Human effector/initiator gene sets that regulate myometrial contractility during term and preterm labor

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ASIC SCIENCE: OBSTETRICS

uman effector/initiator gene sets that regulate myometrialontractility during term and preterm labor

arl P. Weiner, MD, MBA; Clifford W. Mason, PhD; Yafeng Dong, PhD;rina A. Buhimschi, MD; Peter W. Swaan, PhD; Catalin S. Buhimschi, MD

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BJECTIVE: Distinct processes govern transition from quiescence toctivation during term (TL) and preterm labor (PTL). We sought geneets that are responsible for TL and PTL, along with the effector geneshat are necessary for labor independent of gestation and underlyingrigger.

TUDY DESIGN: Expression was analyzed in term and preterm with orithout labor (n � 6 subjects/group). Gene sets were generated with

ogic operations.

ESULTS: Thirty-four genes were expressed similarly in PTL/TL but

m J Obstet Gynecol 2010;202:474.e1-20.

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74.e1 American Journal of Obstetrics & Gynecology MAY 2010

ific to PTL (preterm initiator set), and 174 genes were specific to TLterm initiator set). The gene ontogeny processes that comprise termnitiator and effector sets were diverse, although inflammation was rep-esented in 4 of the top 10; inflammation dominated the preterm initia-or set.

ONCLUSION: TL and PTL differ dramatically in initiator profiles. Al-hough inflammation is part of the term initiator and the effector sets, its an overwhelming part of PTL that is associated with intraamnioticnflammation.

ere absent from nonlabor samples (effector set); 49 genes were spe- Key words: effector, initiator, labor, myometrium, preterm birth

ite this article as: Weiner CP, Mason CW, Dong Y, et al. Human effector/initiator gene sets that regulate myometrial contractility during term and preterm labor.

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uccessful parturition requires thesynchronization of uterine events

myometrial activation, myometrialontraction, cervical ripening, cervicalilation, and rupture of the fetal mem-ranes).1 The myometrium specificallyust undergo a series of molecular and

iochemical changes that transition itrom the phase of quiescence, which isharacterized by a loss of responsivenesso contractile agents, to the phase of ac-ivation with subsequent onset of la-or.2,3 Many investigators have assumedhe triggers of parturition are similar, re-ardless of the gestational age (GA),2,4-9

rom the Departments of Obstetrics and Gynolecular and Integrative Physiology (Dr We

ansas City, KS; the Department of Pharmacef Pharmacy, Baltimore, MD (Dr Swaan); andeproductive Sciences, Yale University Schoouhimschi and C. S. Buhimschi).

resented at the 53rd Annual Scientific Meetingoronto, ON, Canada, March 22-25, 2006.

eceived Oct. 21, 2009; revised Jan. 10, 2010;

eprints: Carl P. Weiner, MD, MBA, Departmenansas School of Medicine, 3901 Rainbow [email protected].

upported in part by grants from the Public Hea01HL049041-12; both C.P.W.).

nd have focused on the control of termabor as a surrogate for preterm labor.his approach has not met with great

uccess.Genomics helps expand and charac-

erize the number of molecular pathwayshat potentially define an underlyingondition. Since our initial report in000,10 oligonucleotide/complementaryNA microarrays have been used byany groups for the study of biologic

rocesses that are involved in normalerm labor.11-18 The results of such stud-es with the use of myometrium that isbtained from term laboring women

logy (Drs Weiner, Mason, and Dong) andr), University of Kansas School of Medicine,cal Sciences, University of Maryland Schoole Department of Obstetrics, Gynecology andMedicine, New Haven, CT (Drs I. A.

he Society for Gynecologic Investigation,

epted Feb. 10, 2010.

Obstetrics and Gynecology, University ofS2028, Kansas City, KS 66160.

ervice (U01 DP000187-02 and

c/j.ajog.2010.02.034

uggest that contractile stimulators of la-or include, but are not limited to, hor-one receptors, cell adhesion molecules,

nterleukins (ILs), prostaglandins, andap junctions.10,11,13-15,17 Other investi-ators have attempted to correlate hu-an myometrial transcriptional levels at

erm with samples from idiopathic,pontaneous preterm labor and haveoncluded that the mechanisms under-ying parturition at all stages of preg-ancy are related.1,13,17 This is surprisingecause there are several well acceptedauses of spontaneous preterm birth.19,20

lthough molecules within these groupsay well influence parturition during

ormal labor, there has been little studyf myometrial gene expression inomen with either preterm or dysfunc-

ional term/preterm labor. Array type in-estigations have also been used to ex-lore myometrial gene expression in theregnant rodent.11,18,21 However, the re-ulting conclusions should be inter-reted cautiously because the gestationalnd hormonal patterns of these speciesre not homologous to the human. Al-hough these studies provide insight,hey tend as a group to be biased by theimited number of genes that are in-

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www.AJOG.org Basic Science: Obstetrics Research

enes that are selected for confirmatorytudy.

Rather than compare term labor to 1f several models for preterm birth, we

nvestigated human pregnancy and hy-othesized that there should be a core setf genes the expression of which are nec-ssary for the process of labor indepen-ent of GA and the underlying trigger forhe labor. We further hypothesized thisffector gene set should relate to myome-rial contractility and the cellular activi-ies necessary to sustain it. We also hy-othesized there must be initiator geneets that are responsible for the transi-ion of the myometrium from quies-ence to activation. But unlike the effec-or set that would be unaffected by thenderlying labor stimulus, there shoulde separate initiator gene sets for termnd preterm labor that reflect the under-ying mechanism of labor. The potentialesult of identifying these gene sets coulde the development of alternative treat-ent options that can be targeted at pre-

erm and term or dysfunctional labor.he purpose of this investigation was to

est our hypotheses in a series of womenho were undergoing cesarean delivery

t term or preterm in labor or absentabor.

ATERIALS AND METHODStudy designyometrium was obtained from the up-

er pole of the transverse lower uterineegment incision of 4 groups of womenn � 6 per group) at the time their pri-ary cesarean section at Yale University:

1) preterm not in labor and no inflam-ation (PTNL; mean GA, 28.8 weeks;

ange, 25.4 –32.5 weeks), (2) preterm inabor with inflammation (PTL; meanA, 29.7 weeks; range, 25.1–32.6 weeks),

3) term not in labor (TNL; mean GA,9.3 weeks; range, 38.4 – 41.0 weeks),nd (4) term labor (TL; mean GA, 40.2eeks; range, 39.0 – 41.2 weeks). Theale University Institutional Reviewoard approved the protocol for sampleollection, and all women provided in-ormed written consent. Labor was de-ned by the presence of regular uterineontractions accompanied by progres-
ive cervical dilation. The diagnosis of f
ntraamniotic inflammation was basedn an amniotic fluid mass restrictedcore of 3 or 4 plus �100 white bloodells/�L3 in the context of a positive am-iotic fluid culture in a sample that wasbtained by transabdominal amniocen-esis.22-25 These tests provided the mostccurate tools currently available toaximize the likelihood of sample ho-ogeneity. The mass restricted score

rovides qualitative information regard-ng the presence or absence of intraam-iotic inflammation. Briefly, the scoreanges from 0-4, depending on the pres-nce (assigned a value of 1) or absenceassigned a value of 0) of each of 4 pro-ein biomarkers.25 A score of 3-4 indi-ates inflammation, whereas a score of-2 excludes it. This biomarker pattern isredictive of preterm birth, histologichorioamnionitis, and adverse neonatalutcome. A detailed description of theass restricted method has been pub-

ished previously.22-25 Indications for ce-arean delivery in the PTNL group werell related to preeclampsia and in theNL were all related to breech presenta-

ion. The indication for cesarean deliveryn the TL group was an arrest of cervicalilation at �6 cm. No normal laboringatient at term underwent cesarean sec-ion delivery in this sample. Clinical dataere retrieved from the medical records,

nd statistical analysis of patient demo-raphics was performed using 1-waynalysis of variance, followed by New-an Keuls Post Hoc test. All laboratory

tudies were performed at either theniversity of Kansas School of Mediciner the University of Maryland Baltimorechool of Pharmacy.

solation of RNAotal RNA was isolated using TRIzol Re-gent (Invitrogen, Carlsbad, CA), accord-ng to the manufacturer’s protocol. Theurity and integrity of each RNA sampleas assessed by spectroscopy and formal-ehyde-agarose gel electrophoresis.

icroarray preparationyometrial gene profiling was per-

ormed on each individual RNA sampleith the Affymetrix GeneChip Humanenome U133 Plus 2.0 microarray (Af-

ymetrix, Santa Clara, CA) that con- s

MAY 2010 Americ

ained 38,500 human genes. RNA qualityas reassessed before spotting with thegilent 2100 Bioanalyzer (Agilent Tech-ologies, Palo Alto, CA). Once the qual-

ty was confirmed, biotin-labeled com-lementary RNA was generated, and 20g of the sample was hybridized to theicroarrays, using the manufacturer’s

tandard conditions. Image processingsed an Affymetrix GeneArray 3000canner.

icroarray data processingnd statistical analysisligonucleotide microarrays were ana-

yzed with the Affymetrix Expression Con-ole. Gene expression levels were normal-zed by the R Statistical package andoftware that is available through the Bio-onductor Project (www.bioconductor.rg). The process normalizes gene ex-ression using a background adjustmentrocedure and a sequence-specific ex-ression method as described by Wu etl.26 Normalized microarray data werehen subject to further discriminativenalysis. A detection probability valueas used to make a reliable call of gene

xpression (present, marginal, or ab-ent). Genes that were present in �4 ofhe 6 patient samples were classified asbsent overall and not consideredurther.

