research article gene expression profiling of the...

Research ArticleGene Expression Profiling of the Paracrine Effects of UterineNatural Killer Cells on Human Endometrial Epithelial Cells

Xin Gong1 Zhenzhen Chen2 Yanxia Liu1 Qiudan Lu1 and Zhe Jin1

1 Reproductive Endocrinology Centre Dongfang Hospital of Beijing University of Chinese Medicine No 6 Fangxingyuan 1 QuFengtai District Beijing 100078 China

2 School of Chinese Materia Medica Beijing University of Chinese Medicine No 6 Zhonghuan South Road WangjingChaoyang District Beijing 100102 China

Correspondence should be addressed to Zhe Jin zjin0129163com

Received 19 December 2013 Accepted 18 February 2014 Published 26 March 2014

Academic Editor F Xavier Donadeu

Copyright copy 2014 Xin Gong et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The endometrium contains a population of immune cells that undergo changes during implantation and pregnancy The majorityof these cells are uterine natural killer (uNK) cells however it is unclear how these cells interact with endometrial epithelial cellsTherefore we investigated the paracrine effects of the uNK cell-secretion medium on the gene expression profile of endometrialepithelial cells in vitro through microarray analysis Our results which were verified by qRT-PCR and western blot revealed thatsoluble factors from uNK cells alter the gene expression profiles of epithelial cells The upregulated genes included interleukin-15 (IL-15) and interleukin-15 receptor alpha (IL-15RA) which result in a loop that stimulates uNK cell proliferation In additionvascular endothelial growth factor C (VEGF-C) and chemokine (C-X-C motif) ligand 10 (CXCL-10) were also determined to beupregulated in epithelial cells which suggests that uNK cells work synergistically with epithelial cells to support implantation andpregnancy In addition oriental herbal medicines have been used to treat infertility since ancient times however we failed to findthat Zi Dan Yin can regulate these endometrial paracrine effects

1 Introduction

In the human endometrium uterine leucocytes undergocyclic changes during the menstrual cycle Uterine adap-tation to pregnancy includes highly regulated immune celltrafficking [1] According to recent studies uterine naturalkiller (uNK) cells are an in vivo factor that contributes tothe pathological elongation of the window of endometrialreceptivity [2] The genetic deficiency of uNK cells resultsin a lack of remodeling of the uterine vasculature leadingto hypertrophied vascular media swollen endothelial cellsand narrow vessel lumens [3] Abnormal activation andnumbers of uNK cells have been implicated in pregnancycomplications [4]

uNK cells are the most abundant leucocytes during theimplantation window and early pregnancy [5] This sharpincrease is likely due to the proliferation of existing uNKcells [6ndash8] Hormonal effects may control this proliferationHowever to date progesterone receptors have not been

localized on these cells [9] therefore it has been proposedthat progesteronemay exert its effects on uNK cells indirectlyvia cytokines such as interleukin-15 (IL-15) and other solublefactors [8 10] A previous study indicated that the paracrinecommunication between uterine leucocytes (gt85uNK) anduterine stromal cells can upregulate IL-15 and interleukin-15receptor alpha (IL-15RA) to induce the proliferation of uNKcells and contributes to the support of trophoblast migrationduring implantation [11]

uNK cells are resident throughout the endometrium atthe time of conception and throughout early pregnancyAlthough uNK cells predominantly localize to the uterinestroma and the surrounding areas of the spiral arteriesMatrigel-supported cocultures of endothelial cells and uNKcells have shown that vascular endothelial growth fac-tor C (VEGFC) producing uNK cells induce transporter1 (TAP-1) expression in endothelial cells [12] Furthermorethe endometriumundergoes precisely definedmorphologicaland biochemical changes during implantation and early

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2014 Article ID 393707 15 pageshttpdxdoiorg1011552014393707

2 International Journal of Endocrinology

Table 1 Composition of Zi Dan Yin (ZDY)

Components Ratio(1) Sheng Di (Rehmannia glutinosa (Gaertn) Libosch root) 15(2) Dan Shen (Salviae Miltiorrhizae Bge root) 10(3) Dang gui (Angelica sinensis (Oliv) Diels root) 12(4) Chuan Duan (Dipsacus asperoides C Y Cheng et TMAi root) 15

(5) Du Zhong (Eucommia ulmoides Oliv cortex) 12(6) Shan Yao (Dioscorea oppositaThunb rhizome) 15(7) Mei Gui-hua (Rosa rugosaThunb flower) 6(8) Chuan Xiong (Ligusticum Chuanxiong Hort rhizome) 6(9) Yi Yi-ren (Coix lacryma-jobi L var ma-yuen (Roman)Stapf seed) 12

pregnancy thus there is no doubt that the preparation ofepithelial cells is directed towards these process We hypoth-esize that there similar paracrine communications occurbetween uNK cells and uterine epithelial cells Our resultsrevealed that soluble factors from uNK cells have substantialeffects on endometrial epithelial gene expression uNK cellswere found to upregulate transcript levels in epithelial cellsthat are known to promote uNK cell proliferation and theseupregulated transcripts may contribute to the preparation forimplantation and trophoblast migration and invasion

