An array comparative genomic hybridization approach

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Original article

Genomic answers for recurrent spontaneous abortion in Saudi Arabia:

An array comparative genomic hybridization approach

^$

Sajjad Karim

^a,

*, Hasan Salleh Jamal

^b

, Abdullraheem Rouzi

^b

,

Mohammed Salleh M. Ardawi

^c

, Hans-Juergen Schulten

^a

, Zeenat Mirza

^d

,

Nuha A. Alansari

^a

, Maha M. Al-Quaiti

^a

, Heba Abusamra

^a

, Muhammad Imran Naseer

^a

, Rola Turki

^b,e

, Adeel Gulzar Chaudhary

^a

, Mamdooh Gari

^a

,

Adel Mohammed Abuzenadah

^a,e

, Mohammed Hussain Al-Qhatani

^a,

*

aCenterofExcellenceinGenomicMedicineResearch,FacultyofAppliedMedicalSciences,KingAbdulazizUniversity,Jeddah,SaudiArabia

bDepartmentofGynecology,FacultyofMedicine,KingAbdulazizUniversity,Jeddah,SaudiArabia

cCenterofExcellenceforOsteoporosisResearch,FacultyofMedicine,KingAbdulazizUniversity,POBoxNo.80216,Jeddah21589,SaudiArabia

dKingFahdMedicalResearchCenter,KingAbdulazizUniversity,Jeddah,SaudiArabia

eKACSTInnovationCenterforPersonalizedMedicine,KingAbdulazizUniversity,Jeddah,SaudiArabia

ARTICLE INFO Articlehistory:

Received4November2016

Receivedinrevisedform14March2017 Accepted17March2017

Availableonlinexxx Keywords:

Recurrentspontaneousabortion Array-CGH

Microarray Cytogenetics SaudiArabia

ABSTRACT

Tostudythegenomics/geneticfactorsassociatedwithrecurrentspontaneousabortion(RSA),as50%of RSAareunexplained.However,chromosomeabnormalitieshavebeenreportedtoplaymajorroleinRSA.

Weperformedwholegenomearray-CGHbasedgenomicanalysisoffortyfourSaudiRSApatientsto identifypotentialmolecularandchromosomalabnormalities.Weidentifiedatotalof845alterations, usuallynotdetectedbyclassiccytogeneticmethods,indifferentgenomicregionsusingacutoffvalueof 0.25and0.25forstructurallossandgain,whereas 1.0and0.58wereusedforsinglecopynumber deletion and duplication respectively.We identified frequent(present at leastin 10% of patients) alterationsincluding threemacro-alterationat8p23.1,10q11.21-q11.22and15q11.2aswellaslarge numbers ofmicro-deletions/amplifications withaffected genesincluding 22q11.23 (GSTT1),3p22.2 (CTDSPL),6p21.32(HLA),and8p22(MSR1).PathwayanalysisofgeneslocatedindetectedCNVsregions revealedtheallograftrejectionsignaling,IL-4signaling,andautoimmunethyroiddiseasesignalingasthe mostsignificantcanonicalpathwaysassociatedwithRSA.WholegenomearrayCGHtechniquecanbe used toidentifypotential genes,biofunctionsand chromosomalabnormalities associatedwithRSA whichissupportedbyourfindingsofanumberofnovelCNVs/genes(22q11.23/GSTT1,3p22.2/CTDSPL, 6p21.32/HLA,8p22/MSR1,and14q32.33/AKT1)andpathwaysinpatientsaffectedwithRSA.Toimprove diagnosisandtreatmentofRSA,acomprehensiveprocedureisneededforidentificationandvalidationof causativegenes.

1.Introduction

Human reproductionis a remarkably inefﬁcient process and morethan50%ofallpregnanciesgetabortedbeforetheirclinical recognition [1,2] and up to 3% of couples face recurrent spontaneous abortion (RSA) (3 subsequent pregnancy losses before 22nd weeks of gestation) leading to psychical stress, disappointments,andinadequacyofaffectedcouple[3–8].Ithas beenreportedthatabout15%ofclinicallyrecognizedpregnancies terminatespontaneouslyintheirﬁrsttrimester[9–11].RSAmight beassociated withearly arrestof celldivision,orearly embryo implantationfailurebeforeitsestablishment[12–14].Althougha Abbreviations:RSA,recurrentspontaneousabortion;CGH,comparativegeno-

michybridization; CNVs,copynumber variations; IL,interleukin; SNP,single nucleotidepolymorphism;bp,basepair;GO,geneontology;HLA,humanleukocyte antigen;Th1,T-helper1;Th2,T-helper2;GSTT1,glutathioneS-transferasetheta1;

CTDSPL, CTD small phosphatase-like protein; MSR1, macrophage scavenger receptor;DGV,databaseofgenomicsvariation.

$ Wholegenomearray-CGHtechniquehaspotentialtoidentifymolecularand chromosomal abnormalities associated with recurrent spontaneous abortion complications and improve the diagnoses and treatment after validation of causativegenes.

* Correspondingauthorsat:CenterofExcellenceinGenomicMedicineResearch, KingAbdulazizUniversity,POBOX80216,Jeddah21589,SaudiArabia.

