Insights into the multifactorial nature of preterm birth: proteomic profiling of the maternal serum glycoproteome and maternal serum peptidome among women in preterm labor

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nsights into the multifactorial nature of preterm birth:roteomic profiling of the maternal serum glycoproteome andaternal serum peptidome among women in preterm labor

eonardo Pereira, MD, MCR; Ashok P. Reddy, PhD; Amanda L. Alexander, BS; Xinfang Lu, MS;odi A. Lapidus, PhD; Michael G. Gravett, MD; Srinivasa R. Nagalla, MD

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BJECTIVE: The purpose of this study was to identify peptide classifi-rs that predict spontaneous preterm birth (SPTB) among women inreterm labor (PTL) and to demonstrate specific protein pathways thatre activated in PTL.

TUDY DESIGN: Serum from 110 women with PTL between 20 weeksnd 33 weeks 6 days of gestation was subjected to glycoprotein purifi-ation, matrix-assisted laser desorption ionization time-of-flight masspectrometry peptide profiling, 2-dimensional liquid chromatographyandem mass spectrometry, and pathway analysis. Women were di-ided into 2 groups: delivery at �34 weeks’ gestation (SPTB group) andelivery at �34 weeks’ gestation (PTL group).

ESULTS: Twenty-three peptide masses were identified that dis-

lycoproteome and maternal serum peptidome among women in preterm labor. Am

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002-9378/$36.00 • © 2010 Mosby, Inc. All rights reserved. • doi: 10.1016

resent differentially between PTL and SPTB; 48 of 52 proteinsere classified into 1 of 4 functional pathways that were involvedith PTL: (1) complement/coagulation cascade, (2) inflammation/

mmune response, (3) fetal-placental development, and (4) extra-ellular matrix proteins.

ONCLUSION: Among women in PTL, proteomic analysis of serumeptides and glycoproteins classifies women who will deliver pretermnd identifies specific protein pathways at work among individuals withidiopathic” PTL.

ey words: preterm birth, preterm labor, proteomics, peptide,
riminated PTL from SPTB in 97% of cases. Fifty-two proteins were glycoprotein
ite this article as: Pereira L, Reddy AP, Alexander AL, et al. Insights into the multifactorial nature of preterm birth: proteomic profiling of the maternal serum
J Obstet Gynecol 2010;202:555.e1-10.
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he treatment of preterm labor(PTL) remains one of the greatest

linical challenges confronting obstetri-ians today; preterm birth (PTB) com-licates 12.7% of US deliveries and is re-

rom the Division of Maternal-Fetal MedicinDr Pereira and Ms Alexander), and Divisionnd Preventive Medicine (Dr Lapidus), Oregoiabetomics, LLC, Portland, OR (Drs Reddy

Dr Lapidus and Ms Lu); Division of Maternaynecology, University of Washington Schoo

resented in abstract form at the 28th Annual Mallas, TX, Jan. 28-Feb. 2, 2008.

eceived July 2, 2009; revised Dec. 24, 2009; a

eprints: Leonardo Pereira, MD, Division of Matd., Mailcode L458, Oregon Health & Science U

upported by the Oregon Clinical and Translatiorom the National Center for Research Resourceealth, and National Institutes of Health Roadm

roteoGenix, Inc, provided the laboratory and eregon Health & Science University and Drs Gra

nancial interest in ProteoGenix, Inc, a companyf this research and technology. This potential canagement plan has been approved by the Orrogram Oversight Council and by the Universit

ponsible for 80% of perinatal deaths.1,2

hanges in clinical practice, includinghe use of progesterone supplementa-ion, nutrient supplementation, and cer-ical cerclage have all been implemented

epartment of Obstetrics and Gynecologyiostatistics, Department of Public Healthealth & Science University, Portland, OR;Nagalla); Proteogenix, Inc, Beaverton, ORtal Medicine, Department of Obstetrics andMedicine, Seattle, WA (Dr Gravett).

ing of the Society for Maternal-Fetal Medicine,

pted Feb. 17, 2010.

l Fetal Medicine, 3181 SW Sam Jackson Parkersity, Portland, OR 97239. [email protected].

Research Institute, Grant no. UL1 RR024140component of the National Institutes of

or Medical Research.

ment that made this research possible.t, Nagalla, and Lapidus have a significantt may have a commercial interest in the resultsict of interest has been reviewed and an Health & Science University IntegrityWashington Office of Technology Transfer.

t/j.ajog.2010.02.048

JUNE 2010 Americ

n specific populations as part of aider effort to prevent PTB.3-7 Despiteedical advances, the incidence of

TB continues to increase both nation-lly1 and globally.8,9

Clinically, our inabilities to predictTB and to treat PTL are major obstacles

o reducing prematurity. Multiple strat-gies for the prediction of PTB have beendvocated;10-14 however, none of theseave the predictive accuracy necessary toemonstrate clinical benefits to eitherhe mother or fetus. Likewise, mostreatment trials have failed to demon-trate any difference in the duration ofregnancy with the use of tocolyticgents.15,16 The origin of these deficien-ies lies in our inability to understand theultifactorial nature of PTB and imple-ent a cause-based treatment paradigm

nto clinical practice. Proteomic profil-ng, because of its ability to analyze mul-iple protein signatures concurrently,ses novel methods that ideally areuited for the study of complex condi-