To identify the effector and initiatorene sets, we conducted a series of logicperations based on gene presence (de-ection probability value) and illustratedy the Venn diagram in Figure 1. We firstenerated 4 groups of genes: group A �PTL – PTNL); group C � (TL – TNL);roup B � (group A – TNL); group D �group C – PTNL).

The effector gene set was defined ashose genes common to groups B and. The effector set was further filtered

o exclude genes whose expressionuring PTL changed �2-fold in eitherirection compared with TL, reason-

ng there was something unique to theisease state that influenced gene ex-ression. The preterm initiator gene setas defined as those genes in group A

xclusive to PTL (minus group C).ast, the term initiator gene set was de-ned as those genes in group C exclu-
ive of TL (minus group A).
an Journal of Obstetrics & Gynecology 474.e2

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4

etwork analysis andiologic classificationdentification and visual analysis of geneetworks were performed using Meta-ore analytical suite (version 2.0;eneGo, St. Joseph, MI). There were fewirect interactions identified among the

ndividual proteins of the effector, pre-erm initiator, and term initiator genes.herefore, we analyzed the possible net-orks that could be built from such rel-

tively large gene sets using an algorithmhat generates subnetworks that areighly saturated with input genes fromach dataset (ie, effector set, preterm ini-iator set, and term initiator set). In thisase, the network algorithm takes a list ofoot nodes (ie, genes from effector, pre-erm initiator, and term initiator sets)nd for each 1 creates shortest paths net-orks to other root nodes in the list and

tops the network expansion at a size of0 nodes (standard number predefinedn MetaCore). The resulting networksre evaluated and ranked according toheir statistical significance (probabilityalues). This high trust probability valuealculation was also used to evaluate theetwork’s relevance to gene ontologyGO) biologic processes classification. Ineneral, GO is a method of standardizinghe representation of gene and generoduct attributes across species and da-abases. Three GO functional ontologiesprocesses, molecular functions, and lo-alizations) are used in Metacore soft-are for enrichment analysis. The ad-antage of using this network algorithms that it may find a well-connected clus-er of root nodes without any predefinedestrictions and, as a result, offers moreexibility in the identification of possibleonnections. These interactions werehen assigned to specific biologic pro-esses, cellular components, and/or mo-ecular functions to further characterizehe underlying condition to yield insightnto the underlying mechanisms. We se-ected some genes of interest for confir-

ation by quantitative real time–poly-erase chain reaction (qRT-PCR) based

n their participation in subnetworksith a low probability value. Other genes

hat were selected from the input list
ere selected for qRT-PCR analysis
74.e3 American Journal of Obstetrics & Gynecolo

FIGURE 1Venn diagram

iagram illustrates the logic process for the derivation of the 3 unique gene sets. The number referso the number of genes that were identified in the various subgroups.TL, preterm labor; PTNL, preterm not in labor and no inflammation; TL, term labor; TNL, term not in labor.

einer. Human myometrial effector/initiator gene sets for labor. Am J Obstet Gynecol 2010.

gy MAY 2010

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ased on their statistically significant as-ociation with GO processes.

RT-PCRotal RNA from the original samplessed for the microarrays was used touantify each gene. Primer sequences formplifications were based on previouslyublished complementary DNA se-uences with the Beacon Designer pro-ram (BioRad, Hercules, CA). SYBRreen (BioRad) was used for ampliconetection. All primer sets were tested tonsure the efficiency of amplificationver a wide range of template concentra-ions. PCR reactions were carried out inhe iQ5 RT-PCR detection system (Bio-ad). A melting curve was performed af-

er amplification to ensure all samplesxhibited a single amplicon. Gene ex-ression of each individual myometrialample was first normalized to the ex-ression of 18s ribosomal RNA (internalontrol) by the �CT method27 and thenompared with the average of the corre-ponding control samples. MessengerNA (mRNA) relative expression isqual to 2-��CT and determined by theollowing equations: (1) ��CT � �CT1each individual sample) – �CT2 (con-rol group mean); 920 �CT1 � gene CTalue – 18S CT value of the same sample;nd (3) �CT2 � average of (gene CTalue of control group – 18S CT value ofontrol group). The point at which theuorescence crosses the threshold isalled the CT value and is read by theT-PCR instrument as a specific

TABLE 1Clinical characteristics

GroupGestationalage, wka

Maag

Term not in labor 39 � 1 30...................................................................................................................

Term labor 40 � 1 28...................................................................................................................

Preterm not in labor 29 � 3b,c 26...................................................................................................................

Preterm labor 30 � 3b,c 34

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

NA, not applicable.a Data are presented as mean � SD; b P � .05, based on Ne

Weiner. Human myometrial effector/initiator gene sets fo

arameter. t

ESULTSlinical characteristicsf myometrial sampleshe clinical characteristics of the preg-ancies included are listed in Table 1.here were no statistically significantifferences in GA or birthweight amongomen at term (no labor vs labor) orreterm (no labor vs labor). There wereo statistically significant differences inaternal age as determined by 1-way

nalysis of variance. Thus, the observedifferences in genes in the initiatorroups are most likely the result of laborterm or preterm) rather than any varia-ion in GA or maternal age.

dentification of effector setnd the term initiator andreterm initiator setsixty-seven genes were present in bothTL and TL but were absent from allonlabor samples (n � 6; groups B and). This group was termed the effector setecause the genes comprising it repre-ent a distinct group that might act toustain the labor itself rather than con-ributing to its onset. This effector setas then further filtered to exclude genes

hat showed at least a 2-fold differenceetween PTL and TL based on the as-umption that there was somethingbout the disease process that led to dif-erential regulation. This left 34 genes inhe effector set (Table 2).

Although the effector gene set was byefinition independent of GA, we sus-ected there must exist gene sets that en-bled the initiation of the labor either

nala Birthweight, ga Indication for ces

3490 � 618 Breech.........................................................................................................................

3490 � 333 Failure to progress.........................................................................................................................

971 � 380b,c Preeclampsia.........................................................................................................................

1299 � 344b,c Preterm prematurspontaneous prete

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

n-Keuls test compared with term not in labor; c P � .05, based

or. Am J Obstet Gynecol 2010.

erm or preterm. We detected 49 genes o

MAY 2010 Americ

hat were specific to preterm labor alonepreterm initiator set; Table 3) and 174enes that were specific to TL aloneterm initiator set; Table 4). In light ofhe GAs of the preterm subjects, it isighly likely that the selected gene setsepresent, or at least include, molecularroups that are responsible for initiatinghe transition from myometrial quies-ence to activation in their respectiveategories.

haracterization of initiatornd effector gene setst is not adequate to categorize genesased solely on a given condition.ather, there must be an understandingf how the encoded proteins interact.he result of these interactions is a uni-erse that represents the functionalotential in terms of complex protein as-ociations, such as pathways with succes-ive reactions and transient structuralomplexes. We reasoned that character-zation of networks based on the identi-ed initiator and effector gene sets woulddd insight into the molecular eventshat regulate preterm labor and TL.hus, we enriched the initiator and effec-

or sets using the GO classifications. Un-ike standard GO classification systemshat produce a vague and general under-tanding of the molecular events under-ying the given networks, GeneGo GOlassifies gene groups within a specifiedealm of biologic processes, molecularunction, and cellular components.eneGo processes are network modules

an deliveryHistologicchorioamnionitis

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ewman-Keuls test compared with term labor.

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ted manually on the basis of GO pro-esses and pathway maps.

The specificity of each gene set allowsor a gene to be present in only 1 of the 3roups. The effector set (Figure 2, A) and

TABLE 2Core myometrial genes of labor (ef

Gene symbol Gene name

ABRA Actin-binding...................................................................................................................

ALDH16A1 Aldehyde deh...................................................................................................................

ANKS3 Ankyrin repea...................................................................................................................

ATRIP/TREX1 ATR-interactin...................................................................................................................

BAIAP2L1 Bai1-associat...................................................................................................................

CADM2 Cell adhesion...................................................................................................................

CCDC36 Coiled-coil do...................................................................................................................

CFB Complement...................................................................................................................

CORO2A Coronin, actin...................................................................................................................

D4S234E DNA segment...................................................................................................................

EGFL7 Egf-like-doma...................................................................................................................

FPGS Folylpolygluta...................................................................................................................

GPATCH3 G patch doma...................................................................................................................

H2AFY2 H2a histone f...................................................................................................................

HNT Neurotrimin...................................................................................................................

HTRA4 Htra serine pe...................................................................................................................

IL8RB Interleukin 8...................................................................................................................

KCNAB2 Potassium vo...................................................................................................................

KRTAP8-1 Keratin assoc...................................................................................................................

LY6G5C Lymphocyte a...................................................................................................................

MPP3 Membrane pr...................................................................................................................

P2RY8 Purinergic rec...................................................................................................................

PLAC8L1 Plac8-like 1...................................................................................................................