E-mailaddresses:[email protected](S.Karim),[email protected] (M.H.Al-Qhatani).

http://dx.doi.org/10.1016/j.repbio.2017.03.003

xxx–xxx ContentslistsavailableatScienceDirect

Reproductive Biology

j o u r n a lh o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / r e p b i o

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numberofRSArisk factorslikeuterine anatomicabnormalities, immunologic factors, endocrine abnormalities, maternal age, hormonal disorders, infections, environmental factors, sperm qualityand thrombophilic disorders are known, still in around 50%ofpatientsetiologyofRSAremainsunidentiﬁed[4,15–17].In view of this, detection of genetic abnormalities involved in molecularpathogenesis ofRSAis mandatorytohelpexplaining unknowncausesofrecurrentpregnancyloss.

Contribution of thegenetic predisposition and chromosome abnormalitiestoRSA iswellreportedand classicalcytogenetics techniqueisroutinelyusedtodetectchromosomalabnormalities.

However, it suffers from limitations such as low resolution, frequentfailure of cell culture,contamination of maternalcell, poor chromosome morphology, and is a time consuming procedures[18–21].Fluorescence in situ hybridization(FISH) is another technique that can be used to confirm chromosomal abnormalitiesthat areclinicallysuspected,however, thesescan notbeusedforhithertounknownanomalies.Additionally,single nucleotidepolymorphisms(SNP)basedstudieshavealsoreported morethanhundredRSAcandidategenesthoughwithconflicting results [22–24]. More recently, genetic, epigenetic and gene expressionprofilinghavebeenapplied toidentifychromosome abnormalities associated with RSA [25–28]. Array-comparative genomichybridization(array-CGH)techniquecandetectsubmi- croscopic(microdeletions and microduplications) chromosomal aberrations,withouttheneedtoinitiatecellcultures[29,30].

Inthecurrentgenome-widestudy,weperformedhigh-density arrayCGHanalysistodefinegenomicalterationpatternsand to understandtheroleofCNVsinpredisposingtoRSAamongcouples sufferingfromunexplainedRSAresidinginSaudiArabia.Ourmajor finding wasidentificationof 845genomicalterations,including frequentCNVin3p22.2(CTDSPL),6p21.23(HLA),8p22(MSR1)and 22q11.23(GSTT1)thatintheunaffectedstatusmightbeassociated withhealthypregnancymaintenance.

2.Materialsandmethods 2.1.Patientsandsamples

FortyfourcasesofRSA(16couplesand12mothersonly)were includedinpresentcytogeneticandarrayCGHbasedstudy.The studywasapprovedbyKingAbdulazizUniversitylocalBioethics CommitteeandbytheCEGMRethicalcommittee(No.09-CEGMR- ETH-01).Informedconsentwastakenfromallsubjectsincludedin thepresentstudy.

2.2.Cytogeneticmethods

A standard 72-h lymphocyte culture and GTG banding (G bandingbyTrypsinand Giemsa) wascarried out inmetaphase chromosomes of RSA cases from King Abdulaziz University Hospital,Jeddah.Microscopicexaminationsweredoneinatleast 20metaphasesforeachpatient.Incasewithsuspectedmosaicism, thisnumberwasexpandedtoonehundredmetaphases.Chromo- someswereanalyzedbyusingasemi-automaticappliedimaging karyotyper and karyotyping software (Applied Imaging, Santa Clara, CA) and International System for Human Cytogenomic Nomenclature(ISCN,2013)wasusedtodescribekaryotypes[31].

2.3.Arraycomparativegenomichybridizationproﬁling

Thearray-CGHanalysiswasperformedaspermanufacturer’s protocolusingAgilentsureprintG3HumanCGH2400Karrays, Agilentlabelingkit(AgilentTechnologies,USA).

(i) DNA Preparation: Genomic DNA was extracted from peripheral blood using QIAamp DNA blood mini kit (Qiagen,

USA), and was quantiﬁed by using a NanoDrop ND-1000 Spectrophotometer. (ii) Genomic DNA Fragmentation: Patients DNA(500ng)andreferenceDNA(Promega,USA)fromthesame sexweredigestedat37CbyRsaIandAluI(Promega,USA)for2h.

ThereferenceDNAwasheat-fragmentedfor10minat95C.(iii) Fluorescent Labeling, Purification and Hybridization: Patient and reference DNA were labeled with Cy5-dUTP and Cy3-dUTP respectively. Labeled samples werepurified by using Microcon YM-30filterunits(Millipore,Billerica,Massachusetts,USA).Cot-1 DNA(Invitrogen,Carlsbad,California, USA),hybridizationbuffer andblockingagentweremixedwithlabeledDNAanddenaturation wasperformedat95C beforehybridizationat65C for40hat 20rpm.(iv)MicroarrayWashing,ScanningandFeatureExtraction:

Firstly microarray slides and gaskets were disassembled and washedfor10mininwashbuffer1(Agilent,cat#5188–5221),then were shifted to wash buffer 2 (Agilent, cat# 5188–5222) and agitatedat37Cfor2–3min.Slideswerewashedwithanhydrous acetonitrile. Chip scanning, image analysis and data extraction wereperformedonan Agilent Scanner(G2505C),and Agilent’s Feature Extraction software (V.1.5.1.0) respectively. (v) Data Analysis: Array CGH proﬁling was done using Agilent CytoGe- nomicsv2.7softwaretovisualize,detectandanalyzeaberrations.

All detected alterations were identiﬁed by using log2 ratiofor diploidcases-test(cy5)/reference(cy3)ratio(2n:2n)isequalto1 on linear scale but 0.0 on log₂ scale respectively, however for multiple cases the standard deviation of log2 ratios in an oligonucleotidearrayisontheorderof0.25,i.eanythingbeyond 0.00.25 is considered gain (+0.25) or loss (-0.25). Thus, quantitativelossorgainincopynumberweredetectedbyshift values in the log2 ratio from zero. One set copy number gain (3n:2n)orloss(1n:2n)wasmeasuredbybothlinearscale(1.5and 0.5)andlog2scale(+0.58and 1)respectively.