In China oriental herbal medicines have been used totreat infertility since ancient times [13] Zi Dan Yin (ZDY)a compound of nine herbs (Table 1) was found to improvethe morphology of the endometrium in mice [14] Althoughthe treatment effects of ZDY on mice have been confirmedit is unclear whether ZDY contributes to the local immuneresponses However our results do not support the hypothe-sis that ZDY can improve the paracrine effects between uNKand endometrial epithelial cells

2 Materials and Methods

21 Ethics Statement All of the subjects understood andsigned the informed consent form before participation Theexperimental protocolswere approved by the Ethics Commit-tee of the Dongfang Hospital Human Ethics Committee (no2011090201)

22 Collection of Material Decidual tissues were obtainedfrom healthy women undergoing elective termination ofa normal pregnancy between 7 and 8 weeks of gestationThe endometrial tissues were collected from endometrialbiopsies taken during the proliferative phase of the cyclefrom women undergoing laparoscopy (Dongfang Hospital ofBeijing University of Chinese Medicine China) for benigndisease The average age was 30 plusmn 13 years and the bodymass index (BMI) was 221 plusmn 08 kgm2 All of the ninewomen were Chinese of Han ethnicity The exclusion criteriawere hormonal stimulation cancerous lesions and irregularmenstrual bleeding

23 Isolation of the Uterine NK (uNK) Cells The uNK cellswere isolated as previously described [15 16] Briefly thedecidual tissues were extensively washed with Ca2+- andMg2+-free Hankrsquos balanced salt solution (HBSS) containingantibiotics minced thoroughly between two scalpels intofragments of 1-2mm3 and digested for 1 h at 37∘Cwith gentleagitation in HBSS with 01 (wv) collagenase I (GibcoUSA)The cell suspensions were layered over Ficoll-Hypaquemedium (General Electric USA) and centrifuged at 800timesgand room temperature for 25min The cells at the interfacewere washed twice with Roswell Park Memorial Institute(RPMI)-1640 media with 10 fetal calf serum (FCS) andantibiotics After incubation for 20min at 4∘C with anti-CD56 microbeads (Miltenyi Biotec Ltd Germany) thecells were washed with washing buffer (PBS 2mM EDTAand 05BSA (wv)) and then loaded onto a MiniMACSSeparator (MS) column in a MiniMACS magnet (MiltenyiBiotec Ltd Germany) After flushing the MS column threetimes the CD56+ cells were flushed as indicated by the man-ufacturerThe purity of the uNK cells was gt90CD56+CD3minusaccording to the flow cytometric analysis The uNK cellswere cultured in RPMI 1640 media with 1 FCS and IL-15(10 ngmL) (RampD systems USA) in which to maintain theviability of the purified uNK cells [17]

24 Production of uNKCell-SecretionMedium TheuNKcell-secretion medium was prepared using 200120583L of RPMI 1640media with 1 FCS and IL-15 (10 ngmL) containing 5times105 ofthe purified uNK cells The cell suspension was placed in theupper chamber of a 04 120583m pore hanging cell culture insert(Millipore USA) in a 24-well tissue plate and 1300 120583L of thesamemediawithout cells was placed in the lower chamber Asa result only the soluble molecules from the uNK cells canpass through the filter into the lower chamber The controlmedium was 1500120583L of the RPMI 1640 media with 1 FCSand IL-15 (10 ngmL) and this control medium was alsoused in the subsequent experiments After incubation for24 h at 37∘C the uNK cell-secretion medium (in the lowerchamber) and the control medium were collected and frozenat minus80∘C The cells in the upper chamber were collected andthe cell viability was measured using a livedead viability kit(Invitrogen USA) Only the conditional medium from uNKpopulations with less than 35 dead cells after overnightincubation was used in the subsequent experiments

25 Human Endometrial Epithelial Cell Culture Humanendometrial tissue was cut using scissors and dissociated intosingle-cell suspensions using 01 (wv) collagenase I for 50ndash60min Every 15min the digests were pipetted and their dis-sociation was monitored by microscopyThe cell suspensionswere filtered using a 40-mesh screen to separate the singlecells from undigested tissue To remove the erythrocytes thecells were resuspended in 4mL ofDulbeccorsquosmodified Eaglersquosmedium and Hamrsquos nutrient F12 (DMEMF12) with 1 FCSlayered over Ficoll-Paque PLUS and centrifuged for 25minat 800timesg The leukocytes were removed with CD45-coatedDynabeads (Dynal Biotech USA)The purified epithelial cellsuspensions were then obtained through a further round of