PLCXD1 Phosphatidyli...................................................................................................................

POLR3D Polymerase (R...................................................................................................................

RRAD Ras-related a...................................................................................................................

SIRPB2 Signal-regula...................................................................................................................

SKIV2L Superkiller vi...................................................................................................................

SLPI Secretory leu...................................................................................................................

ST3GAL3 St3 beta-gala...................................................................................................................

ZNF552 Zinc finger pr...................................................................................................................





he term initiator set (Figure 3, A) were t


ach dominated by inflammatory GOrocesses, although they were distinctlyifferent from each other. Each had 4ignificant processes (based on probabil-ty value and false discovery rate calcula-

tor set), excluding hypothetic and ot

activating protein.........................................................................................................................

ogenase 16 family, member a1.........................................................................................................................

d sterile alpha motif domain containing 3.........................................................................................................................

rotein/Three prime repair exonuclease 1.........................................................................................................................

protein 2-like 1.........................................................................................................................

lecule 2 precursor.........................................................................................................................

in containing 36.........................................................................................................................

or b.........................................................................................................................

ding protein, 2a.........................................................................................................................

chromosome 4 (unique) 234 expressed sequen.........................................................................................................................

multiple 7.........................................................................................................................

te synthase.........................................................................................................................

ontaining 3.........................................................................................................................

ly, member y2.........................................................................................................................

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

ase 4.........................................................................................................................

ptor, beta.........................................................................................................................

e-gated channel, shaker-related subfamily, beta.........................................................................................................................

d protein 8-1.........................................................................................................................

en 6 complex, locus g5c.........................................................................................................................

n, palmitoylated 3 (maguk p55 subfamily memb.........................................................................................................................

or p2y, g-protein coupled, 8.........................................................................................................................

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

itol-specific phospholipase c, x domain containin.........................................................................................................................

) iii (DNA directed) polypeptide d, 44 kd.........................................................................................................................

ciated with diabetes mellitus.........................................................................................................................

protein beta 2.........................................................................................................................

idic activity 2-like (s. Cerevisiae).........................................................................................................................

yte peptidase inhibitor.........................................................................................................................

ide alpha-2,3-sialyltransferase 3.........................................................................................................................

n 552.........................................................................................................................

n 718.........................................................................................................................

n 74 (cos52).........................................................................................................................

n 788.........................................................................................................................


ion) within the top 10 GeneGo pro- t

gy MAY 2010

esses that were identified were involvedith inflammation. These findings

learly support the concept that inflam-ation plays an important role in TL.Perhaps not surprisingly, considering

r unidentified/unnamed proteinsEntrezidentification no.

137735..................................................................................................................

126133..................................................................................................................

124401..................................................................................................................

84126..................................................................................................................

55971..................................................................................................................

253559..................................................................................................................

339834..................................................................................................................

629..................................................................................................................

7464..................................................................................................................

27065..................................................................................................................

51162..................................................................................................................

2356..................................................................................................................

63906..................................................................................................................

55506..................................................................................................................

50863..................................................................................................................

203100..................................................................................................................

3579..................................................................................................................

mber 2 8514..................................................................................................................

337879..................................................................................................................

80741..................................................................................................................

) 4356..................................................................................................................

286530..................................................................................................................

153770..................................................................................................................

55344..................................................................................................................

661..................................................................................................................

6236..................................................................................................................

284759..................................................................................................................

6499..................................................................................................................

6590..................................................................................................................

6487..................................................................................................................

79818..................................................................................................................

255403..................................................................................................................

7625..................................................................................................................

388507..................................................................................................................

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otei......... .........

he patient selection criterion, the pre-


TABLE 3Myometrial genes that initiate preterm labor (preterm initiator set),excluding hypothetic and other unidentified/unnamed proteins

Genes symbol Gene nameEntrezidentification no.

ABCA3 ATP-binding cassette, sub-family a (abc1), member 3 21................................................................................................................................................................................................................................................................................................................................................................................

ABP1 Amiloride binding protein 1 (amine oxidase [copper-containing]) 26................................................................................................................................................................................................................................................................................................................................................................................

ARSA Arylsulfatase a 410................................................................................................................................................................................................................................................................................................................................................................................

CD3E Cd3e antigen, epsilon polypeptide (tit3 complex) 916................................................................................................................................................................................................................................................................................................................................................................................

DGKQ Diacylglycerol kinase, theta 110 kd 1609................................................................................................................................................................................................................................................................................................................................................................................

GJA3 Gap junction protein, alpha 3, 46 kd (connexin 46) 2700................................................................................................................................................................................................................................................................................................................................................................................

GSTT2 Glutathione s-transferase theta 2 2953................................................................................................................................................................................................................................................................................................................................................................................

HLA-DOA Major histocompatibility complex, class ii, do alpha 3111................................................................................................................................................................................................................................................................................................................................................................................

HYAL1 Hyaluronoglucosaminidase 1 3373................................................................................................................................................................................................................................................................................................................................................................................

IFNG Interferon, gamma 3458................................................................................................................................................................................................................................................................................................................................................................................

IRF5 Interferon regulatory factor 5 3663................................................................................................................................................................................................................................................................................................................................................................................

LLGL1 Lethal giant larvae homolog 1 (drosophila) 3996................................................................................................................................................................................................................................................................................................................................................................................

LYL1 Lymphoblastic leukemia derived sequence 1 4066................................................................................................................................................................................................................................................................................................................................................................................

MYCL1 V-myc myelocytomatosis viral oncogene homolog 1, lung carcinoma derived (avian) 4610................................................................................................................................................................................................................................................................................................................................................................................

PDE1C Phosphodiesterase 1c, calmodulin-dependent 70 kd 5137................................................................................................................................................................................................................................................................................................................................................................................

MAPK11 Mitogen-activated protein kinase 11 5600................................................................................................................................................................................................................................................................................................................................................................................

PTPRZ1 Protein tyrosine phosphatase, receptor-type, z polypeptide 1 5803................................................................................................................................................................................................................................................................................................................................................................................

ZNF208 Zinc finger protein 208 7757................................................................................................................................................................................................................................................................................................................................................................................

ABCA7 ATP-binding cassette, sub-family a (abc1), member 7 10347................................................................................................................................................................................................................................................................................................................................................................................

CXCL13 Chemokine (c-x-c motif) ligand 13 (b-cell chemoattractant) 10563................................................................................................................................................................................................................................................................................................................................................................................

OGFR Opioid growth factor receptor 11054................................................................................................................................................................................................................................................................................................................................................................................

ELOVL2 Elongation of very long chain fatty acids (fen1/elo2, sur4/elo3, yeast)-like 2 54898................................................................................................................................................................................................................................................................................................................................................................................

CPXM Carboxypeptidase x (m14 family) 56265................................................................................................................................................................................................................................................................................................................................................................................

AARSL Alanyl-tRNA synthetase like 57505................................................................................................................................................................................................................................................................................................................................................................................

TINAGL1 Tubulointerstitial nephritis antigen-like 1 64129................................................................................................................................................................................................................................................................................................................................................................................

DIO3OS Deiodinase, iodothyronine, type iii opposite strand 64150................................................................................................................................................................................................................................................................................................................................................................................

NDRG4 Ndrg family member 4 65009................................................................................................................................................................................................................................................................................................................................................................................

VPS33A Vacuolar protein sorting 33a (yeast) 65082................................................................................................................................................................................................................................................................................................................................................................................

LRFN4 Leucine rich repeat and fibronectin type iii domain containing 4 78999................................................................................................................................................................................................................................................................................................................................................................................

TLE6 Transducin-like enhancer of split 6 (e[sp1]) homolog, drosophila) 79816................................................................................................................................................................................................................................................................................................................................................................................

TIGD6 Tigger transposable element derived 6 81789................................................................................................................................................................................................................................................................................................................................................................................

KRTAP4-8 Keratin associated protein 4-8 83898................................................................................................................................................................................................................................................................................................................................................................................

CHCHD6 Coiled-coil-helix-coiled-coil-helix domain containing 6 84303................................................................................................................................................................................................................................................................................................................................................................................


CCDC97 Coiled-coil domain-containing protein 97 90324................................................................................................................................................................................................................................................................................................................................................................................

STK11IP Serine/threonine kinase 11 interacting protein 114790................................................................................................................................................................................................................................................................................................................................................................................

SORCS1 Sortilin-related vps10 domain containing receptor 1 114815................................................................................................................................................................................................................................................................................................................................................................................

CATSPER2 Cation channel, sperm associated 2 117155................................................................................................................................................................................................................................................................................................................................................................................

Weiner. Human myometrial effector/initiator gene sets for labor. Am J Obstet Gynecol 2010. (continued )

MAY 2010 American Journal of Obstetrics & Gynecology 474.e6

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4

erm initiator set was also associatedeavily with inflammation; all but 2 ofhe top 10 GeneGo processes (Figure 4,). Of interest was the appearance ofypoxia and oxidative stress (no. 5 inigure 4, A) because we have demon-trated recently that chronic hypoxialone stimulates a response that is clini-ally the same as the fetal inflammatoryesponse syndrome.28

isualization of term initiator, pretermnitiator, and effector networkshe traditional approach to gene arraynalyses includes a list with the genes thatre clustered based on biologic similarityTables 2-4). Although this method pro-ides insight into the transcriptional activ-ty of major biologic processes, it fails toxpress how the encoded proteins mightnteract with each other and surroundingroteins. We previously demonstratedhat the identification of gene networksrovides a more robust tool by which toxtract information from the microarrayshan gene lists.16 These networks incorpo-ate genomic data and represent the en-oded proteins in the form of predicted in-eractions and reactions that are compiledrom different experiments and experi-

ental conditions.There were few direct interactions iden-

TABLE 3Myometrial genes that initiate pretexcluding hypothetic and other uni

Genes symbol Gene name

LRGUK Leucine-rich rep...................................................................................................................

