- tion, receptivity and implantation. Proper communication and interactionbetween maternal decidualcells and the embryois essential for the establishment of a functional fetal–maternal interface,whichischaracterizedbythebreakdownandreorgani- zationoftheECM.Theextensivedegradationandremodelingof theECMdependsontheactivityofMMPsandonthebalanceof MMPswithTIMPs.Recentstudieshave confirmedthat URSAis associatedwithabnormalitiesintheremodelingoftheendome- trialextracellularmatrixandwithaberrantMMPgeneexpression inendometriumandchorionicvilliofURSAwomen;MMP2-735 C/Tand MMP9-1562C/T functionalgenepolymorphismsmight be associated with an increased risk of URSA in women [8,11,22,30–32].
Inthedevelopinggonads,primordialgermcells(PGCs)migrate fromtheiroriginallocationsinextra-embryonicsitestowardtheir finaldestinations.MMPand TIMPgenesplay somerolesinthe
Table2
GenotypeandallelefrequenciesoftheTIMPgeneSNPs’.
SNP Female Male
URSA (n=84)
Control (n=69)
Chi- square(x2)
Pvalue OR(95%CI) URSA (n=84)
Control (n=69)
Chi- square(x2)
Pvalue OR(95%CI)
TIMP-1rs4898#1
MAF#2 79(47.0) 68(49.3) 0.154 0.695 0.914(0.582–1.434)
TT 21(25.0) 20(29.0) 2.589 0.274
TC 47(56.0) 30(43.5)
CC 16(19.0) 19(27.5)
TIMP-2rs2277698
MAF 38(22.6) 33(23.9) 0.071 0.79 0.930(0.546–1.584) 40(23.8) 31(22.5) 0.077 0.781 1.079(0.632–1.841)
GG 49(58.3) 41(59.4) 1.225 0.542 48(57.1) 42(60.9) 0.406 0.816
GA 32(38.1) 23(33.3) 32(38.1) 23(33.3)
AA 3(3.6) 5(7.3) 4(4.8) 4(5.8)
TIMP-3rs2234921
MAF 13(7.7) 10(7.2) 0.026 0.871 1.074(0.456–2.529) 10(6.0) 11(8.0) 0.483 0.487 .731(0.301–1.775)
AA 72(85.7) 60(87.0) 0.095 0.954 74(88.1) 58(84.1) 0.521 0.47 1.403(0.558–3.531)
AG 11(13.1) 8(11.6) 10(11.9) 11(15.9)
GG 1(1.2) 1(1.4) 0 0
TIMP-3rs5749511
MAF 4(2.4) 2(1.4) 0.324 0.559 1.659(0.299–9.193) 1(0.6) 3(2.2) 1.464 0.226 0.269(0.028–2.620) CC 80(95.2) 67(97.1) 0.349 0.555 0.597(0.106–3.361) 83(98.8) 66(95.7) 1.483 0.223 3.773(0.384–37.112)
CT 4(4.8) 2(2.9) 1(1.2) 3(4.3)
TT 0 0 0 0
TIMP-4rs17035945
MAF 21(12.5) 24(17.4) 1.445 0.229 0.679(0.36–1.28) 27(16.1) 20(14.5) 0.145 0.703 1.130(0.603–2.117)
CC 63(75.0) 47(68.1) 2.909 0.234 58(69.0) 52(75.4) 2.988 0.224
CT 21(25.0) 20(29.0) 25(29.8) 14(20.3)
TT 0 2(2.9) 1(1.2) 3(4.3)
#1rs4898inTIMP-1islocatedontheXchromosomeandisthusnotpresentinmen.
#2MAF:minorallelefrequency.
activemigrationofPGCs,andthelevelsofTIMPgeneexpression arealwayshigherinpost-migratingPGCsthaninmigratingPGCs;
i.e.,TIMP-3levelsare3.4-foldhigher,TIMP-1levelsare2.4-fold higher,and TIMP-2 levelsare 1.8-foldhigher [33]. Thepresent study further proved that MMP genes and TIMP gene have importantinfluencesonthenormalgrowthofembryos.
Allfour TIMPs mRNAs have been found tobe expressed in humanfetalmembranecells(amniochorionanddecidua),thecells ofthedevelopinghumanplacentaandintheendometriumduring allphases,andthesemRNAlevelsincreaseduringthesecretory phase [23,34–36]. TIMP-1, -2 and -3 have been found in trophoblasts and in matrix-producing decidual stromal cells (DSC)ofthefirsttrimesterplacentalbeds[37].Furthermore,TIMP genetic variations have been associated with different human
pregnancypathologies.TIMP-1hasbeenshowntobeacandidate geneforembryoimplantationinanimalmodels[38,39].TIMP-2 copynumbervariationshavebeenfoundinURSApatientswith normalkaryotypes[40],andrs2277698inTIMP-2hasbeenfound strongly influence the preterm period regardless of membrane ruptures[41,42].
Importantly, many studies have found that TIMPs play important roles across the entire period of gestation [8,12–
14,17,18,23].In ourstudyofHanChinesecouplesinShandong, whether the subjects lived in Han Chinese habitations was examine, and no significant difference in the TIMP gene SNPs werefoundbetweentheURSApatientsandthecontrolgroup.