International Journal of Endocrinology 3

Table 2 Sequences of primers used in the quantitative real-time PCR analysis

Gene Forward primer Reverse primer AmpliconCXCL-10 CTTTCTGACTCTAAGTGGCATTC CACCCTTCTTTTTCATTGTAGCAA 176 bpIL-15 TGGCTGCTGGAAACCC CACAAGTAGCACTGGATGGAAAT 123 bpICAM-1 GAGGAAGGAGCAAGACTCAA AGCATACCCAATAGGCAGCAAG 141 bpNLRC-5 AAACTTGATGACTCCTCCCTTACTT TTAGACCTGGCTTTGTCCCTTAC 120 bpIRF-1 CCAGAAAAGCATAACACCAATCC CCACTTTCCTTCACATTTCACTG 144 bpGAPDH GAGCCAAAAGGGTCATCATCT AGGGGCCATCCACAGTCTTC 231 bp

magnetic bead sorting using Collection Epithelial Dynabeads(Dynal Biotech USA) The epithelial cell preparations weregt95 pure

The endometrial epithelial cells were cultured in serum-free bronchial epithelial growth medium (BEGM CloneticsCrop) containing 2mL of bovine pituitary extract (BPE)05mL of insulin 05mL of hydrocortisone (HC) 05mL ofgentamicin sulfate and amphotericin-B (GA-1000) 05mLof retinoic acid 05mL of transferrin 05mL of triiodothy-ronine 05mL of epinephrine and 05mL of recombinanthuman epidermal growth factor (hEGF) (all supplied byClonetics Corp USA) The media was changed every fivedays After two weeks the epithelial cells were passed fromsix-well plates into 25 cm2 cell culture flasks

26 ZDY Preparation All of the crude drugs of ZDY wereobtained from the Pharmacy Department of Dongfang Hos-pital of Beijing University of Chinese Medicine (BeijingProvince China)The quality of the raw herbs was controlledaccording to the requirement of the Pharmacopoeia of ChinaThe aqueous extract of ZDY was prepared as follows Inbrief nine medicinal materials were mixed at the appropriateproportion macerated for 1 h with eight volumes of distilledwater and then decocted for 2 h The cooled extract was thenfiltered This extraction procedure was repeated twice Theextracts were then combined and concentrated by boiling toa final volume of 100mL (103 gmL) The ZDY extract con-centration of 2mgmL was further diluted with the epithelialcell culture medium and passed through 02 120583m sterilizationfilters for use in subsequent studies The ZDY extract wasstored at 2ndash8∘C until further use The concentration of ZDYwas used according to previous analyses using a cell countingkit-8 (CCK-8)

27 Treatment of Epithelial Cells with uNK Cell-SecretionMedium The endometrial epithelial cells were allocated intothree groups control uNK and ZDY groupsThe cells in thecontrol and uNK groups were treated with DMEM mediumwith 10 FBS for 24 h and the cells in the ZDY groupwere treated with 2mgmL ZDY for 24 h All of the cellswere then washed twice with PBS and placed in serum-free DMEM for 24 h prior to the subsequent experimentsThe uNK cell-secretion medium was pooled from differentbatches to decrease the variability The DMEM in the ZDYand uNK groups was then replaced with 80 uNK cell-secretion medium and 20 serum-free DMEM The control

groupwas treated with 80 controlmedium and 20 serum-free DMEM After 6 h of incubation the cells from the threegroups were collected

28 Microarray Experiments The total RNA from endome-trial epithelial cells from the control uNK and ZDY groupswas extracted using Trizol (Invitrogen USA) The RNAwas purified using the RNeasy Mini Kit (Qiagen Germany)according to the manufacturerrsquos instructions quantified byspectrophotometry The microarray analysis was performedusing the GeneChip 31015840 IVT Express Kit (Affymetrix) Brieflythe total RNA was subjected to reverse transcription first-strand cDNA synthesis double-strand DNA in vitro tran-scription and fragmentation The samples were hybridizedonto the GeneChip PrimeView Human Gene ExpressionArray (Affymetrix USA) which covers more than 36000transcripts and variants After 16 h of hybridization at 45∘Cthe arrays were washed on a Fluidics Station 450 (AffymetrixUSA) and then scanned with a Scanner 3000 (AffymetrixUSA) to obtain the quantitative gene expression levels Theendometrial epithelial cells from the control uNK andZDY groups were processed simultaneously throughout theexperimental process and three chips were used for eachgroup

29 Quantitative Real-Time PCR (qRT-PCR) Analysis Toverify the results obtained from the microarray experi-ments qRT-PCR verification was performed for five geneschemokine (C-X-C motif) ligand 10 (CXCL-10) interleukin-15 (IL-15) intercellular adhesion molecule 1 (ICAM-1) NLRfamily CARDdomain containing 5 (NLRC-5) and interferonregulatory factor 1 (IRF-1) The cells from the control uNKand ZDY groups were washed twice with PBS and thetotal RNA was then extracted using Trizol The RNA wasquantified by spectrophotometry Reverse transcription wasperformed with 8120583L of total RNA in a 20120583L reactionvolume using a standard cDNA Synthesis Kit (Takara BIOJapan) The qRT-PCR primer sequences for the target geneswere self-designed and the PCR primers were ordered fromInvitrogen The primer sequences for target genes are shownin Table 2