FAM69B Family with sequ...................................................................................................................

BEAN Brain expressed,...................................................................................................................

GRASP Grp1 (general re...................................................................................................................

ZNF579 Zinc finger prote...................................................................................................................

DENND1B Denn/madd dom...................................................................................................................

SAMD14 Sterile alpha mo...................................................................................................................

ALS2CL Als2 c-terminal l...................................................................................................................

STK32C Serine/threonine...................................................................................................................

CATSPER2P1 Cation channel,...................................................................................................................

GSTT2B Similar to glutath...................................................................................................................

Entrez (Entrez Global Query Cross-Database Search System) i


ified among the proteins of the 34 effector p


enes, the 49 preterm initiator genes, and74 term initiator genes. This supports ourupposition of gene set uniqueness. Wehen analyzed the possible networks thatould be built from such gene sets with these of an algorithm that generates subnet-orks that are highly saturated with the se-

ected objects (Figures 2, B; 3, B, and 4, B).e selected subnetworks based on their

robability values. Figures 2, B; 3, B, and 4,show the top ranked identified subnet-orks in the effector (n � 9), preterm ini-

iator (n�11), and term initiator sets (n�0), respectively. The inward and outwardound lines extending from the selectedenes (genes in experimental sets) illus-rate the interactive environment that sur-ounds the selected genes and can be usedo show how their encoded proteins are af-ected (positively, negatively, unknown)pstream or downstream within the net-ork pathway. This information couldave significant implications for the devel-pment and design of therapeutic entitiesargeted at inhibiting or stimulating the ac-ivity of the targeted gene or protein in theathway.

alidation of initiator andffector gene setshe high sensitivity of qRT-PCR makes

t likely that small changes in gene ex-

labor (preterm initiator set),tified/unnamed proteins (continued)

and guanylate kinase domain containing.........................................................................................................................

e similarity 69, member b.........................................................................................................................

sociated with nedd4.........................................................................................................................

tor for phosphoinositides 1)-associated scaffold.........................................................................................................................

79.........................................................................................................................

containing 1b.........................................................................................................................

omain containing 14.........................................................................................................................

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

ase 32c.........................................................................................................................

rm associated 2 pseudogene.........................................................................................................................

e s-transferase theta 2 (gst class-theta 2).........................................................................................................................

integrated search and retrieval system at the National Center for


ression exist that were undetected by 6

gy MAY 2010

he microarray analyses. Although wessume for microarray analyses that rel-vant genes were described by gene ex-ression above a set expression thresh-ld, we recognize that the sensitivity ofRT-PCR might reveal enhanced ex-ression in genes that were unselected inhe microarray analyses. Thus, we fo-used on subnetwork genes with highRNA levels.The idea that a unique group of en-

oded proteins is responsible for sustain-ng myometrial contractility during la-or irrespective of GA (Effector set) hasot been investigated previously. Basedn our analyses, we selected HNTneutrimin precursor), KCNAB2 (volt-ge-gated potassium channel, shaker-re-ated subfamily), and RRAD (Ras-re-ated associated with diabetes mellitus;igure 5). The expression of these genesas not been localized previously to theyometrium, nor have these genes been

ssociated with labor. The microarraynalyses and the logic process were con-rmed to identify genes that were in sim-

larly increased during labor indepen-ent of GA and mechanism.By applying a similar analytic ap-

roach, we selected 5 genes for confir-ation in the term initiator set (Figure

Entrezidentification no.

136332..................................................................................................................

138311..................................................................................................................

146227..................................................................................................................

tein 160622..................................................................................................................

163033..................................................................................................................

163486..................................................................................................................

201191..................................................................................................................

259173..................................................................................................................

282974..................................................................................................................

440278..................................................................................................................

653689..................................................................................................................

chnology Information (NCBI) website.

ermden

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enc......... .........

as......... .........

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ain......... .........

tif d......... .........

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kin......... .........

spe......... .........

ion......... .........

s an Biote

): PROK1 (prokineticin 1), IL-9R (IL 9


TABLE 4Genes that initiate term labor (term initiator set) in myometrium,excluding hypothetic and other unidentified/unnamed proteins

Gene symbol Gene nameEntrezidentification no.

A1BG Alpha-1-b glycoprotein 1................................................................................................................................................................................................................................................................................................................................................................................

ADCYAP1R1 Adenylate cyclase activating polypeptide 1 (pituitary) receptor type i 117................................................................................................................................................................................................................................................................................................................................................................................

ALDOC Aldolase c, fructose-bisphosphate 230................................................................................................................................................................................................................................................................................................................................................................................

AQP9 Aquaporin 9 366................................................................................................................................................................................................................................................................................................................................................................................

AREG Amphiregulin (schwannoma-derived growth factor) 374................................................................................................................................................................................................................................................................................................................................................................................

ARHGAP4 Rho gtpase activating protein 4 393................................................................................................................................................................................................................................................................................................................................................................................

BCL2A1 Bcl2-related protein a1 597................................................................................................................................................................................................................................................................................................................................................................................

BDKRB1 Bradykinin receptor b1 623................................................................................................................................................................................................................................................................................................................................................................................

BUB1 Bub1 budding uninhibited by benzimidazoles 1 homolog (yeast) 699................................................................................................................................................................................................................................................................................................................................................................................

BUB1B Bub1 budding uninhibited by benzimidazoles 1 homolog beta (yeast) 701................................................................................................................................................................................................................................................................................................................................................................................

CALCA Calcitonin/calcitonin-related polypeptide, alpha 796................................................................................................................................................................................................................................................................................................................................................................................

RUNX3 Runt-related transcription factor 3 864................................................................................................................................................................................................................................................................................................................................................................................

CCNE1 Cyclin e1 898................................................................................................................................................................................................................................................................................................................................................................................

CD48 Cd48 antigen (b-cell membrane protein) 962................................................................................................................................................................................................................................................................................................................................................................................

CDC20 Cdc20 cell division cycle 20 homolog (s. Cerevisiae) 991................................................................................................................................................................................................................................................................................................................................................................................

CHI3L1 Chitinase 3-like 1 (cartilage glycoprotein-39) 1116................................................................................................................................................................................................................................................................................................................................................................................

CSF3 Colony stimulating factor 3 (granulocyte) 1440................................................................................................................................................................................................................................................................................................................................................................................

CSN2 Casein beta 1447................................................................................................................................................................................................................................................................................................................................................................................

DDIT3 DNA-damage-inducible transcript 3 1649................................................................................................................................................................................................................................................................................................................................................................................

EFNB1 Ephrin-b1 1947................................................................................................................................................................................................................................................................................................................................................................................

EREG Epiregulin 2069................................................................................................................................................................................................................................................................................................................................................................................

EYA4 Eyes absent homolog 4 (drosophila) 2070................................................................................................................................................................................................................................................................................................................................................................................

ESRRA Estrogen-related receptor alpha 2101................................................................................................................................................................................................................................................................................................................................................................................

FPRL1 Formyl peptide receptor-like 1 2358................................................................................................................................................................................................................................................................................................................................................................................

GALE Udp-galactose-4-epimerase 2582................................................................................................................................................................................................................................................................................................................................................................................

GCHFR Gtp cyclohydrolase i feedback regulator 2644................................................................................................................................................................................................................................................................................................................................................................................

GK Glycerol kinase 2710................................................................................................................................................................................................................................................................................................................................................................................

GRIN2D Glutamate receptor, ionotropic, n-methyl d-aspartate 2d 2906................................................................................................................................................................................................................................................................................................................................................................................

NRG1 Neuregulin 1 3084................................................................................................................................................................................................................................................................................................................................................................................

HK2 Hexokinase 2 3099................................................................................................................................................................................................................................................................................................................................................................................

NR4A1 Nuclear receptor subfamily 4, group a, member 1 3164................................................................................................................................................................................................................................................................................................................................................................................

IL1B Interleukin 1, beta 3553................................................................................................................................................................................................................................................................................................................................................................................

IL9R Interleukin 9 receptor 3581................................................................................................................................................................................................................................................................................................................................................................................

IL13RA2 Interleukin 13 receptor, alpha 2 3598................................................................................................................................................................................................................................................................................................................................................................................

INHBB Inhibin, beta b (activin ab beta polypeptide) 3625................................................................................................................................................................................................................................................................................................................................................................................

KRT7 Keratin 7 3855................................................................................................................................................................................................................................................................................................................................................................................

LIF Leukemia inhibitory factor (cholinergic differentiation factor) 3976................................................................................................................................................................................................................................................................................................................................................................................

MMP3 Matrix metallopeptidase 3 (stromelysin 1, progelatinase) 4314................................................................................................................................................................................................................................................................................................................................................................................