2.4.Functionalenrichmentanalysis

Wederivedthegenessymbol,p-valueandfoldchangevalues fromalteredchromosomallocusanduploadedthesedataintothe Ingenuity Pathways Analysis software(Ingenuity Systems,Red- woodCity,CA,USA;IPAhttp://www.ingenuity.com/).TheIngenu- ityPathwaysKnowledgeBase,whichisalargenetworkdatabaseof curated molecular interactions and pathways, was used to generate gene maps of interest in order toidentify significant biological associations including, interaction and functional framework.Networksorsub-networkswerepresentedgraphically using direct and indirect molecular relationships. The gene ontologymethodpredictedthesignificanceofoverrepresentation of biological processes, byassesingthe number of significantly associatedgeneinrelationtothecuratedbackgroundgenes.The impactof the linkbetweenthe aberration/expressiondataand canonicalpathwayswerecomputedbyusingBenjamini-corrected modifiedFisher’sexacttestandestimatedbypvalues(significance

<0.05).

3.Results

3.1.Cytogeneticstudy

We report the chromosomal analysis of the 44 recurrent spontaneousabortionpatients.Theageofthesubjectsrangedfrom 24to48years(meanage32.17)andthenumberofabortionsper couplevariedfrom3to15(mean4.22).Mostofthedocumented pregnanciesinthiscohort,gotterminatedintheirﬁrsttrimester (79.50%)followedbysecond(16%)andthird(4.5%)trimester.Fifty seven percent of RSA patients were able to achieve viable pregnancieswithatleastonelivebirthswhereasremaining43%

hadtwoormultiplepregnancyterminationexperienceswithout xxx–xxx

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Table1

Clinicopathologicalinformationandcytogeneticresultsoffortyfourrecurrentspontaneousabortioncases-sevencoupleshadconsanguineousmarriageandthreehadfamily historyofrecurrentabortion.

PatientNo BiobankNo Age ClinicalHistory Sex Preg Misc Trim Rel Family History

Nation Cytogeneticresults

RSA1 BL-0588-11 28 RecurrentAbortion F 3 3 1 Yes Yes Saudi 46,XX,rob(13:14)

RSA2 BL-0589-11 37 RecurrentAbortion F 7 4 1 Yes Yes Saudi 46,XX

RSA3 BL-0590-11 39 Hus,RA589 M 7 4 1 Yes Normal Saudi 46,XY

RSA4 BL-0609- 12

29 RecurrentAbortion&

Thrombophilia

F 5 4 3 Yes Normal Saudi 46,XX

RSA5 BL-0614-12 37 RecurrentAbortion&

Thrombophilia

F 3 3 1 No Normal Saudi 46,XX

RSA6 BL-0806-11 35 RecurrentabortionandIVPD. F 11 8 1 No Normal Saudi 46,XX

RSA7 BL-0817-11 28 RecurrentAbortion F 6 6 1 No Normal Saudi 46,XX

RSA8 BL-0824-11 34 RecurrentAbortion F 3 3 1 No Normal Saudi 46,XX

RSA9 BL-0841-11 40 Recurrentabortion F 9 9 1 No Normal Saudi 46,XX

RSA10 BL-0878-11 33 Hus,RA M 6 4 1 No Normal Palestinian 46,XY,t(11;22)(q23;q11)

RSA11 BL-0937-11 32 Recurrentabortion F 3 3 1 No Normal Indian 46,XX

RSA12 BL-0938-11 36 Hus,RA938 M 3 3 1 No Normal Indian 46,XY

RSA13 BL-0990- 09D

36 Hus,RA991 M 10 6 1 Yes Normal Saudi 46,XY

RSA14 BL-0991- 09D

36 RecurrentAbortion F 10 6 1 Yes Normal Saudi 46,XX

RSA15 BL-1024-11 45 Recurrentabortion(hormone therapy)

RSA16 BL-1099- 13D

Thrombophilia

RSA17 BL-1106- 10D

42 Hus,RA1107 M 12 11 1 No Normal Egyptian 46,XY

RSA18 BL-1111- 11D

RSA19 BL-1113- 11D

Thrombophilia

F 3 3 1 Yes Normal Saudi 46,XX

RSA20 BL-1562- 10D

31 RecurrentAbortion F 6 5 1 No Normal Saudi 46,XX

RSA21 BL-1640- 10D

34 RecurrentAbortion&FS F 4 4 1&

2

Yes Normal Saudi 46,XX[96]/45,X[2]/37-42,XX,-X,t(7;14) (q34;p10)