ThedifferencesinTIMPpolymorphismsbetween149couples withhistoriesofthreeormoreURSAsand149coupleswithnormal fertilities and no histories of pregnancy pathologies were examined by Slovenian researchers. Using PCR and restriction fragmentlengthpolymorphismmethods,theseresearchersana- lyzed the genotypesand allelefrequencies of TIMP-1372 C/T, TIMP-2303C/T,TIMP-3915A/G,TIMP-31296C/T,andTIMP-430- UTRC/Tindetail.Theyfoundnoevidenceforassociationsbetween TIMPsandURSAinthisSlovenianpopulation[24].Inthepresent study,wealsofoundnoevidenceforassociationsbetweenTIMPs andURSAinaChinesepopulation.
The relationships between TIMP polymorphisms and URSA remainproblematic.However,increasinginvestigationsofother SNPsites in TIMPgenesutilizingincreasedsample sizesmight revealmoremeaningfulassociations.
Condensation
In this study, we didn’tfoundtheassociation of TIMPgene polymorphismsandURSA.
Table3
Oddsratios(ORs)and95%confidenceintervals(CIs)fortheriskofURSAaccordingtodifferentgeneticmodels.
SNP Model Female Male
OR(95%CI) Pvalue OR(95%CI) Pvalue
TIMP-1rs4898#1 Dominant TT+CTvs.CC 1.615(0.756–3.449) 0.214
Recessive TTvs.CT+TT 0.817(.399–1.673) 0.58
Codominant TTvs.CC 1.247(0.505–3.079) 0.632
TTvs.CT 0.670(0.312–1.440) 0.304
CCvs.CT 0.538(0.240–1.205) 0.13
Tvs.C 1.094(0.697–1.718) 0.695 0.716(0.454–1.129) 0.15
TIMP-2rs2277698 Dominant GG+GAvs.AA 2.109(0.486–9.160) 0.31 1.231(0.296–5.112) 0.775
Recessive GGvs.GA+AA 0.956(0.500–1.827) 0.892 0.857(0.448–1.639) 0.641
Codominant GGvs.AA 1.992(0.449–8.847) 0.357 1.143(0.269–4.855) 0.856
GGvs.AG 0.859(0.436–1.692) 0.66 0.821(0.417–1.617) 0.569
AAvs.AG 0.431(0.094–1.988) 0.271 0.719(0.163–3.176) 0.662
Gvs.A 1.075(0.631–1.831) 0.79 0.927(0.543–1.582) 0.781
TIMP-3rs2234921 Dominant AA+AGvs.GG 1.221(0.075–19.878) 0.888
Recessive AAvs.AG+GG 0.90(0.355–2.28) 0.824 1.403(0.558–3.531) 0.47
Codominant AAvs.GG 1.2(0.073–19.594) 0.898
AAvs.AG 0.873(0.33-2.309) 0.784 1.403(0.558–3.531) 0.47
GGvs.AG 0.727(0.039–13.452) 0.83
Avs.G 0.931(0.395–2.195) 0.871 1.369(0.563–3.325) 0.487
TIMP-3rs5749511 Dominant CC+CTvs.TT
Recessive CCvs.CT+TT 0.597(0.106–3.361) 0.555 3.773(0.384-37.112) 0.223 Codominant CCvs.TT
CCvs.CT 0.597(0.106–3.361) 0.555 3.773(0.384–37.112) 0.223
TTvs.CT
Cvs.T 0.603(0.109–3.342) 0.559 3.711(0.382–36.084) 0.226
TIMP-4rs17035945 Dominant CC+CTvs.TT 0.444(0.371–0.530) 0.116 3.773(0.384–37.112) 0.223
Recessive CCvs.CT+TT 1.404(0.692–2.848) 0.346 0.729(0.356–1.494) 0.387
Codominant CCvs.TT 0.427(0.344–0.53) 0.106 3.346(0.338–33.173) 0.276
CCvs.CT 1.277(0.622–2.621) 0.505 0.625(0.294–1.327) 0.219
TTvs.CT 0.187(0.018–1.969) 0.128
Cvs.T 1.474(0.781–2.78) 0.229 0.885(0.472–1.658) 0.703
1#rs4898inTIMP-1islocatedontheXchromosomeandnoORsorPvaluedataareavailableforthedifferentmodelsofinheritanceinmen.
Table4
Hardy–Weinbergequilibriumtest.
x2 P#
TIMP-1rs4898
URSA 1.27 0.26
Control 1.17 0.28
TIMP-2rs2277698
URSA 0.79 0.37
Control 0.57 0.45
TIMP-3rs2234921
URSA 0.07 0.80
Control 0.06 0.81
TIMP-3rs5749511
URSA 0.07 0.79
Control 0.04 0.84
TIMP-4rs17035945
URSA 2.3 0.13
Control 0.9 0.34
#P>0.05.
Acknowledgements
This work was supported by the National Basic Research ProgramofChina(973program,2012CB944700,2011CB944502) andScienceandTechnologyDevelopmentPlanningofShandong (2013GGE27001).
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