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5

Current proteomics technologies canrobe proteome-wide expression changesetween different samples in a high-hroughput fashion. Proteins that are dif-erentially present in biologic fluids be-ween groups can then be used to developensitive, accurate, and rapid diagnosticssays.18-21

Our objectives in this study were toharacterize the maternal serum glycop-oteome and maternal serum peptidomen 2 groups of women: those with PTLho delivered at �34 0/7 weeks’ gesta-

ion (spontaneous PTB [SPTB] group)nd those with PTL who delivered at34 0/7 weeks’ gestation (PTL group).haracterization of the maternal serumlycoproteome was performed to iden-ify key peptide classifiers that woulderve as the foundation for a rapid assayo distinguish SPTB from PTL (ie, therediction of SPTB among women inTL). Quantification of differential pep-

ide expression and functional categori-ation of identified proteins was per-ormed to demonstrate specific proteinathways that are activated amongomen with spontaneous PTL in a

ause-specific manner.

ATERIALS AND METHODSubjectsprospective cohort of 309 women in

TL with suspected intraamniotic infec-ion at the University of Washingtonrom 1991-1997 were identified and un-erwent cervical-vaginal fluid collection.rom this larger cohort, 138 women un-erwent serum sampling; of these, 28omen had evidence of intraamniotic

nfection, which left the subset of 110omen with PTL, but no evidence of in-

raamniotic infection, that was used inhis analysis. After informed consent wasiven, we used a standard collectionechnique to obtain serum samples fromll the women. All women had spontane-us PTL and intact amniotic mem-ranes. Serum samples were processedy a standard protocol, centrifuged, fro-en, and stored at – 80°C until analysis.he study was approved by the institu-

ional review boards at the University ofashington where the samples were col-

ected and at Oregon Health & Science g

55.e2 American Journal of Obstetrics & Gynecolo

niversity where sample analysis waserformed.PTL was defined as the presence of reg-

lar uterine contractions accompaniedy cervical dilation or effacement be-ween 20 weeks and 33 weeks 6 days’ ges-ation. Women with PTL who deliveredt �33 weeks 6 days’ gestation were con-idered to have had SPTB; women whoelivered after 33 weeks 6 days’ gestationere considered as having had PTL withelivery �34 weeks’ gestation.Race/ethnic distinctions were self-de-

cribed by study participants. Race wasssessed to minimize possible confound-ng between race and protein expressionetween groups.Comprehensive proteomic profiles

nd total serum glycoprotein profilesere derived in 110 women by total gly-

oprotein purification and digestion andatrix-assisted laser desorption ioniza-

ion time-of-flight mass spectrometryMALDI-TOF-MS) peptide profiling asescribed later. In a subset of 10 womenn � 5 with SPTB; n � 5 with PTL andelivery �34 weeks’ gestation) gesta-ional-age matched serum samples wereooled, depleted of high abundance pro-eins, and subjected to 2-dimensionaliquid chromatography tandem masspectrometry (2D-LC-MS/MS).

ndividual sample analysisglycoprotein purification, digestion,nd MALDI-TOF-MS peptide profiling)otal glycoprotein purification, diges-

ion, and MALDI-TOF-MS peptide pro-ling were was performed with 750 �g of

otal serum protein from each sampleith the use of previously described

echniques.18,19 Discrimination of theesultant serum glycoprotein profiles be-ween SPTB and PTL was performedith ClinProTools (CPT) pattern-rec-gnition software (Bruker Daltronicsnc, Billerica, MA). The recognition ca-ability and cross-validation of the CPTlgorithm to discriminate small changesn peptide expression in a complex digestere determined to be 100% through a

eries of proof-of-principle experiments.After these preliminary experiments,

erum samples were enriched for glyco-roteins and subjected to enzymatic di-
estion. The sensitive (approximately w
gy JUNE 2010

.1 �g per spot) and the high-through-ut nature of MALDI-TOF-MS was usedo obtain peptide profiles on the result-ng glycopeptides. Spectral data fromuadruplicate analyses for each digestedample were imported into CPT pattern-ecognition software, and individualeptide peak statistics were generated ateveral signal-to-noise levels (2, 3, 4, and). The averaged peptide profiles fromoth subject populations (SPTB andTL) were then determined. Identifica-

ion of differentially expressed proteinsas performed by separate quantitative

ime-of-flight analysis.