MYH3 Myosin, heavy polypeptide 3, skeletal muscle, embryonic 4621................................................................................................................................................................................................................................................................................................................................................................................




4

TABLE 4Genes that initiate term labor (term initiator set) in myometrium,excluding hypothetic and other unidentified/unnamed proteins (continued)


NEFH Neurofilament, heavy polypeptide 200 kd 4744................................................................................................................................................................................................................................................................................................................................................................................

NEK2 Nima (never in mitosis gene a)-related kinase 2 4751................................................................................................................................................................................................................................................................................................................................................................................

NPPB Natriuretic peptide precursor b 4879................................................................................................................................................................................................................................................................................................................................................................................

SERPINB2 Serpin peptidase inhibitor, clade b (ovalbumin), member 2 5055................................................................................................................................................................................................................................................................................................................................................................................

PCSK1 Proprotein convertase subtilisin/kexin type 1 5122................................................................................................................................................................................................................................................................................................................................................................................

PFKFB4 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 5210................................................................................................................................................................................................................................................................................................................................................................................

PGK2 Phosphoglycerate kinase 2 5232................................................................................................................................................................................................................................................................................................................................................................................

SERPINF2 Serpin peptidase inhibitor, clade f (alpha-2 antiplasmin, pigment epithelium derived factor),member 2

5345

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

POLE Polymerase (DNA directed), epsilon 5426................................................................................................................................................................................................................................................................................................................................................................................

PRKCD Protein kinase c, delta 5580................................................................................................................................................................................................................................................................................................................................................................................

S100P S100 calcium binding protein p 6286................................................................................................................................................................................................................................................................................................................................................................................

CCL11 Chemokine (c-c motif) ligand 11 6356................................................................................................................................................................................................................................................................................................................................................................................


CXCL6 Chemokine (c-x-c motif) ligand 6 (granulocyte chemotactic protein 2) 6372................................................................................................................................................................................................................................................................................................................................................................................

SELE Selectin e (endothelial adhesion molecule 1) 6401................................................................................................................................................................................................................................................................................................................................................................................

ST3GAL1 St3 beta-galactoside alpha-2,3-sialyltransferase 1 6482................................................................................................................................................................................................................................................................................................................................................................................

SLAMF1 Signaling lymphocytic activation molecule family member 1 6504................................................................................................................................................................................................................................................................................................................................................................................

SLC9A2 Solute carrier family 9 (sodium/hydrogen exchanger), member 2 6549................................................................................................................................................................................................................................................................................................................................................................................

SPAG4 Sperm associated antigen 4 6676................................................................................................................................................................................................................................................................................................................................................................................

STXBP2 Syntaxin binding protein 2 6813................................................................................................................................................................................................................................................................................................................................................................................

TAC1 Tachykinin, precursor 1 (substance k, substance p, neurokinin 1, neurokinin 2, neuromedin l,neurokinin alpha, neuropeptide k, neuropeptide gamma)

6863

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

VAV1 Vav 1 oncogene 7409................................................................................................................................................................................................................................................................................................................................................................................

VIL1 Villin 1 7429................................................................................................................................................................................................................................................................................................................................................................................

ZNF31 Zinc finger protein 31 (kox 29) 7579................................................................................................................................................................................................................................................................................................................................................................................

GCS1 Glucosidase i 7841................................................................................................................................................................................................................................................................................................................................................................................

PLA2G7 Phospholipase a2, group vii (platelet-activating factor acetylhydrolase, plasma) 7941................................................................................................................................................................................................................................................................................................................................................................................

NR4A3 Nuclear receptor subfamily 4, group a, member 3 8013................................................................................................................................................................................................................................................................................................................................................................................

FOSL1 Fos-like antigen 1 8061................................................................................................................................................................................................................................................................................................................................................................................

GPR68 G protein-coupled receptor 68 8111................................................................................................................................................................................................................................................................................................................................................................................

SLC7A5 Solute carrier family 7 (cationic amino acid transporter, y� system), member 5 8140................................................................................................................................................................................................................................................................................................................................................................................

ENC1 Ectodermal-neural cortex (with btb-like domain) 8507................................................................................................................................................................................................................................................................................................................................................................................

CPZ Carboxypeptidase z 8532................................................................................................................................................................................................................................................................................................................................................................................

TNFRSF6B Tumor necrosis factor receptor superfamily, member 6b, decoy 8771................................................................................................................................................................................................................................................................................................................................................................................

PROM1 Prominin 1 8842................................................................................................................................................................................................................................................................................................................................................................................

RRP9 RRP9, small subunit (SSU) processome component, homolog (yeast) 9136................................................................................................................................................................................................................................................................................................................................................................................

MSC Musculin (activated b-cell factor-1) 9242................................................................................................................................................................................................................................................................................................................................................................................

CYP7B1 Cytochrome p450, family 7, subfamily b, polypeptide 1 9420................................................................................................................................................................................................................................................................................................................................................................................






IL27RA Interleukin 27 receptor, alpha 9466................................................................................................................................................................................................................................................................................................................................................................................

IKBKE Inhibitor of kappa light polypeptide gene enhancer in b-cells, kinase epsilon 9641................................................................................................................................................................................................................................................................................................................................................................................

DHX34 Deah (asp-glu-ala-his) box polypeptide 34 9704................................................................................................................................................................................................................................................................................................................................................................................

SEC14L5 Sec14-like 5 (s. Cerevisiae) 9717................................................................................................................................................................................................................................................................................................................................................................................

CENTB1 Centaurin, beta 1 9744................................................................................................................................................................................................................................................................................................................................................................................

CLSTN3 Calsyntenin 3 9746................................................................................................................................................................................................................................................................................................................................................................................

DHRS2 Dehydrogenase/reductase (sdr family) member 2 10202................................................................................................................................................................................................................................................................................................................................................................................

TACC3 Transforming, acidic coiled-coil containing protein 3 10460................................................................................................................................................................................................................................................................................................................................................................................

DPYSL4 Dihydropyrimidinase-like 4 10570................................................................................................................................................................................................................................................................................................................................................................................

IL24 Interleukin 24 11009................................................................................................................................................................................................................................................................................................................................................................................

LILRA1 Leukocyte immunoglobulin-like receptor, subfamily a (with tm domain), member 1 11024................................................................................................................................................................................................................................................................................................................................................................................

CIT Citron (rho-interacting, serine/threonine kinase 21) 11113................................................................................................................................................................................................................................................................................................................................................................................

KPTN Kaptin (actin binding protein) 11133................................................................................................................................................................................................................................................................................................................................................................................

RASSF1 Ras association (ralgds/af-6) domain family 1 11186................................................................................................................................................................................................................................................................................................................................................................................

IRAK3 Interleukin-1 receptor-associated kinase 3 11213................................................................................................................................................................................................................................................................................................................................................................................

ATXN2L Ataxin 2-like 11273................................................................................................................................................................................................................................................................................................................................................................................

RRP12 Ribosomal RNA processing 12 homolog (S cerevisiae) 23223................................................................................................................................................................................................................................................................................................................................................................................

SMG6 Smg-6 homolog, nonsense mediated mRNA decay factor (C elegans) 23293................................................................................................................................................................................................................................................................................................................................................................................

NUP188 Nucleoporin 188 kd 23511................................................................................................................................................................................................................................................................................................................................................................................

CLEC5A C-type lectin domain family 5, member a 23601................................................................................................................................................................................................................................................................................................................................................................................

CLEC4E C-type lectin domain family 4, member e 26253................................................................................................................................................................................................................................................................................................................................................................................

SIGLEC7 Sialic acid binding ig-like lectin 7 27036................................................................................................................................................................................................................................................................................................................................................................................

SULT1B1 Sulfotransferase family, cytosolic, 1b, member 1 27284................................................................................................................................................................................................................................................................................................................................................................................

CECR2 Cat eye syndrome chromosome region, candidate 2 27443................................................................................................................................................................................................................................................................................................................................................................................

DKFZP434P211 Pom121-like protein 29774................................................................................................................................................................................................................................................................................................................................................................................

EMR2 Egf-like module containing, mucin-like, hormone receptor-like 2 30817................................................................................................................................................................................................................................................................................................................................................................................

ANGPTL4 Angiopoietin-like 4 51129................................................................................................................................................................................................................................................................................................................................................................................

ABI3 Abi gene family, member 3 51225................................................................................................................................................................................................................................................................................................................................................................................

RTEL1 Regulator of telomere elongation helicase 1 51750................................................................................................................................................................................................................................................................................................................................................................................

P2RY13 Purinergic receptor p2y, g-protein coupled, 13 53829................................................................................................................................................................................................................................................................................................................................................................................

GPR84 G protein-coupled receptor 84 53831................................................................................................................................................................................................................................................................................................................................................................................

SOX18 Sry (sex determining region y)-box 18 54345................................................................................................................................................................................................................................................................................................................................................................................

SDK2 Sidekick homolog 2 (chicken) 54549................................................................................................................................................................................................................................................................................................................................................................................

RAB39 Rab39, member ras oncogene family 54734................................................................................................................................................................................................................................................................................................................................................................................

GRAMD1C Gram domain containing 1c 54762................................................................................................................................................................................................................................................................................................................................................................................