RSA22 BL-1659- 10D

43 RecurrentAbortion F 6 4 1 Yes Normal Eritrean 46,XX

RSA23 BL-1660- 10D

48 Hus,RA1659 M 6 4 1 Yes Normal Eritrean 46,XY

RSA24 BL-1757- 10D

35 RecurrentAbortion F 3 3 1&

2

No Normal Saudi 46,XX

RSA25 BL-1758- 10D

36 Hus,RA1757 M 3 3 1&

2

No Normal Saudi 46,XY

RSA26 BL-1784- 10D

41 RecurrentAbortion F 5 5 2&

3

No Normal Saudi 46,XX

RSA27 BL-1814- 10D

RSA28 BL-1833- 10D

RSA29 BL-1834- 10D

41 Hus,RA1833 M 4 3 1 No Normal Saudi 46,XY

RSA30 BL-1865- 10D

RSA31 BL-1898- 10D

37 RecurrentAbortion F 7 5 1 Yes Yes Yemeni 46,XX

RSA32 BL-1899- 10D

39 Hus,RA1898 M 7 5 1 Yes Normal Yemeni 46,XY

RSA33 BL-1917- 10D

28 RecurrentAbortion F 4 3 1 Yes Yes Saudi 46,XX

RSA34 BL-1927- 10D

RSA35 BL-1928- 10D

RSA36 BL-1949- 10D

RSA37 BL-2010- 10D

29 RecurrentAbortion F 9 6 1 Yes Normal Yemeni 46,XX

RSA38 BL-2051- 10D

RSA39 BL-2082- 10D

29 Hus,RA2084 M 3 3 1‘ No Normal Saudi 46,XY

RSA40 BL-2084- 10D

33 RecurrentAbortion F 3 3 1‘ No Yes Saudi 46,XX

RSA41 BL-2363- 10D

37 Hus,RA M 4 4 1 No Normal Egyptian 46,XY

RSA42 BL-2842- 10D

27 Hus,RA M 5 4 1 No Normal Yemeni 46,XY

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livebirth.PatientswereconsideredasRSAcaseswhentheyfailed to complete pregnancies and had experienced spontaneous abortionfor at least three times. Therewere 70.5% cases with upto5abortionsand29.5%patientswithmorethan5abortions.

SevencouplehadconsanguineousmarriageandthreeRSApatients hadfamilyhistoryofrecurrentabortion.Therewerethreecases with chromosomal abnormalities: 46,XX rob(13:14), 46,XY, t (11;22)q23;q11) and 46,XX/45,X/37-42,X/37XX,t(7;14)(q34;p10) [31][Table1][Fig.S1].

3.2.ArrayCGHstudy

Weidentiﬁedinthecohortof44RSApatientsmorethan845 alterations,rarelyseenbyclassiccytogeneticmethods,indifferent genomicregionsusingacutoffvalue0.25forstructuralgainor loss, 1.0and0.58forsinglecopynumberdeletionorduplication.

Wedetectedthreemacro-and33micro-alterationsconsistingof 25gainsand11lossesinmorethan10%ofthecases.Thethree macro-alterationwerelocatedat8p23.1[Fig.1],10q11.21-q11.22, and15q11.2 [Fig.2]. Theampliﬁedregions of15q11.2 harbored following genes BCL8, GOLGA6L6, GOLGA8C, LOC646214, LOC646396, CXADRP2, POTEB, NF1P1, LOC727924, OR4M2, OR4N4andOR4N3P.The33micro-deletions/ampliﬁcationswere locatedat1q21.3(affectedgene,LCE3C),1q24.2(NME7),3p22.2 (CTDSPL), 4q13.2 (UGT2B17), 6p21.32 (HLA-DRB5,HLA-DRB6), 7p15.2 (SKAP2), 7p14.1(TARP), 7q34 (MGAM, PRSS1, PRSS2, MTRNR2L6, TRY6), 8p23.2 (CSMD1), 8p22 (MSR1), 8p11.23 p11.22 (ADAM5P, ADAM3A), 10q11.22 (PPYR1, GPRIN2), 11q11 (OR4C11, OR4P4, OR4S2, OR4C6), 12p13.2 (PRH1, TAS2R46, TAS2R43, PRR4), 14q11.1-q11.2 (OR11H12, POTEG, POTEM, OR4Q3, OR4M1, OR4N2, OR4K2, OR4K5, OR4K1) [Fig. 3], 14q32.33 (KIAA0125, ADAM6, NCRNA00226), 20p13 (SIRPB1), 22q11.22 (MIR650, IGLL5), and 22q11.23 (LOC391322, GSTT1, GSTTP2)[Table2][32–34].Themostcommongainswereobserved at14q32.33(77%),followedby15q11.2(68%),14q11.1-11.2(57%), 8p11.23-22(55%)and22q11.23(52%),whilemostcommonlosses were observed at 20p13 (47%) and 1q24.2 (39%). Our result indicatescorrelationbetweennumberofCNVsandmiscarriagesas wefoundaverageCNVsof10.6,11.75,12.5,11.5and14for3,4,5,6 and>7miscarriagesrespectively.Furthermore,wecategorizedthe samplesbasedonthenumberofmiscarriagestothreegroups;3, 4–6, and >=7 and found direct associations of number of alterationswithincreasingnumbersofmiscarriages[Fig.4].

3.3.Pathwayanalysis

PathwayanalysisofgeneslocatedonCNVsrevealedanumber ofcanonicalpathwayssigniﬁcantlyassociatedwithRSA[Table3]

whichwereIL-4signaling,Bcelldevelopment,CD28signalinginT helpercells,antigenpresentationpathway,autoimmunethyroid disease signaling, allograft rejection signaling, role of NFAT in regulationoftheimmuneresponse,IL-17Asignalinginairwaycells and calcium-induced T lymphocyte apoptosis. Further analysis revealedsigniﬁcantfunctionalannotationsincludingrelaxationof uterus,bindingof oocytes,and adhesionofgranulosacells that mightbeplayingacriticalroleinpregnancy[Table4].