ooled sample analysis2D-LC MS/MS)otal proteome characterization of in-ividual serum samples with 2D-LCS/MS is a time-consuming process.ence, 2 pooled samples, 1 for each con-

ition, were created with a small cohortf serum samples from women with PTLn � 5) and SPTB (n � 5). Serum wasmmunodepleted of the 12 most abun-ant proteins with the use of a Genwaymmunoglobin Y-12 column (GenWayiotech Inc, San Diego, CA). Proteinoncentration was determined with a de-ergent compatible protein assay kitBio-Rad, Hercules, CA). After proteinssay, samples were digested with tryp-in, and the digested peptides were sepa-ated by strong cation exchange chroma-ography, as previously published.18,19

C-MS/MS analysis was performed onhe cation exchange fractions using a ly-ine tyrosylquinone (Thermo Fisher Sci-ntific Inc, Waltham, MA) connected ton Agilent 1100 LC (Agilent Technolo-ies, Inc, Santa Clara, CA).

rotein identificationass spectra were searched against a

omposite protein database that con-ained forward and reversed entries ofwiss-Prot database (version 51.3; Swissnstitute of Bioinformatics, Geneva, Swit-erland). Peptide identifications with ateast a probability of 0.8 and without anynknown and unexpected modificationsre considered as likely to be present inhe sample. Protein identifications witht least 2 unique peptide identifications
ere considered to be present in a sam-

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www.AJOG.org Obstetrics Research

le. Resulting protein identifications andheir spectral counts were considered forabel-free quantitation.

eptide quantitationhe total number of tandem mass spec-

ra that were matched to a protein (spec-ral counting) is considered a label-free,ensitive, and semiquantitative measureor the estimation of its abundance inomplex mixtures.22,23 The differenceetween spectral counts of a protein haseen used to quantitate its relative expres-ion changes between complex sam-les.18-20 To reduce the false-positive rate,rotein entries were further curated beforeeing subjected to label-free quantitation.urated proteins were then subjected toair-wise comparisons.

tatistical methodsll proteins and their corresponding

pectral counts were subjected to label-ree quantitation. Quantitative analysisas performed through pair-wise com-arisons between PTL and SPTB samplesith either a 2 � 2 �2 or Fisher’s exact

est. Normalization of spectral counts toccount for experimental variability wasuilt into the pair-wise comparisons.he method was automated with an SAS

oftware program (version 9.1; SAS In-titute Inc, Cary, NC), and all proteinsere independently tested. A proteinas considered to be significantly differ-

ntially expressed between the samples ifhe hypothesis has a probability value of

.05 in either the �2 or Fisher’s exactest. The fold expression change of dif-erentially expressed proteins was quan-ified with previously established meth-ds.22,24 To reduce false positives from

abel-free quantitation, only proteinsith at least 3 unique peptide identifica-

ions in 1 of the samples and showing anxpression change of at least �1.5-foldetween the samples were considered to be

ikely candidates. Analyses of MALDI-OF-MS glycoproteome spectral dataere performed with ClinProTools (ver-

ion 2.0.319; Bruker Daltonics Inc.,illerica, MA) as we have previouslyescribed.18

athway analysishe differentially expressed proteins
ere uploaded into Ingenuity Pathway a
nalysis (2007 Ingenuity Systems, http://ww.ingenuity.com) to categorize theroteins into related functional path-ays and networks based on the associa-

ion between the input proteins and pro-eins that were present in the Ingenuityathway analysis database. The proteins

n the networks that were not identifiedn this study were removed from the net-orks for better visualization. The net-orks are displayed graphically as nodes

individual proteins) and edges (the bi-logic relationships between the nodes).

ESULTSrom the existing cohort, serum samplesrom 110 women between 20 weeks and3 weeks 6 days gestation with PTL

TABLE 1Maternal and pregnancy characterwomen in spontaneous preterm lab

Variable

Mean maternal age, y...................................................................................................................

Maternal race, n (%)..........................................................................................................

White..........................................................................................................

African American..........................................................................................................

Other...................................................................................................................

Plurality of current pregnancy, n (%)..........................................................................................................

Singleton..........................................................................................................

Twins...................................................................................................................

Previous delivery �34 wk, n (%)..........................................................................................................

No, parous..........................................................................................................

Yes, parous..........................................................................................................

Nulliparous...................................................................................................................

Previous pregnancy complications, n (%)...................................................................................................................

Diabetes mellitus at admission, n (%)...................................................................................................................

Uterine anomaly, n (%)...................................................................................................................

Hypertension at admission, n (%)...................................................................................................................

Mean gestational age, wka

..........................................................................................................

At enrollment..........................................................................................................

At delivery...................................................................................................................

Mean days between enrollment anddelivery...................................................................................................................

