Expression of CPPED1 in human trophoblasts is associated with timing of term birth

Exp ession o CPPED1 in human ophoblas s is associa ed wi h iming o e m bi h An i M. Haapalainen a, b, * , Minna K. Ka jalainen a, b , Ra ind a Daddali a, b , S e en Ohlmeie c , Julia An onen a, b , Tomi A. M€ a€ a € a a, b , Annama i Salminen a, b , Ma i Mahlman a, b , Ul ich Be gmann c , Kaa in M€ akikallio a, d, e , Ma ja Ojaniemi a, b , Mikko Hallman a, b, # , Mika R€ ame a, b, , # a PEDEGO Resea ch Uni and Medical Resea ch Cen e Oulu, Uni e si y o Oulu, Oulu, Finland b Depa men o Child en and Adolescen s, Oulu Uni e si y Hospi al, Oulu, Finland c P o eomics Co e Facili y, Biocen e Oulu, Facul y o Biochemis y and Molecula Medicine, Uni e si y o Oulu, Oulu, Finland d Depa men o Obs e ics and Gynecology, Oulu Uni e si y Hospi al, Oulu, Finland e Depa men o Obs e ics and Gynecology, Tu ku Uni e si y Hospi al and Uni e si y o Tu ku, Tu ku, Finland BioMediTech Ins i u e and Facul y o Medical and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland Recei ed: Decembe 2, 2016; Accep ed: Augus 29, 2017 Abs ac Unde s anding o iming o human pa u i ion is incomple e. The e o e, we ca ied ou p o eomic analyses o ull- e m placen as om uncompli- ca ed p egnancies o iden i y p o ein signa u es associa ed wi h he onse o spon aneous deli e y. We ound quan i a i e associa ions o 10 p o eins wi h spon aneous e m bi h, e iden ei he in he basal o in he cho ionic pla es o in bo h. Addi ional 18 p o eins we e associa ed acco ding o he loca ion wi hin placen a indica ing local a ia ions in p o ein amoun s. Calcineu in-like phosphoes e ase domain-con aining 1 (CPPED1), a phospha ase p e iously sugges ed dephospho yla ing AKT1/PKB, was one o he iden i ied p o eins. qRT-PCR e ealed he mRNA le el o CPPED1 was highe in elec i e caesa ean deli e ies han in spon aneous bi hs, while immunohis ochemis y showed CPPED1 in cy o ophoblas s, syncy io ophoblas s and ex a illous ophoblas s. No ewo hy, phospho yla ion s a us o AKT1 did no di e be ween pla- cen as om elec i e caesa ean and spon aneous deli e ies. Addi ionally, analyses o samples om in an s indica ed ha single-nucleo ide poly- mo phisms s11643593 and s8048866 o CPPED1 we e associa ed wi h du a ion o e m p egnancy. Finally, pos - ansc ip ional silencing o CPPED1 in cul u ed HTR8/SVneo cells by siRNAs a ec ed gene exp ession in pa hways associa ed wi h in lamma ion and blood essel de elop- men . We pos ula e ha unc ions egula ed by CPPED1 in ophoblas s a cho iodecidual in e phase ha e a ole in he induc ion o e m labou , bu i may be independen o AKT1. Keywo ds: CPPED1  gene ics  placen a  p o eomics  ophoblas In oduc ion Despi e ex ensi e esea ch, ou unde s anding o he iming o human pa u i ion a he molecula le el emains incomple e. Mul iple ac o s may ini ia e labou [1], and he signal can o igina e om he mo he , oe us, u e oplacen al uni o a combina ion o hese. Some ho mones a e known o in luence he main enance o p egnancy. These include s e oid ho mones such as p oges e one, oes ogens, and and ogens, as well as he pep ide ho mones cho ionic gonado opin, cho ionic soma omammo opin, elaxin and oxy ocin, which u he in e ac wi h he immune sys em o p omo e p egnancy [2, 3]. P oges e one is essen ial o u e ine quiescence; oes ogen, on he o he hand, inc eases myome ial sensi i i y o oxy ocin, he eby inducing con- ac ions [2]. S ong e idence om animal s udies also sugges s ha he oe al hypo halamic–pi ui a y–ad enal (HPA) axis con ols sec e- ion o co ico ophin- eleasing ho mone (CRH), leading o inc eased induc ion o oe al ma u i y by co icos e oids and inc eased sec e ion o labou -inducing ho mones [4]. In he placen a, CRH exp ession is nega i ely egula ed by p oges e one and oes ogen and posi i ely egula ed by a ious neu opep ides [1, 5–7]. O he known ac o s, such as u e ine s e ch, in lamma o y cy okines, p os aglandins and ni ic oxide, ha e known o p oposed oles in he ini ia ion o labou . # These au ho s made equal con ibu ions. *Co espondence o: An i M. HAAPALAINEN E-mail: [email p o ec ed] ª2017 The Au ho s. Jou nal o Cellula and Molecula Medicine published by John Wiley & Sons L d and Founda ion o Cellula and Molecula Medicine. This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. doi: 10.1111/jcmm.13402 J. Cell. Mol. Med. Vol 22, No 2, 2018 pp. 968-981 CRH and oe al ib onec in (FFN) a e addi ional key p o eins ha ha e been used o moni o he iming o pa u i ion in humans [1]. In ma e nal plasma, CRH le els inc ease exponen ially du ing p egnancy [1]. Because he e a e la ge indi idual a ia ions, he a e o inc ease a he han a single CRH measu emen should be de e mined [1, 8]. By con as , FFN is p esen mainly in amnio ic luid and oe al memb anes and in placen al issue. FFN may be eleased in o ce ico aginal sec e- ions h ough mechanical and in ec ion-media ed damage o he oe al memb anes o placen a p io o bi h [9]. Ele a ed FFN le els in ce ical luids indica e an inc eased likelihood o deli e y; hus, hey a e a p e- dic o o e m and p e e m bi h [9, 10]. Unlike in expe imen al animals such as sheep and small oden s, in human p egnancy blood le els o p oges e one inc ease a he han dec ease du ing labou . I has been p oposed ha unc ional wi hd awal o p oges e one upon labou in humans may occu , as exp ession o a low-a ini y a ian o p oges- e one ecep o inc eases owa ds e m [11]. In human placen a, o- phoblas s p oduce and sec e e p oges e one [12]. Some s udies ha e epo ed c oss alk be ween p oges e one and oes ogen ecep o s and RAC-alpha se ine/ h eonine-p o ein kinase (AKT1) in a ious diseases [13]. AKT1 is pa o he phospha idylinos- i ol-3-kinase (PI3K) signalling pa hway [14, 15], and in some cance s, he PI3K/AKT pa hway is hype ac i a ed [13]. Phospho yla ion o Se 473 is equi ed o ull ac i a ion o AKT1; his is accomplished by 3-phosphoinosi ide-dependen p o ein kinase 2 (PDK2) [16–18]. Cal- cineu in-like phosphoes e ase domain-con aining 1 (CPPED1) is a newly cha ac e ized phospha ase. I inac i a es AKT1 by emo ing he c i ical phospha e om Se 473 o AKT1 [19]. Recen epigene ic, ansc ip omic, p o eomic and exosome analy- ses o human placen a and he placen al mic obiome ha e e ealed new da a, no only abou placen al de elopmen bu also abou labou . Placen a-de i ed exosomes, eleased o he ma e nal ci cula ion, appea o ha e oles in egula ing di e se unc ions, including placen- al de elopmen and immuno ole ance [20]. Placen al ansc ip omic di e ences ha e been iden i ied in he decidua, amnion and cho ion, sugges ing a ious unc ionally dis inc sub egions [21, 22]. Mo e- o e , epigene ic a ia ion in he placen a may be associa ed wi h p egnancy ou come [23]. Se e al p o eomic s udies ha e epo ed bioma ke s o e m and p e e m bi hs [10, 24, 25]. O e a hund ed bioma ke s o spon aneous p e e m bi h ha e been iden i ied o a ious ypes o biochemical pa hways [26]. Mo e han hal o hese bioma ke s ha e been classi ied as being in ol ed in immune unc ion and in lamma ion. P o eomic analyses o placen al blood plasma [27] and ce ico aginal luids [28] om no mal e m deli e ies ha e iden i ied p o eins linked o immune and de ence esponses and in lamma ion and oxida i e s ess, espec- i ely. P o eomic analyses o placen a ha e also ocused on oe al g ow h es ic ion [29], ea ly p egnancy loss [30–32], p e-eclampsia [32, 33], down synd ome [34], di e ences be ween i s - and hi d- i- mes e human placen as [35] and low molecula weigh componen s in he cho ionic and basal pla es o placen a [36]. The majo i y o placen al s udies ha e ocused on di e en pa ho- logical condi ions, while only ew p o eomic s udies ha e in es iga ed changes associa ed wi h no mal e m bi h. The aim o his s udy was o iden i y placen al p o eins associa ed wi h ges a ional age and labou in uncomplica ed e m p egnancies. To do so, we analysed he p o eome o he cho ionic and basal pla es o human placen a o iden- i y p o eins in ol ed in