4.Discussion

To investigate the proportion of chromosome aberrations among RSA patients, many cytogenetic studies have been conducted,indicatingvariablefrequencyofchromosomeaberra- tions ranging between approximately 3–13%, [35–41] and our cytogenetics analysis also identiﬁed around 12% chromosome aberrationsinapproximately300recurrentabortionpatients[42].

However, the present array CGH study with 44 RSA patients Table1(Continued)

PatientNo BiobankNo Age ClinicalHistory Sex Preg Misc Trim Rel Family History

Nation Cytogeneticresults

RSA43 BL-2868- 10M

46 Hus,RA2871 M 7 7 1 No Normal Saudi 46,XY

RSA44 BL-2871- 10F

43 RecurrentAbortion F 7 7 1 No No Saudi 46,XX

ListofAbbreviations:M:male;F:female;Hus:Husband,RA:Recurrentabortion;Preg:Pregnancy,Misc:Miscarriage;Trim:Trimester;Rel:Relative;Nation:Nationality.

Fig. 1.Whole genome 2400K oligonucleotide-based microarray analysis showingfrequentalterations(amp/del)inchromosome8(8p23.1=del,8p22=amp, 8p11.23=amp)ofrecurrentspontaneousabortionpatients.Lossorgainincopy numberisdetectedbyshift(0.25)inlog2ratiofromzero(zerovalueindicates equalﬂuorescenceintensityratiobetweenthesampleandreference).Theleftward shiftedratio(red)indicatesloss,whereasrightshiftedratio(blue)indicatesgain.

(Forinterpretationofthereferencestocolourinthisﬁgurelegend,thereaderis referredtothewebversionofthisarticle.)

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Fig.2.Wholegenome2400Koligonucleotide-basedmicroarrayanalysisofthe15q11.2regionthatwasamplifiedin68%ofrecurrentspontaneousabortioncasesand includeBCL8,GOLGA6L6,GOLGA8C,LOC646214,LOC646396,CXADRP2,POTEB,NF1P1,LOC727924,OR4M2,OR4N4andOR4N3P.Lossorgainincopynumberisdetectedby shift(0.25)inlog2ratiofromzero(zerovalueindicatesequalfluorescenceintensityratiobetweenthesampleandreference).Theleftwardshiftedratio(red)indicatesloss, whereastherightshiftedratio(blue)indicatesgain.Theleft-upperwindowshowschromosomalview,right-upperwindowshowsgeneviewandlowerwindowshows detailsofalterationincludingalteredchromosomenumber,startandstopregion,size(bp),probenumber,genenameandtypeofalteration(amp/del).(Forinterpretationof thereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)

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detected845geneticalterationsthatwereinthevastmajoritynot seen by classical cytogenetic techniques applied routinely. We cross checked the detected CNVs with database of genomics variation(DGV)forhealthycontrolstoavoidcommonvariantsin absenceofethniccontrol,andfound257non-overlapCNVs.Inthis

study, we have performed array-CGH, a molecular cytogenetic technique, to address the effect of CNVs and contribution of associated genestobetterunderstandmolecularmechanismof RSA.Array-CGHis ahighlysensitive methodtodetecttheDNA copy number gains and losses across the whole genome at Fig.3. Wholegenome2400Koligonucleotide-basedmicroarrayanalysisdetectedamplificationofthe14q11.1-q11.2regionin57%ofRSApatients.Theregionincludes POTEM,OR4Q3,OR4M1,OR4K2,OR4K5,andOR4K1.Lossorgainincopynumberisdetectedbyshift(0.25)inlog2ratiofromzero(zerovalueindicatesequalfluorescence intensityratiobetweenthesampleandreference).Theleftwardshiftedratio(red)indicatesloss,whereastherightshiftedratio(blue)indicatesgain.Theleft-upperwindow showschromosomalview,right-upperwindowshowsgeneviewandlowerwindowshowsdetailsofalterationincludingalteredchromosomenumber,startandstopregion, size(bp),probenumber,genenameandtypeofalteration(amp/del).(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtotheweb versionofthisarticle.)

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Table2

FrequentlypresentchromosomalabnormalitiesinrecurrentspontaneousabortionpatientsdetectedbyarrayCGHandcomparisonwithrelatedstudyanddatabase.

Chromosome Cyto- band

CNVs probe Change p-

value

Frequency Genes

Start-Stop (bp)