Mean cervical dilation at enrollment, cma

...................................................................................................................a P � .001, comparison of groups by nonparametric analysis o

Pereira. Insights into the multifactorial nature of preterm

nd intact membranes were identified. d

JUNE 2010 Americ

ooled analysis by 2D-LC-MS/MS waserformed on a subset of 10 samples (5PTB, 5 PTL with delivery at �34 weeksf gestation) matched for gestational agemean, 30.3 � 1.8 [SD] weeks; range,8 –33 weeks). Comprehensive pro-eomic profiles and total serum glycop-otein profiles were derived by MALDI-OF-MS peptide profiling for the

emaining 110 women (48 SPTB, 62 PTLith delivery at �34 weeks’ gestation).here were no differences between thePTB and PTL groups in terms of mater-al age, race, underlying medical condi-

ions, and pregnancy history. As ex-ected, women in the SPTB group had aignificantly earlier gestational age at de-ivery and shorter enrollment to delivery

cs of 110

ontaneousterm birth3 wk 6 d (n � 48)

Preterm laborwith delivery at> 34 wk (n � 62)

.5 25.5..................................................................................................................

..................................................................................................................

(62) 35 (56)..................................................................................................................

(15) 12 (19)..................................................................................................................

(23) 15 (24)..................................................................................................................

..................................................................................................................

(81) 60 (97)..................................................................................................................

(19) 2 (3)..................................................................................................................

..................................................................................................................

(31) 26 (42)..................................................................................................................

(27) 11 (18)..................................................................................................................

(42) 25 (40)..................................................................................................................

(31) 13 (21)..................................................................................................................

(2) 4 (6)..................................................................................................................

(4) 3 (5)..................................................................................................................

(6) 0..................................................................................................................

..................................................................................................................

.4 31.2..................................................................................................................

.6 37..................................................................................................................

.6 40.4

..................................................................................................................

.1 3.3..................................................................................................................

nce (continuous variables) or �2 test (categoric variables).

. Am J Obstet Gynecol 2010.

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PTB were evaluated slightly earlier inestation and with more advanced cervi-al dilation (Table 1). Overall, 24.8% ofhe women received antenatal steroids,

ore commonly in the SPTB group12.9% PTL after 40.4% SPTB; P �001); and 95.4% of the women receivedocolytic therapy (93.4% PTL after7.9% SPTB; P � .385).With the use of the total glycopro-

eome digests, the average discrimina-ion power over 16 computed modelsas 97.3% for PTL and 97.0% for SPTB.wenty-three peptide masses that aresed commonly by the CPT classifier toifferentiate between SPTB and PTL arehown in Table 2. Fifty-seven percent of

TABLE 2Glycoproteome digests differentiatPeptide mass P valueb

980.48 � .0001...................................................................................................................

1529.7 � .0001...................................................................................................................

1833.87 � .0001...................................................................................................................

1650.77 .0001...................................................................................................................

1195.53 .0007...................................................................................................................

1129.62 .0012...................................................................................................................

1283.54 .0020...................................................................................................................

1723.92 .0023...................................................................................................................

1213.59 .0023...................................................................................................................

1878.86 .0042...................................................................................................................

1741.78 .0052...................................................................................................................

3282.38 .0140...................................................................................................................

1647.75 .0141...................................................................................................................

1478.71 .0179...................................................................................................................

2833.46 .0203...................................................................................................................

1855.94 .0307...................................................................................................................

1895.88 .0330...................................................................................................................

1743.75 .0360...................................................................................................................

1755.85 .0504

...................................................................................................................

2239.98 .0516...................................................................................................................

2493.2 .0739...................................................................................................................

1323.63 .0872...................................................................................................................

1752.96 .1300...................................................................................................................a Peptide fingerprints determined by matrix-assisted laser deso

and preterm labor.


he CPT peptide classifiers were mapped f


o 8 proteins. The false discovery rate ofhis technique was estimated at 3%. Theveraged MALDI-TOF-MS glycopeptiderofiles from SPTB and PTL samples arehown in Figure 1, A. Magnified exam-les of 2 differentially abundant peptidest 980 dalton (haptoglobin) and 1530alton (serotransferrin) are shown inigure 1, B.A total of 369 proteins with at least 2

nique peptide (probability � .8) iden-ifications were found in both samples.dditional constraints were incorpo-

ated, as described earlier, to reducealse-positive results. A total of 52 candi-ate proteins that satisfied the aforemen-ioned constraints were identified as dif-

preterm labor from spontaneous preeptide sequence Swiss-Prot

GYVSGWGR P00738.........................................................................................................................

PVEEYANCHLAR P02787.........................................................................................................................

FSNGADLSGVTEEAPLK P01009.........................................................................................................................

.........................................................................................................................

SGFQMNQLR P02787.........................................................................................................................

LWWLDLK P02790.........................................................................................................................

GYYGYTGAFR P02787.........................................................................................................................

KVEPLRAELQEGAR P02647.........................................................................................................................

.........................................................................................................................

YVLPSFEVIVEPTEK P01024.........................................................................................................................

.........................................................................................................................

.........................................................................................................................

.........................................................................................................................

YLGYEYVTAIR P02787.........................................................................................................................

.........................................................................................................................

NKPFVFLMIEQNTK P01009.........................................................................................................................

.........................................................................................................................

.........................................................................................................................

DPVTLNVLYGPDLPR P11464

.........................................................................................................................

.........................................................................................................................

.........................................................................................................................

SAHGFLKVPPR P02787.........................................................................................................................