he no mal labou p ocess. We ound ha he ex aemb yonic p o ein CPPED1 is no only a p omising ma ke o spon aneous e m bi h bu ha a polymo phism o he CPPED1 gene is associa ed wi h he leng h o no mal uncomplica ed p egnancy. We also p o ide e idence ha in human e m placen a, CPPED1 le els a e no associa ed wi h he phospho yla ion s a us o AKT1. Ma e ials and me hods Placen a samples The s udy was app o ed by he e hics commi ee o Oulu Uni e si y Hospi al, and all mo he s p o ided w i en in o med consen . Placen a issues we e collec ed a Oulu Uni e si y Hospi al in 2012–2014. Sam- ples we e ha es ed as desc ibed p e iously [37], including issue biop- sies om bo h basal and cho ionic pla es. All placen al specimens we e om uncomplica ed e m p egnancies. O he cases (n=31), 18 we e spon aneous aginal deli e ies and 13 in an s we e deli e ed elec i ely by Caesa ean sec ion wi hou signs o symp oms o labou . O he spon aneous e m deli e ies, he ges a ional age (GA) a ied om 39 weeks +6 days o 41 weeks +3 days (a e age 40 week +1 day GA). In he elec i e caesa ean e m bi h g oup, he placen as we e om p egnancies wi h GAs o 38 weeks +3 days o 42 weeks +0 day (a e age 39 weeks +2 days GA). The leng h o p egnancy was based on oe al ul asound examina ion be o e 16 weeks o p egnancy. Two-dimensional minimal di e ence gel elec opho esis and mass spec ome y Fo he p o eomic s udy, we included placen a samples collec ed a e ei he elec i e caesa ean o spon aneous e m bi h om he cho ionic and basal pla es (n=6 pe g oup). Fo p o ein spo s o be conside ed signi ican , we used a 1.5- old cu -o a io. A P alue o ≤0.05 acco d- ing o S uden ’s - es was conside ed s a is ically signi ican . Theo e ical spo posi ions in he 2D gel acco ding o known p o ein sequences we e calcula ed wi h he Compu e pI/Mw ool (h p://ca.expasy.o g/ ools/ pi_ ool.h ml). Associa ions be ween p o eomic changes and GO biologi- cal p ocesses and molecula unc ions we e iden i ied wi h he Da abase o Anno a ion, Visualiza ion and In eg a ed Disco e y (DAVID), 6.7 [38, 39]. In mass spec ome y, o iden i y p o eins, we an addi ional 2D gels wi h la ge amoun s o unlabelled p o ein (400–800 lg) combined wi h Cyanine dye (Cy) 2-labelled in e nal s anda d. Fu he de ails ega d- ing 2D-gel and MS analyses a e p esen ed in he Appendix S1. S udy popula ion o he gene ic s udy o ges a ional age The s udy popula ion was collec ed p ospec i ely a Oulu Uni e si y Hospi- al in 2004–2007 and 2014. Al oge he , we included 342 in an s bo n spon- aneously a e m (GA om 38 weeks +0days o41weeks+6days;GA mean s anda d de ia ion [S.D.] 40.1 0.9 weeks). All s udy subjec s we e o Finnish o igin. Umbilical co d blood o umbilical co d issue was ª2017 The Au ho s. Jou nal o Cellula and Molecula Medicine published by John Wiley & Sons L d and Founda ion o Cellula and Molecula Medicine. 969 J. Cell. Mol. Med. Vol 22, No 2, 2018 collec ed o DNA ex ac ion. Speci ic inclusion c i e ia a e p esen ed in he Appendix S1. Gene ic s udy and geno yping Genes encoding p o eins ha we iden i ied as up- o down- egula ed in he placen al p o eome om spon aneous e m deli e ies we e analysed o hei associa ion wi h GA. Al oge he , we in es iga ed 77 SNPs: ACTB, h ee SNPs; A2M, nine SNPs; B2M, one SNP; CPPED1, 16 SNPs; CYB5A, 11 SNPs; HBG2, i e SNPs; KRT8, 12 SNPs; KRT19, i e SNPs; PRDX2, h ee SNPs; and SERPINB2, 12 SNPs (Table S4). Associa ions be ween hese SNPs and GA we e assessed by he Wald es wi h PLINK, . 