For each qRT-PCR reaction the typical thermal cyclingconditions included an initial activation step of 95∘C for5min and 45 cycles of 95∘C each for 30 sec 65∘C for 35 secand 72∘C for 5min The PCR reactions were performed onan ABI Prism 7700 Sequence Detection System (AppliedBiosystemsUSA) For comparison of the targetmRNA levels


Table 3 Transcripts that were altered bymore than twofold in endometrial epithelial cells by stimulationwith the uNK cell-secretionmedium(119875 lt 0001) The transcript levels were obtained using a GeneChip PrimeView Human Gene Expression Array

Gene symbol Foldchange

GeneID Description

Upregulated genes 40 transcriptsCytokinesmembraneproteinsimmunological factors

NLRC5 141 84166 NLR family CARD domain containing 5IL15 58 3600 Interleukin-15IFIT3 58 3437 Interferon-induced protein with tetratricopeptide repeats 3CXCL10 31 3627 Chemokine (C-X-C motif) ligand 10IFIH1 29 64135 Interferon induced with helicase C domain 1IFIT5 28 24138 Interferon-induced protein with tetratricopeptide repeats 5IL-15RA 27 3601 Interleukin-15 receptor alphaVEGFC 25 7424 Vascular endothelial growth factor CPDCD1LG2 25 80380 Programmed cell death 1 ligand 2

TransportersTAP1 34 6890 Transporter 1 ATP-binding cassette sub-family B (MDRTAP)

TAP2 26 6891 Transporter 2 ATP-binding cassette sub-family B(MDRTAP)

APOL3 21 80833 Apolipoprotein L 3VLDLR 21 7436 Very low density lipoprotein receptorAPOL2 21 23780 Apolipoprotein L 2

Structural factorsLMNB1 43 4001 Lamin B1

TranscriptionIRF1 11 3659 Interferon regulatory factor 1IRF9 49 10379 Interferon regulatory factor 9STAT1 45 6772 Signal transducer and activator of transcription 1 91 kda

TAF15 21 8148 TAF15 RNA polymerase II TATA box binding protein(TBP)-associated factor 68 kda

Cell-cell adhesionICAM1 51 3383 Intercellular adhesion molecule 1

Ion bindingC1S 32 716 Complement component 1 s subcomponentSP110 29 3431 SP110 nuclear body proteinPARP12 23 64761 Poly (ADP-ribose) polymerase family member 12ZC3HAV1 23 56829 Zinc finger CCCH-type antiviral 1

Tryptophan metabolismIDO1 62 3620 Indoleamine 23-dioxygenase 1WARS 42 7453 Tryptophanyl-trna synthetase

Enzyme activityDTX3L 46 151636 Deltex 3-like (Drosophila)

PSMB9 33 5698 Proteasome (prosome macropain) subunit beta type 9 (largemultifunctional peptidase 2)

MSRB1 25 51734 Methionine sulfoxide reductase B1CTSZ 22 1522 Cathepsin Z

Nucleotide metabolismGBP5 105 115362 Guanylate binding protein 5GBP1 58 2633 Guanylate binding protein 1 interferon-induciblePARP14 52 54625 Poly (ADP-ribose) polymerase family member 14GBP2 32 2634 Guanylate binding protein 2 interferon-inducible


Table 3 Continued


GeneID Description

MX1 25 4599 Myxovirus (influenza virus) resistance 1 interferon-inducibleprotein p78 (mouse)

RAD23B 23 5887 RAD23 homolog B (S Cerevisiae)GBP7 2 388646 Guanylate binding protein 7

OthersFAM117B 36 150864 Family with sequence similarity 117 member BC19orf66 26 55337 Chromosome 19 open reading frame 66NUB1 25 51667 Negative regulator of ubiquitin-like proteins 1

Downregulated genes 129 transcriptsCytokinesmembraneproteinsimmunological factors

IL36G minus34 56300 Interleukin-36 gammaIL33 minus30 90865 Interleukin-33IL1RN minus25 3557 Interleukin-1 receptor antagonistDNAJB14 minus25 79982 Dnaj (Hsp40) homolog subfamily B member 14KITLG minus23 4254 KIT ligandLARP4 minus23 113251 La ribonucleoprotein domain family member 4CLDN4 minus22 1364 Claudin 4CUL5 minus22 8065 Cullin 5

Intracellularsignalling factorsITGB8 minus35 3696 Integrin beta 8RICTOR minus28 253260 RPTOR independent companion of MTOR complex 2IL6ST minus26 3572 Interleukin-6 signal transducer (gp130 oncostatin M receptor)SOCS4 minus25 122809 Suppressor of cytokine signaling 4NRIP1 minus25 8204 Nuclear receptor interacting protein 1

SEMA3A minus25 10371 Sema domain immunoglobulin domain (Ig) short basicdomain secreted (semaphorin) 3A

PLCB4 minus22 5332 Phospholipase C beta 4

COPS2 minus22 9318 COP9 constitutive photomorphogenic homolog subunit 2(Arabidopsis)