VGGQEHFAHLLILR P02763.........................................................................................................................

ionization time-of-flight mass spectrometry; b P value correspond


erentially present between PTL and l

gy JUNE 2010

PTB samples; 38 proteins had probabil-ty values of � .01, and the remaining 14roteins had probability values between

01 and .05. The total number of reversedrotein sequence entries in the final pro-ein list was used to compute a false dis-overy rate of 11% for protein identifica-ions. Most of the identified proteins48/52) were classified broadly into 1 of 4istinct functional classes: (1) comple-ent and coagulation cascade, (2) in-

ammation and immune response, (3)etal-placental development, and (4) ex-racellular matrix (actin cytoskeleton orell movement and assembly proteins;able 3). Four proteins were classified asiscellaneous, either because they be-

rm birtha

ession Protein description

Haptoglobin..................................................................................................................

Serotransferrin..................................................................................................................

�-1-Antitrypsin..................................................................................................................

..................................................................................................................


Hemopexin..................................................................................................................


Apolipoprotein A1..................................................................................................................

..................................................................................................................

Complement C3..................................................................................................................

..................................................................................................................

..................................................................................................................

..................................................................................................................


..................................................................................................................

�-1-Antitrypsin..................................................................................................................

..................................................................................................................

..................................................................................................................

Pregnancy-specific �-1glycoprotein 1

..................................................................................................................

..................................................................................................................

..................................................................................................................


�-1-Acid glycoprotein 1..................................................................................................................

ifferential abundance between spontaneous preterm birth

ing teP acc

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n unknown function. Examples ofathway analysis for protein networks 1complement and coagulation cascade)nd 4 (actin cytoskeleton or cell move-ent and assembly) are displayed in

igure 2. Six of the matched proteinshaptoglobin, serotransferrin, apolipopro-ein A1, alpha-1-acid glycoprotein 1, al-ha-1-antitrypsin, and complement C3)hat were identified in the pooled 2D-LCnalysis corresponded to serum proteinshat were also identified as classifiers ofPTB by MALDI-TOF-MS profiling (Ta-le 2).

OMMENTe used 2 comprehensive proteomic

echniques to characterize maternal se-um proteins among a cohort of womenith PTL and SPTB. Primarily, MALDI-OF-MS peptide profiling of total glyco-roteomic expression resulted in identi-cation of 23 key peptide classifiers and 8roteins (6 of which were also identified

n pooled 2D-LC-MS/MS analysis). Incor-orating these maternal serum peptides,he CPT genetic algorithm was able to clas-ify correctly PTL and SPTB in 97% per-ent of cases, even among a complex back-round of multiple peptides.

Currently, our inability to predict PTBs a major obstacle to delivering optimalare to women with PTL. The effective-ess of tocolytic therapy to allow for an-

enatal steroid administration and ma-ernal transfers has been limited severelyy our inability to accurately predictTB. This has led to numerous investi-ative trials aimed at improving our abil-ty to predict PTB. There are 2 extensivetudies that have evaluation maternal se-um markers for the prediction of SPTB:he Preterm Prediction Study from Ma-

ernal-Fetal Medicine Networks of Na-ional Institutes of Child Health and Hu-

an Development25 and Quad Screenrom the FASTER trial consortium.14

oth studies together assessed the pre-ictive accuracy of fetal fibronectin,-fetoprotein, serum alkaline phospho-

ase, serum granulocyte colony–stimu-ating factor, serum relaxin, serum fer-itin, serum c-reactive protein, humanhorionic gonadotropin, and inhibin A.
ll of the tested maternal serum markers, r
hen used either independently or com-ined, showed low sensitivity and/or spec-

ficity for the prediction of PTB.14,25

Various alternative approaches thatave been advocated for the predictionf SPTB have included a variety of diag-ostic assays that were based on genet-

cs,10,26 environmental stress,11 patient

FIGURE 1Serum total glycoproteomic averag

, Total glycoproteomic peptide profiles of digest48) and preterm labor (PTL; n � 62) cohorts

xamples of 2 of the differentially present peptideirth profiles: 1 at 980 dalton matched to haptoerrin.ereira. Insights into the multifactorial nature of preterm birt

isk factors,27 salivary markers,28 cervi- p

JUNE 2010 Americ

al-vaginal proteins,12,29,30 and trans-aginal cervical length measurements.31

owever, all of these assays, which areimilar to the published maternal serumtudies, have a low sensitivity for the pre-iction of PTB. These findings under-core the need to identify highly sensitivend specific biomarkers for the accurate

peptide profiles

serum from spontaneous preterm birth (SPTB; nt were averaged according to the condition. B,etween preterm labor and spontaneous pretermin and 1 at 1530 dalton matched to serotrans-

m J Obstet Gynecol 2010.

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5

TABLE 3Serum proteins associated with preterm birth organized by functional pathwaya

VariableSwiss-Protaccession Protein name

Spectral count

Pretermlabor

Spontaneouspreterm birthb

Foldchangec P value

Complement andcoagulation cascade

P08709 Coagulation factor VII 1 11 5.40 .0070............................................................................................................................................................................................................................................................................................