size(bp) Our

Data

Nagarnajaetal Migita O, etal.,

DGV

No % Control

%

RM cases

% RM cases%

Control

chr14 q32.33 105403001–

105988274

585,274 11 Amp 2.47E- 43

34 77 – 9 8 U KIAA0125,ADAM6,NCRNA00226

chr15 q11.2 18741716–

20079994

1338279 80 Amp 2.94E- 44

30 68 18 13 – U BCL8,GOLGA6L6,GOLGA8C,LOC646214, CXADRP2,POTEB,NF1P1,LOC727924,OR4M2, OR4N4,OR4N3P,LOC646396

chr14 q11.1–

q11.2

18446762–

19490689

1043928 35 Amp 4.75E- 22

25 57 3 6 – U OR11H12,POTEG,POTEM,OR4Q3,OR4M1, OR4N2,OR4K2,OR4K5,OR4K1

chr8 p11.23–

p11.22

39354149–

39505315

151167 9 Amp 1.04E- 15

24 55 51 58 3 U ADAM5P,ADAM3A

chr22 q11.23 22677959–

22725353

47395 10 Amp 2.71E-

16

23 52 – – – U LOC391322,GSTT1,GSTTP2

chr20 p13 1511715–

1532485

20771 6 Del 5.66E-

24

21 47 – – – * SIRPB1

chr8 p22 15996382–

16069666

73,285 5 Amp 1.66E-

28

18 41 – – – U MSR1

chr22 q11.22 21380410–

21575888

195479 8 Amp 1.04E-

15

18 41 – – – U MIR650,IGLL5

chr1 q24.2 167493768–

167507957

14190 5 Del 1.57E-

12

17 39 – – – U NME7

chr3 p22.2 37955930–

37961169

5240 3 Amp 1.17E-

34

17 39 – 4 – * CTDSPL

chr4 q13.2 69069651–

69165872

96222 12 Amp 1.23E- 25

17 39 18 11 – U UGT2B17

chr12 p13.2 11109511–

11147734

38224 8 Amp 4.82E–

19

16 36 – – – U PRR4,PRH1,TAS2R43

chr7 p14.1 38262701–

38349092

86392 18 Del 9.89E-

27

16 36 – – U TARP

chr1 q21.3 150836481–

150848568

12088 5 Del 5.25E-

32

15 34 – – – U LCE3C

chr14 q24.3 73075500–

73092077

16578 3 Amp 6.42E-

32

15 34 – – – U HEATR4,ACOT1

chr6 p21.32 32558677–

32613597

54921 9 Amp 1.51E-

29

15 34 29 13 0 U HLA-DRB5

chr17 q21.31 41570512–

41706929

136418 17 Amp 3.17E- 11

14 31 – – – U KIAA1267

chr7 p15.2 26855104–

26903240

48137 9 Amp 2.58E-

22

14 31 – – U SKAP2

chr7 q34 141408013–

141438563

30551 7 Amp 2.55E-

14

14 31 – 2 – U MGAM

chr22 q11.21 18073135–

18691763

618629 159 Amp 1.44E- 22

13 29 – – – U SEPT5,GP1BB,TBX1,GNB1L,TXNRD2,COMT, ARVCF,DGCR8,TRMT2A,ZDHHC8,RTN4R, DGCR6L,C22orf29,C22orf25,MIR185, MIR1306,RANBP1,LOC150197,MIR1286

chr8 p23.1 7156900–

7790993

634094 8 Del 4.83E-

13

13 29 7 – 0 U DEFB103A,FAM90A7,FAM90A14,FAM90A13, FAM90A19,FAM90A18,FAM90A8,FAM90A9, FAM90A10,FAM66B,DEFB109P1B,ZNF705G, DEFB103B,SPAG11B,DEFB104A,DEFB104B, DEFB106A,DEFB106B,DEFB105A,DEFB105B, DEFB107A,DEFB107B,SPAG11A,DEFB4A chr10 q11.21

q11.22

45478162–

47172593

1694432 57 Amp 1.70E- 41

12 27 – – 2 U SYT15,PPYR1,ANXA8,ANUBL1,FAM21C, AGAP4,PTPN20B,PTPN20A,FRMPD2L1, BMS1P5,BMS1P1,FAM35B,GPRIN2, LOC643650,LOC728643,ANXA8L1,FAM25G, FAM25C,FAM25B,AGAP9,LOC642826, FAM35B2,ANTXRL

chr22 q11.21 18506870–

19117997

611128 61 Amp 3.10E- 11

11 25 – – 0 U ZDHHC8,RTN4R,DGCR6L,RIMBP3,ZNF74, LOC150197,MIR1286,PI4KAP1,SCARF2

chr11 q11 55124730–

55207364

82635 16 Del 1.46E- 22

10 23 29 16 U OR4C11,OR4P4,OR4S2,OR4C6

chr16 p13.3 511780–

1382861

871082 164 Del 6.24E- 18

10 23 – 2 12 U RAB11FIP3,SOLH,WFIKKN1,

chr9 q34.3 138520642–

139278330

757689 155 Amp 6.05E- 10

10 23 – 2 3 U NOTCH1,MIR126,AGPAT2,LCN10,LCN6,LCN8, C9orf86,PHPT1,C8G,LCN12,PTGDS,CLIC3, ABCA2,FUT7,NPDC1,ENTPD2,C9orf140, GRIN1,ANAPC2,SSNA1,SLC34A3,COBRA1, EGFL7,FAM69B,SNHG7,SNORA43,SNORA17, LOC100128593,LCN15,TMEM141,KIAA1984, LOC100131193,C9orf172,MAMDC4,EDF1, TRAF2,FBXW5,LCNL1,C9orf142,C9orf139, xxx–xxx

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submicroscopiclevelof5–10Kbresolutionwhichallowstoidentify affectedgenesinthespeciﬁcregions.MultipleCNVsand/orCNVs oflargersizemayincreasethelikelihoodofalteringkeycandidate genesorpathwaysinvolvedinearlypregnancymaintenanceand leadingtowardsRSA[43,44].Thegenome-wideproﬁlingofCNVs andtheircorrespondinggenesrevealedrelatedpathwaysinclud- ing IL-4 signaling, Bcell development, antigen presentation pathway,autoimmunethyroiddiseasesignaling,IL-17Asignaling inairwaycellsandallograftrejectionsignalingpathway/interaction.Wereport35mostfrequentCNVsofchromosomalregions

disruptingCTDSPL,GSTT1,AKT1,HLA,andMSR1genesthatmight beassociatedwithearlypregnancymaintenance[45–57].