BMPR2 minus2 659 Bone morphogenetic protein receptor type II(serinethreonine kinase)

TransportersTPR minus34 7175 Translocated promoter region nuclear basket proteinKIAA1033 minus25 23325 Kiaa1033BAZ2B minus25 29994 Bromodomain adjacent to zinc finger domain 2BTMED5 minus22 50999 Transmembrane emp24 protein transport domain containing 5

SLC4A7 minus2 9497 Solute carrier family 4 sodium bicarbonate cotransportermember 7

Structural factorsROCK2 minus29 9475 Rho-associated coiled-coil containing protein kinase 2

Transcription

BDP1 minus36 55814 B double prime 1 subunit of RNA polymerase III transcriptioninitiation factor IIIB

TRIP11 minus34 9321 Thyroid hormone receptor interactor 11ZNF644 minus33 84146 Zinc finger protein 644CHD9 minus32 80205 Chromodomain helicase DNA binding protein 9ZNF480 minus31 147657 Zinc finger protein 480


Table 3 Continued


GeneID Description

ZNF292 minus3 23036 Zinc finger protein 292ZNF148 minus28 7707 Zinc finger protein 148ZNF267 minus28 10308 Zinc finger protein 267ZBTB20 minus27 26137 Zinc finger and BTB domain containing 20CREB1 minus27 1385 Camp responsive element binding protein 1ZNF638 minus26 27332 Zinc finger protein 638ZNF518A minus26 9849 Zinc finger protein 518A

ESF1 minus26 51575 ESF1 nucleolar pre-rrna processing protein homolog (SCerevisiae)

FAR1 minus26 84188 Fatty acyl coa reductase 1ZNF91 minus25 7644 Zinc finger protein 91CENPF minus25 1063 Centromere protein F 350400 kda (mitosin)FOXN2 minus24 3344 Forkhead box N2DNTTIP2 minus22 30836 Deoxynucleotidyl transferase terminal interacting protein 2TMF1 minus22 7110 TATA element modulatory factor 1BCLAF1 minus22 9774 BCL2-associated transcription factor 1CEBPZ minus22 10153 CCAATenhancer binding protein (CEBP) zetaZNF146 minus21 7705 Zinc finger protein 146

HIF1A minus2 3091 Hypoxia inducible factor 1 alpha subunit (basichelix-loop-helix transcription factor)

Cell-cell adhesionDST minus29 667 Dystonin

Ion bindingATRX minus46 546 Alpha thalassemiamental retardation syndrome X-linkedEEA1 minus34 8411 Early endosome antigen 1RSF1 minus26 51773 Remodeling and spacing factor 1NIN minus26 51199 Ninein (GSK3B interacting protein)SCAF11 minus25 9169 SR-related CTD-associated factor 11MIB1 minus2 57534 Mind bomb E3 ubiquitin protein ligase 1TRMT13 minus2 54482 Trna methyltransferase 13 homolog (S Cerevisiae)

KinaseCCDC88A minus31 55704 Coiled-coil domain containing 88AFER minus31 2241 Fer (fpsfes related) tyrosine kinaseKTN1 minus3 3895 Kinectin 1 (kinesin receptor)SCYL2 minus26 55681 SCY1-like 2 (S Cerevisiae)AKAP11 minus25 11215 A kinase (PRKA) anchor protein 11

PIK3C2A minus25 5286 Phosphatidylinositol-4-phosphate 3-kinase catalytic subunittype 2 alpha

CDK6 minus23 1021 Cyclin-dependent kinase 6NEK1 minus23 4750 NIMA (never in mitosis gene a)-related kinase 1SLK minus22 9748 STE20-like kinaseUTRN minus22 7402 UtrophinRB1CC1 minus2 9821 RB1-inducible coiled-coil 1YES1 minus2 7525 V-yes-1 Yamaguchi sarcoma viral oncogene homolog 1

Enzyme activityRANBP2 minus29 5903 RAN binding protein 2JMJD1C minus27 221037 Jumonji domain containing 1C


Table 3 Continued


GeneID Description

RNF6 minus27 6049 Ring finger protein (C3H2C3 type) 6LTN1 minus26 26046 Listerin E3 ubiquitin protein ligase 1RBBP6 minus25 5930 Retinoblastoma binding protein 6PPIG minus25 9360 Peptidylprolyl isomerase G (cyclophilin G)PTAR1 minus24 375743 Protein prenyltransferase alpha subunit repeat containing 1PYROXD1 minus23 79912 Pyridine nucleotide-disulphide oxidoreductase domain 1LIG4 minus23 3981 Ligase IV DNA ATP-dependentRAD50 minus21 10111 RAD50 homolog (S Cerevisiae)DBF4 minus2 10926 DBF4 homolog (S Cerevisiae)