P02787 Serotransferrin 175 651 3.70 � .0001............................................................................................................................................................................................................................................................................................

P05160 Coagulation factor XIII, B chain 12 30 2.30 .0080............................................................................................................................................................................................................................................................................................

P01009 Alpha-1-antitrypsin 939 1808 2.00 � .0001............................................................................................................................................................................................................................................................................................

P05155 Plasma protease C1 inhibitor 62 101 1.60 .0030............................................................................................................................................................................................................................................................................................

P01024 Complement component 3 6093 6104 1.21 � .0001............................................................................................................................................................................................................................................................................................

P01023 Alpha-2-macroglobulin 4440 3588 –1.05 .0490............................................................................................................................................................................................................................................................................................

P00747 Plasminogen 1870 1458 –1.08 .0230............................................................................................................................................................................................................................................................................................

P00751 Complement factor B 1870 1366 –1.16 � .0001............................................................................................................................................................................................................................................................................................

P13671 Complement component 6 435 317 –1.16 .0470............................................................................................................................................................................................................................................................................................


P01031 Complement component 5 1004 615 –1.38 � .0001............................................................................................................................................................................................................................................................................................


P05546 Heparin cofactor 2 561 363 –1.50 � .0001............................................................................................................................................................................................................................................................................................

P00748 Coagulation factor XII 441 234 –1.59 � .0001............................................................................................................................................................................................................................................................................................

P04196 Histidine-rich glycoprotein 183 113 –1.60 � .0001................................................................................................................................................................................................................................................................................................................................................................................

Inflammation and immuneresponse modulators

P06702 Protein S100-A9 2 17 5.60 � .0001............................................................................................................................................................................................................................................................................................

P02647 Apolipoprotein A-I 172 479 2.80 � .0001............................................................................................................................................................................................................................................................................................

P00738d Haptoglobin; Haptoglobin relatedprotein

172 371 2.20 � .0001

............................................................................................................................................................................................................................................................................................

P02765 Alpha-2-HS-glycoprotein 1214 839 –1.22 � .0001............................................................................................................................................................................................................................................................................................

P01019 Angiotensinogen 1174 697 –1.43 � .0001............................................................................................................................................................................................................................................................................................

P04278 Sex hormone-binding globulin 332 209 –1.60 � .0001............................................................................................................................................................................................................................................................................................

O43184 ADAM 12 79 44 –1.80 .0030............................................................................................................................................................................................................................................................................................

P18428 Lipopolysaccharide-binding protein 33 13 –2.40 .0060............................................................................................................................................................................................................................................................................................

P06733 Alpha-enolase 16 1 –6.50 � .0001................................................................................................................................................................................................................................................................................................................................................................................

Fetal-placental developmentproteins

P01344 Insulin-like growth factor II 3 12 3.10 .0270............................................................................................................................................................................................................................................................................................

P08185 Corticosteroid-binding globulin 11 36 3.00 � .0001............................................................................................................................................................................................................................................................................................

P02656 Apolipoprotein C-III 43 101 2.30 � .0001............................................................................................................................................................................................................................................................................................

P02652 Apolipoprotein A-II 37 82 2.20 � .0001............................................................................................................................................................................................................................................................................................

P20742 Pregnancy zone protein 567 861 1.50 � .0001............................................................................................................................................................................................................................................................................................

P11464 Pregnancy-specific beta-1-glycoprotein 1

435 316 –1.16 .0430

............................................................................................................................................................................................................................................................................................

Q13787 Apolipoprotein B-100 4142 2875 –1.23 � .0001............................................................................................................................................................................................................................................................................................

P01243 Chorionic somatomammotropinhormone

65 36 –1.80 .0060

............................................................................................................................................................................................................................................................................................

Q13219 Pregnancy associated plasmaprotein A

478 209 –2.30 � .0001

................................................................................................................................................................................................................................................................................................................................................................................

Pereira. Insights into the multifactorial nature of preterm birth. Am J Obstet Gynecol 2010. (continued )

55.e6 American Journal of Obstetrics & Gynecology JUNE 2010

Tcwipistctmsoct

c

hepLrtcfatfatsotm

spcwrteiaAtwogati

birth


Of course, incorporation of MALDI-OF-MS and cluster analysis into clini-al practice at this time is impracticalith regards to cost and time. To be clin-

cally useful, a serum-based test for therediction of PTB should be rapid and

nexpensive. Identification of a restrictedet of peptides (perhaps 3-5) could fulfillhis purpose. To accomplish this goal, weurrently are enrolling a second prospec-ive cohort of women in PTL to deter-

ine the reproducibility of peptide clas-ifiers for prediction of PTB. Validationf a set of peptides in this setting wouldonstitute the basis for a serum-basedest for the prediction of SPTB.

In addition to glycoproteome-based

TABLE 3Serum proteins associated with pre

VariableSwiss-Protaccession

Extracellular matrix proteins:actin cytoskeleton, cellmovement, cell assembly

P02743...............................