1.07 [40]. Unde s ic Bon e oni co ec ion, he signi icance le el was P<6.5 910 4 . We ocused in dep h on geno ypes o he wo GA-associa ed CPPED1 SNPs s11643593 and s8048866 in he in es iga ed in an s. Fu he de ails a e p esen ed in he Appendix S1. Wes e n blo ing To quan i a e he amoun o AKT1 phospho yla ed a Se 473 compa ed o he o al amoun o AKT1, we used abbi an i-human AKT1 pS473 an ibody (2967S, 1:1000 dilu ion; Cell Signaling Technology, Leiden, The Ne he - lands) and mouse an i-human AKT1 an ibody (9018S, 1:1000 dilu ion; Cell Signaling Technology), espec i ely. Seconda y an ibodies we e goa an i- abbi IgG Dyligh 680 conjuga e (611-144-002-0.5, 1:10000 dilu ion; Rock- land, Lime ick, PA, USA) and goa an imouse IgG Dyligh 800 conjuga e (610-145-002-0.5, 1:10000 dilu ion; Rockland). De ec ion was done wi h he Odyssey In a ed Imaging Sys em (LI-COR Biosciences, Lincoln, NE, USA). We used 87 ng o human ecombinan AKT1 (009-001-P21; Rock- land) as a posi i e con ol. To quan i a e he amoun o FOXO1 phospho yla ed a Se 256 compa ed o he o al amoun o FOXO1, we used abbi an i-human FoxO1 pSe 256 an ibody (NB100-81927, 1:1000 dilu ion; No us Biologicals, Abingdon, Oxon, UK) and mouse an i-human FOXO1 an ibody (F6928, 1:1000 dilu ion; Sigma-Ald ich, S . Louis, MO, USA), espec i ely. The seconda y an ibody and de ec ion me hod we e he same as o AKT1. Fu he de ails a e p e- sen ed in he Appendix S1. Immunohis ochemis y Fo CPPED1 de ec ion, samples we e incuba ed in abbi an i-human CPPED1 an ibody (HPA040938, 1:250 dilu ion; Sigma-Ald ich) o 1 h , ollowed by de ec ion o he bound an ibodies wi h he wo-s ep En i- sion ki (K5007; DAKO). Fo FOXO1 and FOXO3 de ec ion, samples we e incuba ed in abbi an i-human phospho-FOXO1 pSe 256 an ibody (SAB4300094, 1:4000 dilu ion; Sigma-Ald ich) and abbi an i-human phospho-FOXO3 pSe 253 an ibody (PA5-37578, 1:10000 dilu ion; The mo Fishe Scien i ic, Wal ham, MA, USA), espec i ely. Fo nega i e con ols, non-immune abbi IgG was used o con i m he speci ici y o he p ima y an ibody. Fu he de ails a e p esen ed in he Appendix S1. Quan i a i e PCR We included samples om he basal and cho ionic pla es o placen as om 13 elec i e caesa ean and 18 spon aneous e m bi hs o qRT- PCR analysis. RNA isola ion, cDNA syn hesis and quan i a i e PCR anal- yses we e done as desc ibed ea lie [37]. The High Pu e RNA Tissue Ki (Roche, B anchbu g, NJ, USA) and RNeasy Mic o Ki (Qiagen, Hilden, Ge many) we e used o RNA isola ion. Rela i e quan i ica ions o CPPED1 mRNA le els om cho ionic and basal pla es we e done wi h he Ligh Cycle  96 ins umen (Roche). The expe imen s we e designed as in on spanning assays wi h cy och ome c1 (CYC1) mRNA as a e - e ence. Fu he de ails ega ding quan i a i e PCR a e p esen ed in he Appendix S1. T ans ec ion wi h small in e e ing RNAs HTR8/SVneo cells (ATCC, CRL-3271) we e ini ially ans ec ed wi h ei he 30 nM o nega i e con ol siRNA o 100 nM o pooled CPPED1 siRNAs (siRNA pai s 1–3) in suspensions and hen added in o 12-well pla e. A e 24 h s o incuba ion, adhe en cells we e e- ans ec ed wi h siRNA pai s 1–3 a he same concen a ion as ans ec ion pe o med in suspension and cells ha es ed a e 48 h s o second ans ec ion. Fu - he de ails ega ding ans ec ion a e p esen ed in he Appendix S1. T ansc ip omes o siRNA samples P io o ansc ip omic analysis o siRNA samples, he RNA quali y was de e mined by Agilen 2100 Bioanalyze sys em a Biocen e Oulu Sequencing Cen e , Finland. The ansc ip omes o he CPPED1 silenced cells and nega i e con ol cells we e de e mined using Illumina HiSeq high- h oughpu sequencing sys em a he Finnish Func ional Genomics Cen e, Finland. The sequencing da a we e analysed by he Bioin o ma - ics Uni Co e Se ice a he Tu ku Cen e o Bio echnology, Finland. Resul s P o ein signa u es associa ed wi h spon aneous e m deli e ies To explo e he molecula mechanisms associa ed wi h spon aneous e m deli e y, we compa ed p o ein quan i ies in human placen as om uncomplica ed p egnancies wi h spon aneous and elec i e caesa ean e m deli e ies. We likewise in es iga ed di e ences wi hin he placen a p o eome by s udying issue samples collec ed om basal and cho i- onic pla es. Gel-based p o eomics wi h di e ence gel elec opho esis (DIGE) app oach was used o s udy no only p o ein le els bu also modi ica ion and p ocessing o in ac p o eins. In he i s s ep, sample p epa a ion, p o ein labelling and sepa a ion we e op imized o pla- cen a issue, which esul ed in he ep oducible