WefoundampliﬁcationinCTDsmallphosphatase-likeprotein (CTDSPL)genein39%cases.Literaturesearchindicatedtheroleof CTDSPL genes in pregnancy and recurrent abortion. It has phosphataseactivity todephosphorylatetheC-terminaldomain ofRNApolymeraseIIandregulatescellgrowthanddifferentiation.

Gain-and loss-of-functionstudies showedthat both miR-26a/b anditshostgene(CTDSP)directlyorsynergisticallydecreasingthe phosphorylated formof pRb (ppRb), therebyincreasing RB-E2F interactionandblockingthecellcycleprogressionatG1/S-phase [55,58].StudiesalsosuggestthatCTDSPL/RBSP3mightfunctionas atranscriptionalco-repressor,inhibitingtranscriptionofneuronal genesinnon-neuronalcells[56],andmayalsoactasaphosphatase ofSmad1,Smad2andSmad3[57].AlterationinCTDSPLgenehas also been reported to be associated with susceptibility to pregnancy loss throughinteraction withSYCP3, RB, SMAD and TNF-betagenes[53,55,59,57].STRINGv10protein-protein interaction network also displays SMAD1, SMAD2, SMAD3, CDK9, GTF2F1,CTDP1,REST,C3orf35,MKI67IP,andOBSCNaspredicted functional partner [34]. CTDSPL has also been found to be frequentlyalteredandmethylatedin manytumors [60,61].This generegulateschromosomalrecombinationandsegregation,and chromosomalsynapsisbyencoding componentsofthesynapto- nemal complex. Alteration/mutation in CTDSPL gene has been reportedto beassociated withsusceptibility topregnancy loss [53].

Inourstudy, wefoundtheglutathioneS-transferasetheta1 (GSTT1) gene, located on 22q11.23 region to be significantly amplified(p value=2.710 ¹⁶). GSTT1 codes for detoxification enzymethatdecreasestheeffectofoxidativestressonembryo.

Normal GSTT1 is crucial for endometrial differentiation and Table2(Continued)

Chromosome Cyto- band

CNVs probe Change p-

value

Frequency Genes

Start-Stop (bp)

size(bp) Our

Data

Nagarnajaetal Migita O, etal.,

DGV

No % Control

%

RM cases

% RM cases%

Control

UAP1L1,LOC100289341,MAN1B1,DPP7, LRRC26,TPRN,TMEM203,NDOR1,RNF208, C9orf169,LOC643596,TUBB2C,FAM166A, C9orf173 ...

chr12 q24.33 131486691–

131807940

321250 61 Amp 1.85E- 19

9 20 – – 1 U POLE,FBRSL1,P2RX2

chr7 p22.3 1229427–

1976440

747014 123 Amp 7.93E- 10

9 20 – 2 8 U MAFK,MAD1L1,UNCX

chr3 q29 196904349–

196972126

67778 19 Del 9.25E-

12

8 18 – – U MIR570,MUC20,MUC4

chr6 p21.33 29962849–

30021967

59119 10 Del 4.61E-

21

8 18 18 13 15 U HLA-H,HLA-A,HCG2P7

chrX q28 152376791–

153473460

1096670 201 Amp 1.30E- 20

8 18 – – 0 * BGN,ATP2B3,FAM58A

chr10 q11.22 46396163–

46568552

172390 31 Del 2.31E- 17

7 16 – – – U PPYR1,GPRIN2,LOC643650

chr17 q25.3 76662104–

77646444

984341 168 Amp 7.79E- 16

7 16 – – 0 U BAIAP2,AATK,ACTG1

chr10 q26.3 134877109–

135026325

149217 38 Amp 2.11E- 15

6 13 7 – 9 U UTF1,ADAM8,PRAP1

chr22 q13.33 48946249–

49099488

153240 38 Amp 9.26E- 17

6 13 – – – U MAPK12,PLXNB2,PANX2

chr14 q32.33 104311091–

104324592

13,502 4 Amp 9.27E-

14

6 13 – 2 – U AKT1

ListofAbbreviations:CNVs:CopyNumberVariations;bp:BasePair;No:numbers;%:percentage;Amp:ampliﬁcation;Del:deletion;*:Non-overlappedinDGV;U:foundin DGV;–:Notreportedbypreviousstudies.

Fig.4.Graphshowingassociationofgeneticalterations(CNVs)withRSA.

xxx–xxx

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embryonicgrowth;however,itsoverexpressionhasbeenreported toincreasetheriskofpregnancyloss[52].Macrophagescavenger receptor(MSR1) hasbeenshown toregulatematernalimmune tolerancebyactivatingTcellresponsethroughuterinedendritic cells[54].AlterationinMSR1mightchangedendriticcellsproﬁle andTcelltoleranceleadingtomiscarriage.

Large ampliﬁed or deletedCNVs regions are more likely to disruptthekeygenesessentialforearlypregnancymaintenance.

We found both gain and loss of large CNVs regions including 15q11.2, 10q11.21 q11.22,8p23.1,9q34.3,8q24.3, 11p15.5 p15.4, 22q11.21, Xp22.33 p11.1,19p13.3,16p13.3, 20q13.33,17q25.3 presentatleastinfourRSAcases.Geneslocatedontheseregions mayhavepotentialtoincreasetheriskofRSAindependentlyorin combination [62]. Excessive genomic burden of CNVs in an individualmightexhibitincreasedriskforRSA.