Q12805

...............................

P02766...............................

P41222...............................

Q81V18...............................

P06396...............................

P43652...............................

Q14520...............................

P60709...............................

Q96PD5

...............................

P02753...............................

P21333...............................

P24821...............................

O00533

...................................................................................................................

Proteins of miscellaneousclass or unknown function

Q96RL7

...............................

Q96KN2...............................

P80108

...............................

P18669...................................................................................................................a Proteins identified by 2-dimensional liquid chromatography

ordered according to their decrease in the fold changed betwshown as a single entry.


luster analysis, we performed compre- p

ensive characterization of protein-widexpression changes in maternal serumeptides between PTL and SPTB by 2D-C, on a pooled sample of women. Ouresults demonstrate that most differen-ially expressed serum proteins (48/52)ould be characterized into 1 of 4 majorunctional pathways: (1) complementnd coagulation cascade, (2) inflamma-ion and immune response proteins, (3)etal-placental development proteins,nd (4) extracellular matrix proteins (ac-in cytoskeleton, cell movement and as-embly). The major clinical implicationf this finding is that each of these func-ional pathways corresponds to a sche-

atic of the accepted causes of SPTB as

rm birth organized by functional path

Protein name

Spectra

Pretermlabor

Serum amyloid-P component 7.........................................................................................................................

EGF-containing fibulin-likeextracellular matrix

5

.........................................................................................................................

Transthyretin 44.........................................................................................................................

Prostaglandin-H2 D-isomerase 5.........................................................................................................................

Fibronectin 300.........................................................................................................................

Gelsolin 579.........................................................................................................................

Afamin 514.........................................................................................................................

Hyaluronan-binding protein 2 143.........................................................................................................................

Beta actin 142.........................................................................................................................

N-acetylmuramoyl-L-alanineamidase

194

.........................................................................................................................

Plasma retinol-binding protein 141.........................................................................................................................

Filamin-A 8.........................................................................................................................

Tenascin C 6.........................................................................................................................

Cell adhesion molecule L1-likeprotein

7

.........................................................................................................................

Vacuolar protein sorting-associatedprotein 13A

0

.........................................................................................................................

Beta-Ala-His dipeptidase 52.........................................................................................................................

Phosphatidylinositol-glycan specificphospholipase D

112

.........................................................................................................................

Phosphoglycerate mutase 1 6.........................................................................................................................

m mass spectrometry; b Preterm birth spectral counts are scalepreterm labor and spontaneous preterm birth samples; d Protein


ostulated by Lockwood and Kuczyn- c

JUNE 2010 Americ

ki32 and Wang et al.33 Differential ex-ression of complement and coagulationascade proteins could occur in cases inhich PTL is due to decidual hemor-

hage or abruptio placentae. Inflamma-ory and immune response activation isxpected in cases in which PTL is due tonfections either systemically or region-lly or in the intrauterine environment.lterations in protein expression along

he fetal-placental developmental path-ay can be ascribed to PTL becausef placental dysfunction (intrauterinerowth restriction) or cases of prematurectivation of the fetal hypothalamic-pi-uitary-adrenal axis. Finally, alterationsn extracellular matrix proteins, specifi-

aya(continued)

ount

Spontaneouspreterm birthb

Foldchangec P value

36 4.50 � .0001..................................................................................................................

17 2.90 .0150

..................................................................................................................

114 2.60 � .0001..................................................................................................................

14 2.50 .0040..................................................................................................................

479 1.90 � .0001..................................................................................................................

402 –1.22 .0030..................................................................................................................

340 –1.28 � .0001..................................................................................................................

92 –1.31 .0420..................................................................................................................

89 –1.34 .0270..................................................................................................................

110 –1.70 � .0001

..................................................................................................................

71 –2.00 � .0001..................................................................................................................

1 –3.50 .0450..................................................................................................................

0 –4.89 .0350..................................................................................................................

0 –5.60 .0180

..................................................................................................................

8 7.80 .0040

..................................................................................................................

84 1.60 .0080..................................................................................................................

75 –1.50 .0100

..................................................................................................................

0 –4.90 .0350..................................................................................................................

h respect to preterm labor spectral counts; c Proteins aret share significant sequence homology are collapsed and

te wl c

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

......... .........

tande d witeen s tha

ally involving actin cytoskeleton, could


bpncfas

Lgiccw

ug(it

soPccmmcbcocstsmpavPmm

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5

e due to primary cervical disease andremature cervical dilation that may sig-ify cervical insufficiency. Of signifi-ance in our study was a lack of any dif-erential expression of proteins that weressociated with uterine stretch or disten-