Nucleotide metabolismCHD1 minus26 1105 Chromodomain helicase DNA binding protein 1CHML minus25 1122 Choroideremia-like (Rab escort protein 2)ELMOD2 minus21 255520 ELMOCED-12 domain containing 2NAA15 minus2 80155 N(alpha)-acetyltransferase 15 nata auxiliary subunitKIF20B minus2 9585 Kinesin family member 20B

OthersGOLGA4 minus39 2803 Golgin A4

ASPM minus34 259266 Asp (abnormal spindle) homolog microcephaly associated(Drosophila)

CEP350 minus33 9857 Centrosomal protein 350 kdaC10orf118 minus32 55088 Chromosome 10 open reading frame 118

GALNT5 minus32 11227 UDP-N-acetyl-alpha-D-galactosaminepolypeptideN-acetylgalactosaminyltransferase 5 (galnac-T5)

BOD1L1 minus31 259282 Biorientation of chromosomes in cell division 1-like 1ANKRD12 minus31 23253 Ankyrin repeat domain 12RIF1 minus3 55183 RAP1 interacting factor homolog (yeast)KIAA2026 minus3 158358 Kiaa2026FAM111B minus29 374393 Family with sequence similarity 111 member BEIF5B minus28 9669 Eukaryotic translation initiation factor 5BOSBPL8 minus28 114882 Oxysterol binding protein-like 8GOLGB1 minus28 2804 Golgin B1

MALAT1 minus27 378938 Metastasis associated lung adenocarcinoma transcript 1(non-protein coding)

PRPF40A minus27 55660 PRP40 pre-mrna processing factor 40 homolog A (SCerevisiae)

QSER1 minus26 79832 Glutamine and serine rich 1THOC2 minus26 57187 THO complex 2RASSF6 minus26 166824 Ras association (ralgdsAF-6) domain family member 6STAG2 minus26 10735 Stromal antigen 2

NUFIP2 minus25 57532 Nuclear fragile X mental retardation protein interactingprotein 2

SMC2 minus25 10592 Structural maintenance of chromosomes 2SLC30A1 minus25 7779 Solute carrier family 30 (zinc transporter) member 1ZNF268 minus24 10795 Zinc finger protein 268KIAA1109 minus24 84162 Kiaa1109NIPBL minus24 25836 Nipped-B homolog (Drosophila)ANKRD30BP2 minus24 149992 Ankyrin repeat domain 30B pseudogene 2KIF5B minus24 3799 Kinesin family member 5B


Table 3 Continued


GeneID Description

SMC4 minus23 10051 Structural maintenance of chromosomes 4SMC5 minus23 23137 Structural maintenance of chromosomes 5NEMF minus23 9147 Nuclear export mediator factorGCC2 minus23 9648 GRIP and coiled-coil domain containing 2EXOC5 minus23 10640 Exocyst complex component 5FCHO2 minus23 115548 FCH domain only 2SMC6 minus23 79677 Structural maintenance of chromosomes 6HOOK3 minus22 84376 Hook homolog 3 (Drosophila)LGALSL minus21 29094 Lectin galactoside-binding-like

SEMA3C minus21 10512 Sema domain immunoglobulin domain (Ig) short basicdomain secreted (semaphorin) 3C

TMEM106B minus21 54664 Transmembrane protein 106BPCM1 minus21 5108 Pericentriolar material 1KRTAP2-3 minus21 730755 Keratin associated protein 2-3MACC1 minus21 346389 Metastasis associated in colon cancer 1CCDC82 minus21 79780 Coiled-coil domain containing 82TTC37 minus21 9652 Tetratricopeptide repeat domain 37

UACA minus21 55075 Uveal autoantigen with coiled-coil domains and ankyrinrepeats

CEP290 minus2 80184 Centrosomal protein 290 kdaUSP53 minus2 54532 Ubiquitin specific peptidase 53METRNL minus2 284207 Meteorin glial cell differentiation regulator-like

the cDNA concentration was normalized with that of theglyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCRproductThe targetmRNA expression was analyzed using the2-ΔΔCt algorithm

210 Western Blot Analysis of ICAM-1 The cells from thecontrol uNK and ZDY groups were lysed with RIPA lysisbuffer (Applygen China) supplemented with a proteaseinhibitor cocktail (Applygen China) The protein concen-trations were quantified using bicinchoninic acid (BCAApplygen China) The proteins were separated using 10sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and transferred to nitrocelluloseThe blots wereblocked overnight in Tris-buffered-saline with Tween (TBST)containing 5 dried milk washed three times for 5min eachin TBST and then incubated overnight in a rocker at 4∘Cwith a cocktail containing ICAM-1 (Abcam UK at a dilutionof 1 1000) and 2 dried milk in TBST The blots were thenwashed three times for 10min each in TBST and incubatedwith the secondary antibody (Applygen China at a dilutionof 1 10000) for 1 h at room temperature in a rocker Theblots were washed three times for 10min each in TBST andthen visualized with the Super ECL Plus Detection Reagent(Applygen China) The ECL signals were detected with theQuantity One software (Bio-Rad USA) GAPDH (Abcam