de ec ion o abou 2,400 spo s (Fig. S1). The applied so wa e o wo-dimensional (2D) gel analysis allows 100% spo ma ching; he e o e, all spo s we e com- pa ed among he ou s udy g oups, which comp ised spon aneous o elec i e caesa ean bi h wi h samples collec ed om he basal and cho ionic pla es o he same placen a (n=6 pe g oup). Compa isons o bo h deli e y ypes as well as placen a loca ions e ealed 49 signi i- can ly changed spo s (cu -o a io: 1.5- old, P≤0.05; Fig. S1), 970 ª2017 The Au ho s. Jou nal o Cellula and Molecula Medicine published by John Wiley & Sons L d and Founda ion o Cellula and Molecula Medicine. Table 1 Bi h pheno ype- and loca ion-dependen changes in he human placen a p o eome Spo P o ein UniP o -KB Desc ip ion Theo e ical pI/MW (kD) De ec ed pI/MW (kD) Ra io E e sus SBa e sus Ch Ba Ch E S Elec i e (E) e sus spon aneous (S) bi h 46*ACTB P60709 Ac in, cy oplasmic 1 ( agmen ) 5.29/41.7 (5.29/41.6) 4.93 18 1,57 (1,13) 2,17 (1,54) 1 A2M P01023 a-2-mac oglobulin 6.03/163.3 5.51 142 1,57 (1,45) (1,12) (1,22) 2 (5.98/160.8) 5.54 142 1,62 1,59 (1,09) (1,13) 3 5.57 142 1,55 1,67 (1,04) (1,11) 49 B2M P61769 b-2-mic oglobulin 6.06/13.7 (6.07/11.7) 6.07 14 (1,12) 1,59 (1,45) (1,04) 39 CPPED1 Q9BRF8 Se ine/ h eonine-p o ein phospha ase (iso o m 1) 5.79/35.5 5.75 31 1,71 1,79 (1,14) (1,19) 44 CYB5A P00167 Cy och ome b5 (iso o m 1 o 2) 4.86/15.3 1 4.72 18 1,74 1,59 (1,07) (1,17) 47 HBG2 P69892 Haemoglobin subuni c-2 6.64/16.1 6.71 16 2,57 2,26 (1,03) (1,18) 48 (6.71/15.9) 6.54 16 2,33 2,03 (1,13) (1,29) 13 KRT8 P05787 Ke a in, ype II cy oskele al 8 (iso o m 1 o 2) 5.52/53.7 1 5.24 49 (1,16) 1,71 (1,14) (1,30) 14 iso o m 1 o 2 5.52/53.7 1 5.24 47 (1,08) 1,73 (1,29) (1,24) 15 iso o m 1 o 2 5.52/53.7 1 5.33 50 (1,55) 2,09 (1,09) (1,46) 30* agmen o iso o m 1 o 2 5.52/53.7 4.79 37 (1,81) 2,39 (1,59) (2,09) 31* agmen o iso o m 1 o 2 5.52/53.7 4.85 38 (1,88) 2,77 (1,07) (1,56) 21 KRT19 P08727 Ke a in, ype I cy oskele al 19 5.05/44.2 4.85 41 (1,09) 1,59 (1,15) (1,48) 29*C- e minal KRT19 agmen 5.05/44.2 4.74 37 (1,19) 1,62 (1,12) (1,54) 42 PRDX2 P32119 Pe oxi edoxin-2 (iso o m 1) 5.66/21.9 (5.67/21.8) 5.52 22 1,65 (1,49) (1,10) (1,21) 22 SERPINB2 P05120 Plasminogen ac i a o inhibi o 2 5.46/46.6 5.51 42 (1,14) 1,92 (1,18) (1,99) ª2017 The Au ho s. Jou nal o Cellula and Molecula Medicine published by John Wiley & Sons L d and Founda ion o Cellula and Molecula Medicine. 971 J. Cell. Mol. Med. Vol 22, No 2, 2018 Table 1. Con inued Spo P o ein UniP o -KB Desc ip ion Theo e ical pI/MW (kD) De ec ed pI/MW (kD) Ra io E e sus SBa e sus Ch Ba Ch E S Basal pla e (Ba) e sus cho ionic pla e (Ch) 46*ACTB P60709 Ac in, cy oplasmic 1 ( agmen ) 5.29/41.7 (5.29/41.6) 4.93 18 1,57 (1,13) 2,17 (1,54) 38 ANXA3 P12429 Annexin A3 5.62/36.4 (5.63/36.2) 5.58 31 (1,35) (1,39) (1,52) 2,83 45*ANXA5 P08758 Annexin A5 ( agmen ) 4.93/35.9 (4.93/35.8) 4,83 18 (1,39) (1,04) 1,55 (1,08) 41 APCS P02743 Se um amyloid P-componen 6.10/25.4 (6.12/25.3) 5.52 25 (1,13) (1,67) (1,89) 2,75 32 CLU P10909 Clus e in (iso o m 3 o agmen s o iso o ms 1,2,4, 5) 5.88/52.5 1 4.76 36 (1,13) (1,65) (1,98) 2,87 33 iso o m 3 o agmen s o iso o ms 1,2,4, 5 (5.89/50.1 1 ) 4.82 35 (1,13) (1,57) 2,16 2,98 34 iso o m 3 o agmen s o iso o ms 1,2,4, 5 4.88 34 (1,11) (1,38) 2,43 3,69 5 EEF2 P13639 Elonga ion ac o 2 6.41/95.3 (6.42/95.2) 6.42 95 (1,02) (1,77) (1,34) 2,32 23*FGB P02675 Fib inogen b(C- e minal agmen in D dime ) 8.54/55.9 5.51 38 (1,43) (1,57) 2,25 2,45 24*C- e minal agmen in D dime (7.95/50.8) 5.60 39 (1,28) (1,63) 2,49 3,17 25*C- e minal agmen in D dime 5.71 39 (1,16) (1,56) 2,54 3,42 26*C- e minal agmen in D dime 5.85 38 (1,56) (1,61) 1,73 (1,81) 6 FGG P02679 Fib inogen c(iso o m gamma-A o –B) 5.37/51.5 A 5.46 94 (2,55) (1,68) 2,07 (1,36) 7 iso o m c-A o –B (5.24/48.5 A ) 5.54 94 (2,18) (1,56) 2,15 (1,53) 16 iso o m c-A o –B 5.54 50 (1,13) (1,16) 1,58 2,05 43 FTL P02792 Fe i in ligh chain 5.50/20.0 (5.50/19.9) 5.50 20 (1,03) (1,47) (1,97) 2,75 