FIGURE 2Pathway analysis for networks 1 adifferentially expressed proteins

Network 1 (maternal serum): Compleme

Network 4 (maternal serum): Actin Cyto

roteins that demonstrate increased expressionerm labor, appear in red; proteins with decreaseflects the magnitude of fold change in expresstatistically significant levels of differential expreeproduced, with permission, from Ingenuity Pathway Systems, In

ereira. Insights into the multifactorial nature of preterm birt

ion. However, because the pooled 2D- b


C-MS/MS analysis was limited to sin-leton gestations, these results maymply that the mechanism for PTB, be-ause of mechanical uterine stretch, oc-urs primarily in multiple gestations,hich is a conclusion that is supported

4 highlighting

and Coagulation Cascade

eleton and Cell Movement or Assembly

pontaneous preterm birth, compared with pre-expression appear in green. The color intensityProteins that appear without color did not shown in this experiment.edwood City, CA.

m J Obstet Gynecol 2010.

y studies that have demonstrated a fail- p

gy JUNE 2010

re in the reduction of SPTB in multipleestations with cerclage or progesterone2 therapies that demonstrate reductionn PTB in certain populations of single-on pregnancies).3,34,35

These findings highlight anotherhortcoming of clinical practice that isur assumption that PTL is the cause ofTB. Treatment trials of PTL have fo-used almost exclusively on various to-olytic agents, which (despite differentechanisms of action) are aimed pri-arily at achieving uterine quies-

ence.15,16,36,37 In our search for a “silverullet” to arrest contractions, we typi-ally ignore both the multifactorial causef PTL and the realization that uterineontractions are neither necessary norufficient to result in PTB. The concepthat PTL is merely a symptom, which isupported by our findings, is common to

any different disease processes andathways has been postulated by severaluthors32,33,38,39 and validated by the de-elopment of experimental models ofTL, such as the infection-mediated PTLodel that was developed in the rhesusacaque.21

One of the shortcomings of this anal-sis includes the possibility that the pro-eins that were identified in this study asifferentially expressed may not be re-roducible. To minimize the likelihoodf false-positive results among our se-um classifiers, we used conservative sta-istical methods to adjust for multipleomparisons and estimate the false dis-overy rate in this analysis to be 3%. Fur-hermore, we plan to validate these find-ngs in a separate cohort of women toetermine the reproducibility of oureptide classifiers.The assignment of a particular protein

n to 1 functional class is also problem-tic. Many proteins that are listed in Ta-le 2 have multiple functions and argu-bly could be classified differently. Forxample, heparin cofactor 2 is a potenthrombin inhibitor that is not only acti-ated in the complement cascade butlso demonstrates chemotactic activityor both monocytes and neutrophils,hich suggests a possible immunologic

ole. Functional protein pathways them-elves are, by definition, extremely com-

nd

nt

sk

in sedion.ssioc., R

h. A

lex, and there is clear redundancy built

ibrtpmasbv

sSgalPlwgladhpwt

tcaiticimrc“appjiuvda

AWAMl

ta

R1M22aaA3ImpaJ4gtiwcs5sm16NrtlU7tsv28L9eapH1vr1tbH1Up21Tsn1Qn

1Gt11dpn1Ctt61fibR1tde2Pio22Dto2atM2btatA2Msi22aspot22lpd12cnl22ar


nto the system that limits the assurancey which we can assign proteins into cor-ect functional classes. Because our pro-ein identifications are derived fromooled samples, it is also possible thatore than the PTL pathway is activated

t the individual patient level. Thesehortcomings require that our findingse reproduced in a larger cohort, as pre-iously discussed.On the basis of peptide profiling of 110

amples, we observed that women withPTB were sampled at a slightly earlierestational age (29.4 vs 31.2 weeks) andt a more advanced stage of cervical di-ation (4.1 vs 3.3 cm) than women in theTL group. Both of these variables are

ikely important confounders for whiche could not account. Our current on-oing study is powered sufficiently to al-ow us to match women by gestationalge at enrollment and degree of cervicalilation. Future analysis will be en-anced by a further understanding of therotein expression changes that occurith advanced cervical dilation and ges-

ational age.In conclusion, we have demonstrated

hat maternal serum peptide classifiersan discriminate PTL from SPTB amongcohort of women in PTL with a prelim-

nary accuracy of 97%. Reproduction ofhese findings on a separate cohort anddentification of 1-2 proteins from eachlass with the optimal classification abil-ty may lead to the development of a

ultianalyte serum test both to accu-ately predict PTB and to identify theause of contractions among women inidiopathic” PTL. Both of these potentialpplications have significant clinical im-lications. Limitations in our abilities toredict PTB and to treat PTL remain ma-

or challenges. Efforts aimed at clinicalmprovements will likely be ineffectiventil accurate prediction models are de-eloped and a more comprehensive un-erstanding of clinical phenotypes ischieved. f

CKNOWLEDGMENTSe thank Charles T. Roberts Jr, Jane Hitti,rchana Thomas, Thomas Jacob, Johnichaels, Surendra Dasari, and Mathew Rod-

and, whose efforts and support contributed to 2

he development of the data presented in thisrticle.

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Documents

Insights into the multifactorial nature of preterm birth: proteomic profiling of the maternal serum glycoproteome and maternal serum peptidome among women in preterm labor