UK) was used as the internal control to validate the amountof protein

211 Statistical Analysis All of the variables were tested inthree different culture experiments The data were analyzedby ANOVA In the microarray experiments the significancelevel was determined by ANOVA with Benjamini-Hochbergcorrection (119875 lt 0001) We selected the up- and downregu-lated genes that showed a median fold change greater than 2and a119875 lt 0001Thedata from the qRT-PCRandwestern blotanalyses are presented as means plusmn SEM after normalizationwith the average value of the housekeeping gene obtainedfor each group and the significance level was 119875 lt 001 Thegraphs of the data were produced using the Microsoft Excelsoftware

3 Results

31Microarray Experiments Gene expression profiling usinga microarray was used to compare the transcript expres-sion levels in epithelial cells treated with control mediumuNK cell-secretion medium and ZDY+ uNK cell-secretionmedium

This analysis identified 169 transcripts that exhibited astatistically significant change in their median expressionlevel (either upregulated or downregulated) in response to


Table 4 Transcripts that were altered by more than twofold in endometrial epithelial cells by stimulation with ZDY and uNK cell-secretionmedium (119875 lt 0001) The transcript levels were obtained using a GeneChip PrimeView Human Gene Expression Array

Gene symbol Foldchange Gene ID Description

Upregulated genes 18 transcriptsIntracellularsignalling factors

AKR1C2 27 1646Aldo-keto reductase family 1 member C2 (dihydrodioldehydrogenase 2 bile acid binding protein 3-alphahydroxysteroid dehydrogenase type III)

Structural factorsTLN1 27 7094 Talin 1KRT75 20 9119 Keratin 75

TranscriptionNUPR1 26 26471 Nuclear protein transcriptional regulator 1

Ion bindingS100A8 21 6279 S100 calcium binding protein A8

Enzyme activityASNS 52 440 Asparagine synthetase (glutamine-hydrolyzing)

AKR1C1 33 1645Aldo-keto reductase family 1 member C1 (dihydrodioldehydrogenase 1 20-alpha (3-alpha)-hydroxysteroiddehydrogenase)

NQO1 29 1728 NAD(P)H dehydrogenase quinone 1CBS 25 875 Cystathionine-beta-synthase

AKR1C3 23 8644 Aldo-keto reductase family 1 member C3 (3-alphahydroxysteroid dehydrogenase type II)

PSAT1 22 29968 Phosphoserine aminotransferase 1HMOX1 22 3162 Heme oxygenase (decycling) 1GPX2 22 2877 Glutathione peroxidase 2 (gastrointestinal)DHRS3 21 9249 Dehydrogenasereductase (SDR family) member 3H1FX 21 8971 H1 histone family member X

Others

SLC7A11 31 23657 Solute carrier family 7 (anionic amino acid transporter lightchain xc-system) member 11

CTH 24 1491 Cystathionase (cystathionine gamma-lyase)HSPB8 22 26353 Heat shock 22 kda protein 8

Downregulated genes 33 transcriptsCytokinesmembraneproteinssignalling factors

CXCL3 minus21 2921 Chemokine (C-X-C motif) ligand 3CAPRIN2 minus20 65981 Caprin family member 2

TransportersCOG5 minus20 10466 Component of oligomeric golgi complex 5

Structural factorsLMNB1 minus55 4001 Lamin B1PNN minus29 5411 Pinin desmosome associated proteinMFAP5 minus22 8076 Microfibrillar associated protein 5RPL37A minus22 6168 Ribosomal protein l37a

Transcription

HIRA minus36 7290 HIR histone cell cycle regulation defective homolog A (SCerevisiae)

YAP1 minus24 10413 Yes-associated protein 1ZNF226 minus24 7769 Zinc finger protein 226


Table 4 Continued


TAF15 minus21 8148 TAF15 RNA polymerase II TATA box binding protein(TBP)-associated factor 68 kda

NFKBIZ minus21 64332 Nuclear factor of kappa light polypeptide gene enhancer inB-cells inhibitor zeta

Ion bindingZDHHC11 minus22 79844 Zinc finger DHHC-type containing 11RNF145 minus21 153830 Ring finger protein 145

Kinase

AHSA2 minus20 130872 AHA1 activator of heat shock 90 kda protein atpase homolog 2(yeast)

Enzyme activity

TNKS2 minus77 80351 Tankyrase TRF1-interacting ankyrin-related ADP-ribosepolymerase 2

Nucleotide metabolismRAD23B minus20 5887 RAD23 homolog B (S Cerevisiae)RCC1 minus20 1104 Regulator of chromosome condensation 1