MCM10 Mcm10 minichromosome maintenance deficient 10 (S cerevisiae) 55388................................................................................................................................................................................................................................................................................................................................................................................

AJAP1 Adherens junction associated protein 1 55966................................................................................................................................................................................................................................................................................................................................................................................





4



ASAH2 N-acylsphingosine amidohydrolase (non-lysosomal ceramidase) 2 56624................................................................................................................................................................................................................................................................................................................................................................................

PDXP Pyridoxal (pyridoxine, vitamin b6) phosphatase 57026................................................................................................................................................................................................................................................................................................................................................................................

ATP10A Atpase, class v, type 10c 57194................................................................................................................................................................................................................................................................................................................................................................................

LOC57228 Small trans-membrane and glycosylated protein 57228................................................................................................................................................................................................................................................................................................................................................................................

LRRN1 Leucine rich repeat neuronal 1 57633................................................................................................................................................................................................................................................................................................................................................................................

NLRC4 NLR family, CARD domain containing 4 58484................................................................................................................................................................................................................................................................................................................................................................................

PROK2 Prokineticin 2 60675................................................................................................................................................................................................................................................................................................................................................................................

CARD9 Caspase recruitment domain family, member 9 64170................................................................................................................................................................................................................................................................................................................................................................................

CDCP1 Cub domain containing protein 1 64866................................................................................................................................................................................................................................................................................................................................................................................



IQCA Iq motif containing with aaa domain 79781................................................................................................................................................................................................................................................................................................................................................................................

VASH2 Vasohibin 2 79805................................................................................................................................................................................................................................................................................................................................................................................

DENND2D Denn/madd domain containing 2d 79961................................................................................................................................................................................................................................................................................................................................................................................

LRRC8E Leucine rich repeat containing 8 family, member e 80131................................................................................................................................................................................................................................................................................................................................................................................

ZC3H12A Zinc finger ccch-type containing 12a 80149................................................................................................................................................................................................................................................................................................................................................................................

DEPDC2 Dep domain containing 2 80243................................................................................................................................................................................................................................................................................................................................................................................

CLPB Clpb caseinolytic peptidase b homolog (e. Coli) 81570................................................................................................................................................................................................................................................................................................................................................................................

PPP1R14C Protein phosphatase 1, regulatory (inhibitor) subunit 14c 81706................................................................................................................................................................................................................................................................................................................................................................................

KIF18A Kinesin family member 18a 81930................................................................................................................................................................................................................................................................................................................................................................................

TMUB1 Transmembrane and ubiquitin-like domain containing 1 83590................................................................................................................................................................................................................................................................................................................................................................................

URP2 Unc-112 related protein 2 83706................................................................................................................................................................................................................................................................................................................................................................................

MND1 Meiotic nuclear divisions 1 homolog (S cerevisiae) 84057................................................................................................................................................................................................................................................................................................................................................................................

PROK1 Prokineticin 1 84432................................................................................................................................................................................................................................................................................................................................................................................

DOT1L Dot1-like, histone h3 methyltransferase (S cerevisiae) 84444................................................................................................................................................................................................................................................................................................................................................................................

SLC12A8 Solute carrier family 12 (potassium/chloride transporters), member 8 84561................................................................................................................................................................................................................................................................................................................................................................................

IGSF21 Immunoglobin superfamily, member 21 84966................................................................................................................................................................................................................................................................................................................................................................................

PNMA6A Paraneoplastic antigen like 6a 84968................................................................................................................................................................................................................................................................................................................................................................................

TSLP Thymic stromal lymphopoietin 85480................................................................................................................................................................................................................................................................................................................................................................................

PPP1R3F Protein phosphatase 1, regulatory (inhibitor) subunit 3f 89801................................................................................................................................................................................................................................................................................................................................................................................

MTG1 Mitochondrial gtpase 1 homolog (S cerevisiae) 92170................................................................................................................................................................................................................................................................................................................................................................................

SLC38A5 Solute carrier family 38, member 5 92745................................................................................................................................................................................................................................................................................................................................................................................

DDIT4L DNA-damage-inducible transcript 4-like 115265................................................................................................................................................................................................................................................................................................................................................................................

ZMYND19 Zinc finger, mynd-type containing 19 116225................................................................................................................................................................................................................................................................................................................................................................................

SLC16A10 Solute carrier family 16, member 10 (aromatic amino acid transporter) 117247................................................................................................................................................................................................................................................................................................................................................................................

OSCAR Osteoclast-associated receptor 126014................................................................................................................................................................................................................................................................................................................................................................................

WTIP Wilms tumor 1 interacting protein 126374................................................................................................................................................................................................................................................................................................................................................................................

PUSL1 Pseudouridylate synthase-like 1 126789................................................................................................................................................................................................................................................................................................................................................................................



r((Pb(wIllfidfcfBTap

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eceptor), EREG (epiregulin), BDKRB1bradykinin receptor B1), and IL-13RA2IL 13 receptor, alpha 2). Of the 5, onlyROK1 previously has been reported toe expressed in the reproductive tractendometrial glands) of nonpregnantomen.29 In each instance except for

L-9R (which declined significantly withabor), the microarray analyses and theogic process confirmed that the identi-ed genes were expressed more highlyuring TL, although each was also af-

ected by preterm labor but to a signifi-antly smaller extent. There was a �4-old increase in the expression ofDKRB1, EREG, and IL-13RA2 duringL compared with PTL, and both EREGnd BDKRB1 were increased in TL com-

TABLE 4Genes that initiate term labor (termexcluding hypothetic and other uni

Gene symbol Gene name

B3GNTL1 Udp-glcnac:betagal b...................................................................................................................

FAM132B Family with sequence...................................................................................................................

SGOL1 Shugoshin-like 1 (S p...................................................................................................................

PRELID2 PRELI domain contain...................................................................................................................

CRYGN Crystallin, gamma n...................................................................................................................

TMEM30B Transmembrane prot...................................................................................................................

CCDC138 Coiled-coil domain co...................................................................................................................

SLC30A8 Solute carrier family...................................................................................................................

PPAPDC1A Phosphatidic acid ph...................................................................................................................

ABHD7 Abhydrolase domain...................................................................................................................

OFCC1 Orofacial cleft 1 cand...................................................................................................................

VMO1 Vitelline membrane o...................................................................................................................

GLDN Gliomedin...................................................................................................................

FMN1 Formin 1...................................................................................................................

AADACL2 Arylacetamide deace...................................................................................................................

CA13 Carbonic anhydrase x...................................................................................................................

RASL11A Ras-like, family 11, m...................................................................................................................

APOBEC4 Apolipoprotein b mRN...................................................................................................................

SPINK6 Serine peptidase inhi...................................................................................................................

SNORD68 Small nucleolar RNA,...................................................................................................................

FAM110C Family with sequence...................................................................................................................

PHLDB3 Similar to pleckstrin...................................................................................................................

Entrez (Entrez Global Query Cross-Database Search System) i


ared with TNL. c

The genes that were selected for vali-ation in the preterm initiator genesere CATSPER2 (cation channel, sperm

ssociated 2 pseudogene 1) and PTPRZ1protein tyrosine phosphatase, receptor-ype, Z polypeptide 1). The existence ofhe latter in rodent myometrium haseen suggested previously.30,31 We con-rm both the presence of mRNA fromoth genes in human myometrium andhat they are increased specifically in pre-erm labor associated with inflammationFigure 7).

OMMENTodels of labor that have been proposed

reviously include endocrine and para-

itiator set) in myometrium,tified/unnamed proteins (continued)

-1,3-n-acetylglucosaminyltransferase-like 1.........................................................................................................................

ilarity 132, member B.........................................................................................................................

be).........................................................................................................................

2.........................................................................................................................

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

30b.........................................................................................................................

ining 138.........................................................................................................................

zinc transporter), member 8.........................................................................................................................

hatase type 2 domain containing 1a.........................................................................................................................

taining 7.........................................................................................................................

te 1.........................................................................................................................

r layer 1 homolog (chicken).........................................................................................................................

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

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

se-like 2.........................................................................................................................

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

ber a.........................................................................................................................

diting enzyme, catalytic polypeptide-like 4 (puta.........................................................................................................................

r, kazal type 6.........................................................................................................................

box 68.........................................................................................................................

ilarity 110, member C.........................................................................................................................

ology-like domain, family b, member 1.........................................................................................................................

integrated search and retrieval system at the National Center for


rine regulation, calcium signaling, in- n

MAY 2010 American

ammation caused by infection. Ratherhan compare TL to 1 of many models ofreterm birth, we hypothesized that la-or is defined by a core set of genes thexpression of which is unaltered by GAnd independent of the underlying laborrigger. We applied functional gene arrayrofiling of myometrium that was ob-ained from preterm and term nonlabor-ng and laboring women. Although thiss not the first time that gene array tech-ology has been used to map the molec-lar networks of labor (we first pub-

ished this in 200616), it is apparently therst attempt to differentiate genomically

he molecular events that initiate andaintain myometrial activity in human

abor at term and preterm. In our origi-

Entrezidentification no.

146712..................................................................................................................

151176..................................................................................................................

151648..................................................................................................................

153768..................................................................................................................

155051..................................................................................................................

161291..................................................................................................................

165055..................................................................................................................

169026..................................................................................................................

196051..................................................................................................................

253152..................................................................................................................