4 GSN P06396 Gelsolin (iso o m 1) 5.90/85.7 (5.72/82.9) 5.82 95 (1,58) (1,37) (1,19) 2,59 40 HIBADH P31937 3-hyd oxyisobu y a e dehyd ogenase 8.38/35.3 (5.54/31.5) 5.62 29 (1,01) (1,42) (1,15) 1,65 972 ª2017 The Au ho s. Jou nal o Cellula and Molecula Medicine published by John Wiley & Sons L d and Founda ion o Cellula and Molecula Medicine. Table 1. Con inued Spo P o ein UniP o -KB Desc ip ion Theo e ical pI/MW (kD) De ec ed pI/MW (kD) Ra io E e sus SBa e sus Ch Ba Ch E S 11 HNRNPK P61978 He e ogeneous nuclea ibonucleop o ein K (iso o m 1, 2 o 3) 5.39/50.9 1 5.39 56 (1,27) (1,74) (1,10) 2,45 27 HSD17B1 P14061 Es adiol 17-b-dehyd ogenase 1 5.46/34.9 5.46 35 (1,11) (1,40) (1,25) 1,96 28 (5.47/34.8) 5.59 35 (1,09) (1,43) (1,32) 2,05 10 LMNB1 P20700 Lamin-B1 5.11/66.4 (5.11/65.9) 5.11 66 (1,01) (1,36) (1,13) 1,53 17 LUM P51884 Lumican 6.16/38.4 4.45 53 (1,21) (1,48) 2,81 5,02 18 (6.17/36.7) 4.51 53 (1,24) (1,52) (2,38) 4,52 19 4.61 53 (1,29) (2,22) (2,65) 7,59 20 4.71 52 (1,01) (2,34) (2,25) 5,23 36 TPM1 P09493 T opomyosin a-1 chain (iso o m 8) 4.71/32.8 4.71 33 (1,06) (1,36) 2,10 3,03 37 iso o m 1 o 9 4.69/32.7 1 4.69 33 (1,09) (1,32) (2,01) 2,90 35 TPM2 P07951 T opomyosin bchain (iso o m 2) 4.63/32.9 4.62 35 (1,04) (1,54) 2,45 3,91 8 VPS35 Q96QK1 Vacuola p o ein so ing-associa ed p o ein 35 5.32/91.7 5.36 82 (1,02) (1,91) (1,60) 2,99 9 5.41 82 (1,02) (1,54) (1,38) 2,17 12 WARS P23381 T yp ophan- RNA ligase (iso o m 1 o 2) 5.83/53.2 1 (5.83/53.0 1 ) 5.83 55 (1,09) (1,45) (1,42) 2,29 Spo numbe s a e acco ding o Fig. S1. Desc ip ions and UniP o accession numbe s o he iden i ied spo s a e shown. Spo s belonging o he same p o ein a e clus e ed. P o ein ag- men s a e indica ed wi h as e isks. Speci ic p o ein iso o ms we e alida ed acco ding o spo posi ions in he gel and he co e ed p o ein sequence as well as iso o m-speci ic pep ides ob ained by mass spec ome y. Theo e ical spo iden i ica ions we e calcula ed acco ding o ull o ma u e (in pa en heses) p o ein sequences, whe eas de ec ed spo posi ions in he gel we e de e mined acco ding o selec ed ma ke spo s. I he iden i ied spo allowed o he p esence o se e al iso o ms, heo e ical alues we e indica ed o he mos common iso- o m 1 o A. Ra io shows he change in mean no malized spo olumes be ween elec i e (E) and spon aneous (S) e m bi h o be ween basal (Ba) and cho ionic (Ch) pla es o he pla- cen a. Changes wi h a oo-low a io (<1.5- old) o no signi icance (P>0.05) a e deno ed by pa en heses. Only he amoun o one p o ein (ACTB) changed signi ican ly in bo h compa isons (E e sus S and Ba e sus Ch). Fo mo e de ails abou p o ein le els, s a is ical signi icance and p o ein iden i ica ion, see Tables S1 and S2. ª2017 The Au ho s. Jou nal o Cellula and Molecula Medicine published by John Wiley & Sons L d and Founda ion o Cellula and Molecula Medicine. 973 J. Cell. Mol. Med. Vol 22, No 2, 2018 which we e iden i ied by mass spec ome y (MS) analyses o co e- spond wi h 28 p o eins (Tables 1 and S1). Ten p o eins changed based on he mode o deli e y (Fig 1; Table 1). Spon aneous deli e y compa ed o elec i e caesa ean bi h was associa ed wi h up- egula ion o ou p o eins: ACTB, b-2-mic o- globulin (B2M), ke a in ype II cy oskele al 8 (KRT8) and 19 (KRT19). Six p o eins we e down- egula ed in spon aneous deli e y compa ed o elec i e caesa ean bi h: a-2-mac oglobulin (A2M), CPPED1, cy o- ch ome b5 (CYB5A), haemoglobin subuni c-2 (HBG2), pe oxi e- doxin-2 (PRDX2) and plasminogen ac i a o inhibi o 2 (SERPINB2). We de ec ed s a is ically signi ican di e ences o A2M, CPPED1, CYB5A and HGB2 in bo h he cho ionic and he basal pla es. By con- as , ACTB and PRDX2 we e only signi ican ly changed in he basal pla e and B2M, KRT8, KRT19 and SERPINB2 we e only signi ican ly changed in he cho ionic pla e (Fig. 1). Di e en p o eomic p o iles in placen al cho ionic and basal pla es We ook samples o placen a om wo opposi e loca ions wi hin he same issue (cho ionic and basal pla es) o