Inourpathwayenrichmentanalysisofaffectedgenesderived fromselectedCNVs regions,we foundimmune signalingto be associatedwiththeRSAstudygroup.Ingeneral,maternalimmune systemmighttargetthepaternalgeneticmaterialandinherited alloantigens in the fetus considering it foreign, but sperm’s histocompatibilitylocus antigens (HLA)interacts withmother’s

HLAandstimulateastrongerprotectiveresponseinthemother’s body toaccepttheconception. Incase ofweak andinsufﬁcient protectiveresponsethewhitebloodcellsofmother’sbodyattack on new cells of embryo resulting in termination of the early pregnancy[45–47].TheHLAgenelocus,presenton6p21.32region, wasampliﬁedinaroundhalfofourregisteredcases.HLAandits alleles (DQA1,DQB1, DRB5, DRB6) are known for theirrole in allograftrejection,additionalstudiessuggestedthemasriskalleles forunexplainedrecurrentabortionandgainingattentionasRSA markers[48–50].

Pro-inflammatoryimmunecell activationis vitalfor embryo implantation,placentation,andparturition;howevertheirdysre- gulation canleadtodetrimentalpregnancyoutcomesincluding spontaneous abortion,fetal growth restriction, maternalhyper- tensive disorders, and even maternal death. During pregnancy, inflammation is tightly controlled and is largely mediated by immunecellsthatproduceinterleukin(IL)-4andIL-10.Persistent excessive maternalinflammatory responsesare associatedwith adverse pregnancycomplications and outcomes like idiopathic miscarriages. Down-regulated IL-4 and IL-10 signaling has detrimentaleffectsonfetalandmaternalphysiologylikeinfertility, spontaneousabortion,pretermbirth,andhypertensivedisorders ofpregnancy[51].

ThebalanceofT-helper1(Th1)andT-helper2(Th2)cellsduring pregnancyplayacriticalrole,Th1-typecytokinesinduceallograft rejectionwhileTh2-type cytokinespromotesallograft tolerance andincreasesfetalsurvivalrates[63–67].PositiveroleofTh1-type cytokines in pregnancy depends on the concentration and gestationstage[68].

Oversecretion ofTh2cytokinesanddownregulationofTh1 cytokines by feto-placental unit redirect the cell mediated maternal immunity to humoral immunity [69]. MEF2–NFAT complex playsan importantrole in T-lymphocyte apoptosisby regulating Nur77 expression[70,71].Suppression of thesignal- transducing–chaininTlymphocytesisamustforfetussurvival, Table3

ListoftopcanonicalpathwaysderivedfromalteredgeneslocatedondetectedCNVsamongrecurrentspontaneousabortioncases.

CanonicalPathways Pvalue Molecules

IL-4Signaling 1.20E-03 AKT1,HLA-DQA1,HLA-DRB5

BCellDevelopment 2.63E-03 HLA-DQA1,HLA-DRB5

CD28SignalinginTHelperCells 4.57E-03 AKT1,HLA-DQA1,HLA-DRB5

AntigenPresentationPathway 4.90E-03 HLA-DQA1,HLA-DRB5

AutoimmuneThyroidDiseaseSignaling 6.92E-03 HLA-DQA1,HLA-DRB5

AllograftRejectionSignaling 8.51E-03 HLA-DQA1,HLA-DRB5

RoleofNFATinRegulationoftheImmuneResponse 1.29E-02 AKT1,HLA-DQA1,HLA-DRB5

Calcium-inducedTLymphocyteApoptosis 1.35E-02 HLA-DQA1,HLA-DRB5

IL-17ASignalinginAirwayCells 1.51E-02 AKT1,DEFB4A/DEFB4B

THelperCellDifferentiation 1.58E-02 HLA-DQA1,HLA-DRB5

TREM1Signaling 1.74E-02 AKT1,DEFB4A/DEFB4B

IL-17Signaling 1.78E-02 AKT1,DEFB4A/DEFB4B

TR/RXRActivation 2.82E-02 AKT1,TBL1Y

RoleofIL-17AinPsoriasis 3.63E-02 DEFB4A/DEFB4B

Acyl-CoAHydrolysis 3.63E-02 ACOT1

GlycogenDegradationIII 5.01E-02 MGAM

DNAdamage-induced14-3-3sSignaling 5.25E-02 AKT1

RegulationofeIF4andp70S6KSignaling 6.46E-02 RPS4Y1,AKT1

GlutathioneRedoxReactionsI 6.92E-02 GSTT1

EIF2Signaling 8.91E-02 RPS4Y1,AKT1

NRF2-mediatedOxidativeStressResponse 8.91E-02 AKT1,GSTT1

Glutathione-mediatedDetoxiﬁcation 9.55E-02 GSTT1

InhibitionofAngiogenesisbyTSP1 1.00E-01 AKT1

mTORSignaling 1.00E-01 RPS4Y1,AKT1

PrimaryImmunodeﬁciencySignaling 1.17E-01 IGLL1/IGLL5

phagosomeformation 2.52E-01 MSR1

HumanEmbryonicStemCellPluripotency 3.21E-01 AKT1

Table4

DiseasesorfunctionsenrichmentofCNVgenesusingIPA.

DiseasesorFunctionsAnnotation p-Value Molecules differentiationoftestis 9.19E-05 SCX,SOX3,SRY differentiationofSertolicells 2.04E-03 SCX,SRY adhesionofgranulosacells 4.52E-02 PTPN20 associationofgonadalcelllines 4.52E-02 MSR1

bindingofoocytes 4.52E-02 ADAM15

morphogenesisoftestis 4.52E-02 SRY

relaxationofuterus 4.52E-02 RLN1

xxx–xxx