266553..................................................................................................................

284013..................................................................................................................

342035..................................................................................................................

342184..................................................................................................................

344752..................................................................................................................

377677..................................................................................................................

387496..................................................................................................................

) 403314..................................................................................................................

404203..................................................................................................................

606500..................................................................................................................

642273..................................................................................................................

653583..................................................................................................................

chnology Information (NCBI) website.

inden

eta......... .........

sim......... .........

om......... .........

ing......... .........

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

ein......... .........

nta......... .........

30 (......... .........

osp......... .........

con......... .........

ida......... .........

ute......... .........

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

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

tyla......... .........

iii......... .........

em......... .........

A e tive......... .........

bito......... .........

C/D......... .........

sim......... .........

hom......... .........

s an Biote

al report, we noted how genomic net-

Journal of Obstetrics & Gynecology 474.e12

A1bfisdpas


4

FIGURE 2Gene ontology table and effector gene set interactive network

, The gene ontology table was generated from the genes of the effector set in the MetaCore analytic suite (version 2.0; GeneGo, St. Joseph, MI). The top0 GeneGo gene ontology pathways are listed based on the number of selected genes that saturate each biologic area. These processes are prioritizedy MetaCore software based on their statistical significance (–log [probability value]) with respect to genes expressed in the dataset. A false discovery ratelter that was set at 10% was also used to illustrate significant processes. The bars that represent experiment mappings that do not pass through thisignificance level are semitransparent. B, Interactive network of the effector gene set was determined with the analyze network algorithm. This algorithmesigns subnetworks that are highly saturated with genes of the effector set. Selection of the network was based on significance (probability value). Therobability value calculation was used to evaluate the network’s relevance to gene ontology biologic processes classification. The network illustrates genend gene interactions within the effector set. Canonic pathways are illustrated by a thick light blue line and represent linear stretches of carefully definedets of consecutive signals that were confirmed as a whole by experimental data. The small colored hexagons on vectors (lines) between nodes


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orks could be used to profile myome-rial events during pregnancy and hereinpply more advanced methods to gener-te signature gene networks.

It is often assumed that the triggersr initiators of human labor are sim-

lar, regardless of gestation, hence an in-estigative focus on TL as a model tonderstand preterm labor. Numerousesearchers have shown that TL is char-cterized by an inflammatory responsend that, at least, most spontaneous pre-erm births are associated with inflam-

ation.8,32-35 The findings of the presenttudy support the premise that inflam-

ation is a central mechanism at termecause such genes were representedeavily in the effector and the term initi-tor gene sets. Although many clinicianshose a tocolytic agent that targets in-ammation (eg, indomethacin), this ap-roach has failed to reduce the preva-

ence of spontaneous preterm birth orysfunctional labor. The results of theresent investigation are more than aimple cataloging of genomic events. Wedentify activity along several pathwayshat have not been known previously toe involved that are part of the effectornd initiator gene sets. In fact, a largeercentage of the involved genes thatere identified in the current investiga-

ion have not been described previouslyn myometrium, much less shown to beltered by disorders of labor. We recog-ize that this is a first step. It remainsow to explore the functional roles of the

dentified pathways to see how alteringhem affects the process of labor.

The effector gene set (Figure 2, A) en-ompasses genes the products of whichre involved in chemotaxis, cell adhe-ion, reproduction and neurohormoneignaling, signal transduction, muscleontraction, vascularization and, asoted earlier, inflammation. Microarrayndings were confirmed for HNT,CNAB2, and RRAD. HNT is grouped

describe positive (green), negative (red), or unsper protein) and is marked with a graphic symbolhe large red circle next to the network object cof this network according to their subcellular loceiner. Human myometrial effector/initiator gene sets for la

n GO processes that are related to bio-ogic and cell adhesion and cell recogni-ion, all of which are logical needs to sus-ain labor. The protein that is encoded is

member of the immunoglobulin do-ain that contains glycosylphosphatidy-

inositol-anchoring adhesion molecules.s such, HNT may play a role in the co-rdination of myometrial cell contrac-ions. Its expression has not been shownreviously in human myometrium or toe affected by labor.We previously demonstrated that

regnancy and sex hormones (estrogennd progesterone) alter G-protein–me-iated signaling,36 at least in part byodulating myometrial GTPase activity.RAD encodes a GTPase molecule thatas identified previously in the myocar-iocyte and shown to interact with cal-odulin, tropomyosin, and CaM kinase

I among others. Its expression in laboringyometrium is a new finding. Analyses

laced it into the node with the most sig-ificant likelihood of not having occurredt random (gScore, 50.44). As a member ofhe effector gene set, it could play a roleoordinating and modulating the strengthf the myometrial contraction.KCNAB2 is another effector gene setember of interest. This voltage-gated

otassium channel represents the mostomplex class of voltage-gated ion chan-els from both functional and structuraltandpoints and fell into the secondighest GO group (gScore, 33.6). Inther anatomic structures, they are in-olved in the regulation of the contrac-ion rate in regularly beating muscles. Aumber of investigators have concluded

hat voltage-gated potassium channelsre involved fundamentally in the regu-ation of rodent myometrial contractil-ty.37-39 Other voltage-gated potassiumhannels are regulated by sex hor-ones.40,41 We previously demon-

trated that the human chorion pro-uces a factor or factors that inhibit

ed (black) interactions or logical relationships (blureflects the type of the network object that is re

ponds to those genes detected in the effector setion. The localization sectors are nucleus, cytop

Am J Obstet Gynecol 2010.

MAY 2010 American

yometrial contractility through thepening of a large conductance calcium-ctivated potassium channels.42 KCNAB2ay help generate the increased frequency

f myometrial contractions that are asso-iated with labor, regardless of GA.

Most of the pregnancy passes with theyometrium in a state of quiescence,hich is a genomically distinct phase

hat renders it relatively unresponsive toontractile agonists. Labor follows myo-etrial activation, possibly because of

omplex alterations in the signalingechanisms. We hypothesized that, in

ontrast to the effector set, there mustxist specific initiator gene sets that areesponsible for that transition from qui-scence to activation that reflect the spe-ific underlying stimulus. The results ofhe current study support that workingypothesis. For example, the cytokineetwork is in sensitive balance through-ut uncomplicated pregnancy, and weuspect a similar balance exists for en-oded cytokines during pregnancy.owever, despite the presence of severalO processes in the effector gene set that

eflect inflammation, ILs are not seen inhat grouping except for IL- 8 receptor,eta. In myocardium, IL-8 is up-regu-

ated in areas of infarction and may in-uce neutrophil infiltration.43 In con-rast to the effector gene set, severalytokine genes are present in the termnitiator gene set, which include IL-1 re-eptor-associated kinase 3, IL-24, IL-beta, IL-13RA2, and IL-9R (the latteras the only conflict between microarray

nd qRT-PCR). Not found in any of theene sets was IL-10, which has been im-licated as an antiinflammatory modu-

ator of cytokine synthesis that is initi-ted by intrauterine infection.44-48 Itsbsence suggests it does not have a sig-ificant role in either inflammation pre-

erm labor or TL.We have previously noted that myome-

rial quiescence and activation are associ-

A node correspondsto a network object (ie, genesented (ie, enzyme, transcription factor, ligand).e network objects are placed in specific vectors, membrane, extracellular, and unspecified.

cifi e).that prerres t. Thaliza lasmbor.


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FIGURE 3Gene Ontology table and term initiator subnetwork

, Similar to Figure 2, A, this gene ontology table is representative of the term initiator gene set. The genes that are included are more varied than thosef the preterm initiator set. B, Similar to Figure 2, B, this term initiator subnetwork was generated with the analyze network algorithm (Metacore analyticuite, version 2.0; GeneGo, St. Joseph, MI). The large red circle next to the network object corresponds to those genes that were detected in the termnitiator group. Selection of the network was based on significance (probability value).IF, macrophage inhibitory factor.