addi ionally de e mine whe he he e we e local a ia ions in p o ein le els independen o bi h pheno ype. To ally, 19 p o eins we e al e ed based on loca ion in he cho ionic and basal pla es o he same placen a a e spon aneous and elec i e caesa ean e m deli e y (n=6 pe g oup; Fig. 2; Table 1). We ound highe p o ein le els in he cho ionic pla e compa ed o he basal pla e o 11 p o eins: ACTB, annexin A3 (ANXA3), annexin A5 (ANXA5), se um amyloid P-componen (APCS), clus e in (CLU), ib inogen b(FGB), ib inogen c(FGG), gelsolin (GSN), lumican (LUM), opomyosin a-1 chain (TPM1) and opomyosin bchain (TPM2). The change was s a is ically signi ican a e spon aneous and elec i e caesa ean deli e y o CLU, FGB, FGG, LUM and TPM1 and 2. ACTB and ANXA5 we e signi ican ly changed a e elec i e cae- sa ean deli e y, and ANXA3, APCS and GSN we e signi ican ly chan- ged a e spon aneous deli e y. Eigh p o eins we e p esen a lowe le els in he cho ionic pla e compa ed o he basal pla e: elonga ion ac o 2 (EEF2), e i in ligh chain (FTL), 3-hyd oxyisobu y a e dehyd ogenase (HIBADH), he e ogeneous nuclea ibonucleop o ein K (HNRNPK), es adiol 17-b-dehyd ogenase 1 (HSD17B1), lamin-B1 (LMNB1), acuola p o ein so ing-associa ed p o ein 35 (VPS35) and yp ophan- RNA ligase (WARS). These changes we e only signi ican a e spon aneous deli e y (Fig. 2; Table 1). Ou indings indica e ha he e a e unc ionally di e en sub egions wi hin he placen a, Fig. 1 P o eomic changes be ween elec i e (E) and spon aneous (S) e m bi h in basal (Ba) and cho ionic (Ch) pla es o he placen a. Sec ions o he gel ep esen he exac spo posi ions and exp ession p o iles o he p o ein le el acco ding o he mean no malized spo olumes o he spo s shown in Fig. S1. Spo s co esponding o he same p o ein a e clus e ed wi h a e ical line. P o ein agmen s a e indica ed wi h as e isks. S a is i- cally signi ican changes (P<0.05) in exp ession p o iles a e shown by ho izon al b acke s. In he compa isons, six placen a samples we e included pe g oup. 974 ª2017 The Au ho s. Jou nal o Cellula and Molecula Medicine published by John Wiley & Sons L d and Founda ion o Cellula and Molecula Medicine. highligh ing he impo ance o ca e ully collec ing samples om he same loca ion. Deli e y ype and issue loca ion al e speci ic p o ein a ian s We de ec ed se e al p o eins in mul iple spo s and/o a unexpec ed posi ions in he 2D gel. This sugges s ha addi ional egula o y mechanisms such as al e na i e splicing, p o ein p ocessing, ag- men a ion and o he modi ica ions a e aking place. The e o e, he heo e ical spo posi ions calcula ed wi h Compu e pI/Mw and he de ec ed spo posi ions in he gel (Fig. S1; Table 1) we e compa ed. In addi ion, sequence co e age and spo -speci ic pep ides ob ained by MS (Table S2) we e also examined. We ound speci ic iso o ms associa ed wi h he deli e y ype (e.g. CPPED1 iso o m 1 and PRDX2 iso o m 1) and placen a loca ion (e.g. GSN iso o m 1, TPM1 iso o m 8 and TPM2 iso o m 2). Likewise, nume ous p o ein agmen s we e Fig. 2 P o eomic changes be ween basal (Ba) and cho ionic (Ch) pla es o he placen a. Samples we e collec ed a e elec i e (E) o spon aneous (S) e m bi h. Gel pa s ep esen he exac spo posi ions and exp ession p o iles o he p o ein le el acco ding o he mean no malized spo ol- umes o he spo s shown in Fig. S1. Fo spo s 17–20, he exac posi ions a e shown in gel pa s ep esen a i e o he cho ionic pla e. Spo s co e- sponding o he same p o ein a e clus e ed wi h a e ical line. P o ein agmen s a e indica ed wi h as e isks. S a is ically signi ican changes (P<0.05) in exp ession p o iles a e shown by ho izon al b acke s. In he compa isons, six placen a samples we e included pe g oup. ª2017 The Au ho s. Jou nal o Cellula and Molecula Medicine published by John Wiley & Sons L d and Founda ion o Cellula and Molecula Medicine. 975 J. Cell. Mol. Med. Vol 22, No 2, 2018 associa ed wi h deli e y ype (KRT8 and KRT19), placen a loca ion (ANXA5 and FGB) o bo h (ACTB). Howe e , all mul iple spo s o A2M, HBG2, KRT8, KRT19, as well as CLU, FGB, FGG, HSD17B1, LUM, TPM1 and VPS35 showed changes simila o hose o he co - esponding p o ein. Thus, he de ec ed p o ein signa u es a e likely a esul o al e ed gene exp ession a he han speci ic p ocessing. P o ein changes du ing spon aneous e m deli e y mos ly associa ed wi h immune sys em and s uc u al/cy oskele al o ganiza ion We u he clus e ed p o eins associa ed wi h he deli e y ype (Table 1) acco ding o hei gene on ology (GO) biological p ocesses and molecula unc ions (Table S3). The majo i y o he GO e ms we e linked o ei he he immune sys em o o s uc u al/cy oskele al o ganiza ion. Biological p ocesses comp ising a leas h ee changed p o eins included esponses o ino ganic subs ance (ACTB, A2M and PRDX2), o ganic subs ance (A2M, B2M, KRT19 and PRDX2) and wounding (A2M, PRDX2 and SERPINB2). S uc u al molecule ac i i y (ACTB, KRT8 and KRT19) ep esen ed he majo molecula unc ion. In addi ion, A2M and SERPINB2 we e in he same blood coagula ion pa hway. CPPED1 was no associa ed wi h any o he en iched GO e ms. As a phospha ase, CPPED1 is hough o be in ol ed in block- ing cell cycle p og ession, p omo ing apop osis and a ec ing glucose me abolism [19, 41]. CPPED1 polymo phism associa ed wi h ges a ional age Changes in p o ein le els a e expec ed du ing labou . To iden i y p o- eins ha a e impo an in ini ia ing labou a he han a ec ed by i , we in es iga ed whe he single-nucleo ide polymo phisms (SNPs) wi hin 20 kb o he genes encoding he a o emen ioned p o eins (ACTB,A2M,B2M,CPPED1,CYB5A,HBG2,KRT8,KRT19,PRDX2 and SERPINB2) we e associa ed wi h ges a ional age (GA) in in an s bo n a e m. We analysed 77 SNPs om 342 samples om in an s bo n be ween 38 and 42 weeks o ges a ion. O all he analysed SNPs, only wo in onic SNPs wi hin CPPED1 we e associa ed wi h GA a he Bon e oni-co ec ed signi icance le el (P<6.5 910 4 ): s11643593 (P=2.7 910 4 ) and s8048866 (P=3.3 910 4 ; Table S4). The mino allele o s11643593 (A) and majo allele o s8048866 (A) we e associa ed wi h highe GA (Table S5). This sug- ges s ha CPPED1 may be in ol ed in he ini ia ion o e m deli e y and p omp ed us o s udy i s exp ession in he placen a du ing labou in mo e de ail. CPPED1 exp essed in ophoblas s o ull- e m placen as To de e mine which cell ypes exp ess CPPED1 in he placen a, we immunos ained placen as om spon aneous and elec i e caesa ean e m deli e ies wi h an i-human CPPED1 an ibody. As shown in Figu e 3, a CPPED1 signal was ob ained om samples o bo h basal and cho ionic pla es o spon aneous and elec i e e m placen as. CPPED1 was de ec ed in he cy oplasm and cell memb anes o cy o ophoblas s and syncy io ophoblas s (Fig. 3). CPPED1 s aining was also posi i e in he epi helium o he amnio ic memb anes. In he decidua, ex a illous ophoblas s we e posi i e o CPPED1. The cap- illa y endo helia o he illi showed only ain and pa chy s aining, whe eas in acapilla y and in e illous leucocy es we e highly posi i e (Fig. 3). Lowe le els o CPPED1 mRNA in spon aneous e m deli e ies To in es iga e whe he he dec eased le els o CPPED1 p o ein ha we obse ed in spon aneous deli e y we e he esul o al e ed an- sc ip ion, we pe o med quan i a i e PCR (qPCR) analysis. Figu e 4 Fig. 3 Placen al localiza ion o CPPED1 in spon aneous and elec i e bi hs. Immunos aining o CPPED1 in ull- e m placen as om spon a- neous (n=6) o elec i e bi hs (n=6). Rep esen a i e placen as immunos ained wi h an i-human CPPED1 an ibodies. Immunos aining is indica ed by illed la ge a ows in cy o ophoblas s, un illed la ge a ows in syncy io ophoblas s, illed small a ows in endo helium, un illed a owheads in leucocy es and illed a owheads in decidual o- phoblas s. O iginal magni ica ion is 209in all igu es. Con ol ep e- sen s iso ype con ols o immunos aining. Scale ba , 100 lm. 976 ª2017 The Au ho s. Jou nal o Cellula and Molecula Medicine published by John Wiley & Sons L d and Founda ion o Cellula and Molecula Medicine.