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ted with altered G protein expression andTPase activity in both the guinea pig anduman.16,36 In addition to the expressionf RRAD in the effector gene set, thereere several G-protein related proteins in-

FIGURE 4Gene ontology table and preterm in

, The gene ontology table shows the major genflammation and the preterm initiator gene set.nalytic suite; version 2.0; GeneGo, St. Joseph, Mreterm initiator group. Selection of the network

FN, interferon; IL-10, interleukin 10; TCR, T-cell receptor; TREM1

einer. Human myometrial effector/initiator gene sets for la

luded in the term initiator gene set in- t

luding G protein-coupled receptor 84GPR84), rab39 member of ras oncogeneamily (RAB39), ras-like family 11 mem-er A (RASL11A), and ras association do-ain family 1 (RASSF1), most of which

ator set subnetwork

ontology pathways underlying the preterm initiaThe preterm initiator set subnetwork was generThe large red circle next to the network object cos based on significance (probability value).gering receptor expressed on myeloid cells.


here is little known and not previously c

MAY 2010 American

ocumented to be expressed in theyometrium.The presence of BDKRB1 and EREG

xpression in the term initiator set areoth provocative. There was �4-fold in-

gene set. There is a high correlation betweenwith the analyze network algorithm (MetaCore

sponds to those genes that were detected in the

iti

ne torB, atedI). rre

wa, trig

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rease in expression of BDKRB1 and


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REG compared with PTL (Figure 6),hich suggests preferential involvement

n TL. Contractile responses of nonpreg-ant human myometrium to bradykininre reduced during the luteal phase.49

assdal et al50 noted that bradykininontracted rodent myometrium throughhe same pathway as did oxytocin. In cul-ured decidua-derived cells, bradykinintimulates the release of arachidoniccid, IL-6, and IL-8. These effects arerevented by the specific B2R antagonistoe 140.51 Further, human decidua-de-

ived cells express the B2R; its expressions up-regulated in response to IL-�, andradykinin stimulates the secretion ofurther mediators by these cells. Finally,radykinin is purported to trigger the re-

ease of prostaglandin E2 from humanyometrium.52 Each of these character-

stics of bradykinin could facilitate thenset of TL. Epiregulin is expressed inyometrium from pregnant mice but

ot myometrium of pseudo pregnantice.53 Induction of its synthesis has

een linked to the RAF/MEK/ERKathway.54

In accordance with our current work-ng hypothesis, we found that genevents that are associated with myome-rial activation and TL are not the sames those that are associated with pretermabor. Not surprisingly in light of the pa-ient sample that was chosen for study,he preterm initiator gene set was domi-ated by inflammatory processes. Theresent study focused on inflammation-ediated preterm labor because it is theost plentiful and the best described. Al-

hough not tested, our hypothesis re-uires that the initiator set differ inomen who experience preterm labor

hat is unassociated with inflammationeg, over distension with multipleestation).Despite the strong weighting of GeneGo

rocesses toward inflammation in the pre-erm initiator gene set, there were a num-er of other genes that were worthy ofollow-up evaluation in the future. For ex-mple, glutathione S-transferase theta 2 ismember of a superfamily of proteins thatatalyze the conjugation of reduced gluta-hione to a variety of electrophilic and hy-rophobic compounds. Human GSTs are
ubdivided into 5 classes: alpha, mu, pi, t
74.e17 American Journal of Obstetrics & Gyneco

heta, and zeta. The theta members GSTT1nd GSTT2 share 55% amino acid se-uence identity and are both thought toave an important role in human carcino-enesis. An increase in its protein could re-ect an increase in oxygen free radical pro-uction. We previously demonstrated that

nflammation-related preterm labor is as-ociated with a prooxidant state in theewborn infant that is characterized by aecrease in reduced glutathione.55

The preterm initiator set also includedhe transporter, ATP-binding cassette,ub-family A member 3 (ABCA3) whoserotein is a member of the ABC1 sub-

amily. Members of the ABC1 subfamilyomprise the only major ABC subfamilyhat is found exclusively in multicellularukaryotes and may be involved in theevelopment of resistance to xenobioticsnd engulfment during programmed celleath. The function of ABCA3 in myome-

FIGURE 5Effector set

he effector set represents genes that sustain myNT, KCNAB2, and RRAD genes were selected fNA (mRNA) levels were determined in the myomreterm with inflammation (n � 6; 30.9 weeksot-in-labor (term: n � 6 [39.6 weeks of gestatuantitative real-time polymerase chain reactionnd Methods, qRT-PCR). Microarray findings werndings confirm both the microarray findings and

abor, regardless of its timing.TL, preterm labor; PTNL, preterm not in labor and no inflammati

einer. Human myometrial effector/initiator gene sets for la

rium is unknown, but 1 possible role in l

logy MAY 2010

he initiation of inflammation-associatedreterm labor could involve the exposurend presentation of potential antigens thatrigger inflammation as a secondary re-ponse to the initiating event. The presenttudy is the first report of its expression in

yometrium and the impact of spontane-us preterm labor on it.It is becoming increasingly clear that

he pattern of cytokine expression differsn preterm labor and TL.56 Therefore,ytokine expression may also differmong various tissues (ie, myometrium)t different gestations. Increased proteinnd/or mRNA concentrations of IL-1�,umor necrosis factor alpha (TNF-�),nd IL-6 in myometrium have each pre-iously been associated with labor.44 Inhe present study, none were included inhe preterm initiator gene set, which sug-ests that the production is a down-tream event or not specific to preterm

trial contractility and are unaffected by stimulus.tudy to verify the microarray results. Messengerum of term (n � 6; 39.7 weeks of gestation) andgestation) pregnant women in active labor and; preterm: n � 6 [28.9 weeks of gestation]) byh the use of the Livak method (refer to Materialsnfirmed for all but 1 gene tested. In general, thepresence of core genes that are associated with

L, term labor; TNL, term not in labor.


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MAY 2010 American

ndings of previous investigators whoound that the levels of cytokines (suchs IL-1�, IL-6, and IL-8) were higher inL, compared with PTL myometrial

amples.57 The results of the currenttudy do not support the use of TNF-� asbiomarker for inflammation that spe-

ifically is associated with preterm labor.he TNF-� gene encodes a multifunc-

ional proinflammatory cytokine thatelongs to the TNF superfamily. It is in-olved in the regulation of a wide spec-rum of biologic processes that includeell proliferation, differentiation, apo-tosis, lipid metabolism, and coagula-ion. Elevated transcription and accu-

ulation of TNF-� and other cytokinesn gestational tissues are proposed to ac-ivate prostaglandin synthesis that, inurn, leads to the myometrial contrac-ions.58,59 Although not present in thereterm initiator gene set, it is a compo-ent of the term initiator gene set and

hus may play a role with normal labor.Regional differences in gene expres-

ion between the fundus, lower segment,nd cervix were reported in a previoustudy.12 In the current study, we used bi-psy specimens from the upper edge ofhe lower uterine segment and empha-ize that our findings must be inter-reted in this context. However, we pre-iously determined that the collagen/uscle ratio is 20%/80% in lower

egment biopsy specimens that were col-ected from the upper lip of the inci-ion.60 Thus, we believe that the muscleomponent to our biopsy specimens isignificant. Importantly, we found a sig-ificant increase in the contribution of

he muscular component in laboring tis-ues that indicate that the lower segmentarticipates morphologically in the pro-ess of labor. In the current study, ouronreliance on differences in levels ofene expression to derive biologic signif-cance, but rather by intersecting theresence of genes in biologically relevant

nstances, should circumvent minor dif-erences in regionalization of the myo-

etrium vs the collagen componentith either GA or labor.Finally, it is interesting to look at the

enes that are not included on any of thegene sets described herein. It is striking

FIGURE 6Term initiator set

erm initiator set genes EREG, BDKRB1, IL13RA2 messenger RNA (mRNA) levels were significantly greatert term labor (TL; open bar) than preterm labor (PTL; closed bar) in myometrium. Messenger RNA levelsere determined in the myometrium of term (n � 6; 39.7 weeks of gestation) and preterm with inflam-ation (n � 6; 30.9 weeks of gestation) pregnant women in active labor and not-in-labor (term: n � 6

39.6 weeks of gestation]; preterm: n � 6 [28.9 weeks of gestation]) by quantitative real-time polymerasehain reaction with the use of the Livak method (refer to Materials and Methods, qRT-PCR). Microarrayndings were confirmed for all but 1 gene tested. EREG and BDKRB1 messenger RNA levels were signif-

cantly higher at labor (open bars) than nonlabor (closed bars) in term myometrium. Only interleukin 9RIL9R ) quantitative real-time polymerase chain reaction results were different from the microarray.TNL, preterm not in labor and no inflammation; TNL, term not in labor.


FIGURE 7Preterm initiator set

essenger RNA (mRNA) levels were determined as indicated previously. Changes in the messengerNA levels for genes Catsper2 and Ptprz1 were consistent with the array data.einer. Human myometrial effector/initiator gene sets for labor. Am J Obstet Gynecol 2010.

hat no list includes oxytocin or an oxy-


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ocin receptor. Because it is recognizedhat oxytocin can induce its own recep-or in rodent myometrium, it is surpris-ng neither gene was present in the effec-or set, nor does 1 of the 3 lists includeepresentation of the eicosanoids or theirelated enzymes. Although there are nu-erous cytokines or cytokine pathway

lements included in 1 of the 3 sets, IL-6,ts receptor, TNF-� or its receptor, orL-10 is not included. Their absence doesot suggest that these compounds areot involved in human parturition.ather, it suggests their primacy in therocess is other than anticipated and thatheir actions may be downstream events.lternatively, there is no uniqueness in

heir expression during spontaneouserm or inflammation-associated pre-erm labor.

In conclusion, we have identified,hrough gene profiling, a specific set ofabor activation/repression genes (the ef-ector gene set) whose expression is un-ffected by the timing of the labor. Fu-ure searches for new tocolytic agents

ight benefit from a focus on thesenique gene products. Although the ef-

ector gene set is unchanged by the ges-ation of the labor, preterm labor and TLiffer dramatically in their initiator geneets. This finding is consistent with theuggestion that there are alternativeathways for preterm labor and TL thatrigger a common phenotype. Last, weonfirm that TL and preterm labor thatre associated with inflammation areharacterized by an inflammatory re-ponse but that they are not the same re-ponses and they do not include severalreviously “in vogue” genes. Thus, thelassic evidence for inflammation as aause of spontaneous preterm labor mayell be a downstream event from myo-etrial gene activation. f

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Human effector/initiator gene sets that regulate myometrial contractility during term and preterm labor