OthersGOLGA8A minus54 23015 Golgin A8 family member ALUC7L3 minus38 51747 LUC7-like 3 (S Cerevisiae)HNRNPA2B1 minus29 3181 Heterogeneous nuclear ribonucleoprotein A2B1XIST minus28 7503 X (inactive)-specific transcript (non-protein coding)SNORA33 minus26 594839 Small nucleolar RNA HACA box 33SNHG12 minus25 85028 Small nucleolar RNA host gene 12 (non-protein coding)C20orf197 minus24 284756 Chromosome 20 open reading frame 197EML2 minus24 24139 Echinoderm microtubule associated protein like 2PNISR minus23 25957 PNN-interacting serinearginine-rich proteinKIAA0020 minus23 9933 Kiaa0020HMMR minus22 3161 Hyaluronan-mediated motility receptor (RHAMM)NOP58 minus22 51602 NOP58 ribonucleoprotein homolog (yeast)PILRB minus2 29990 Paired immunoglobin-like type 2 receptor betaNCAPG minus2 64151 Non-SMC condensin I complex subunit GPICALM minus2 8301 Phosphatidylinositol binding clathrin assembly protein

uNK cell-secretion medium compared with their controllevel These transcripts are listed in Table 3 A total of 40genes were found upregulated and 129 genes were down-regulated The largest group of upregulated genes includedimmunomodulatory gene regulators cytokines such asinterleukin-15 (IL-15) chemokine (C-X-C motif) ligand 10(CXCL-10) and vascular endothelial growth factor C (VEGF-C) and cytokine receptors such as interleukin-15 receptoralpha (IL-15RA) Additionally the upregulated transcriptsencode proteins associated with transport structuration andtranscription The level of adhesion molecule intercellularadhesion molecule 1 (ICAM-1) was also altered ICAM-1binds to the leucocyte integrins LFA-1 and Mac-1 [18 19]which are expressed on uNK cells Thus this change inthe level of ICAM-1 should increase the localized interac-tions between uNK cells and epithelial cells A number oftranscripts regulating enzyme activity such as the deltex

3-like (Drosophila) (DYX3L) and the proteasome (pro-some macropain) subunit beta type 9 (large multifunctionalpeptidase 2) (PSMB9) and several transcripts regulatingnucleotide metabolism genes such as guanylate bindingprotein 5 (GBP5) and guanylate binding protein 1 interferon-inducible (GBP1) were also upregulated In addition geneswith various functions such as the icon binding complementcomponent 1S subcomponent (C1S) SP110 nuclear bodyprotein (SP110) tryptophan metabolism indoleamine 23-dioxygenase 1 (IDO1) and tryptophanyl-tRNA synthetase(WARS) and several genes with unknown function werefound to be upregulated The largest group of downregulatedgenes encodes transcription factors such as B double prime 1subunit of RNA polymerase III transcription initiation factorIIIB (BDP1) and thyroid hormone receptor interactor 11(TRIP11) The levels of immunomodulatory gene regulatorsincluding interleukin-36 gamma (IL-36G) and interleukin-33


02468

101214161820

Control group uNK group ZDY group

mRN

A le

vel o

f CXC

L-10

a

b

(a)

0

2

4

6

8

10

12

14


mRN

A le

vel o

f IL-

15

ab

(b)

0

2

4

6

8

10

12

14

16


mRN

A le

vel o

f ICA

M-1

a

b

(c)

0

5

10

15

20

25

30

35


mRN

A le

vel o

f NLR

C-5

a b

(d)

0

10

20


mRN

A le

vel o

f IRF

-1

ab

18

16

14

12

8

6

4

2

(e)

Figure 1 Transcription levels of (a) CXCL-10 (b) IL-15 (c) ICAM-1 (d) NLRC-5 and (e) IRF-1 determined by qRT-PCR The relative foldchanges are shown in Section 3 The qRT-PCR results were consistent with the microarray analysis The data are expressed as means plusmn SEMa119875 lt 0001 for the comparison between the uNK and control groups b

119875 lt 0001 for the comparison between the ZDY and control groups

(IL-33) were also altered The levels of transcripts encodingmolecules responsible for signaling transport structurationand cell-cell adhesion were also downregulated In additiongenes with various functions such as ion binding kinaseenzyme activity and nucleotide metabolism were found tobe downregulated

The microarray analysis identified 51 transcripts inepithelial cells that exhibited a statistically significant changein their median expression level (either upregulated ordownregulated) in response to pretreatment with ZDY andtreatment with the uNK cell-secretion medium comparedwith their level in response to treatment with uNK cell-secretion medium (Table 4) Of these genes 33 were down-regulated and 18 were upregulated The largest group of

upregulated genes includes the following enzyme asparaginesynthetase (glutamine-hydrolyzing) (ASNS) and aldo-ketoreductase family 1 member C1 (dihydrodiol dehydroge-nase 1 20-alpha (3-alpha)-hydroxysteroid dehydrogenase)(AKR1C1) The transcripts encoding molecules responsiblefor signal transduction namely aldo-keto reductase family 1member C2 (dihydrodiol dehydrogenase 2 bile acid bindingprotein 3-alpha hydroxysteroid dehydrogenase type III)(AKR1C2) and structuration such as talin 1 (TLN 1) andkeratin 75 (KRT75) were also upregulated The levels oftranscripts encoding proteins that regulate transcription andion binding such as nuclear protein transcriptional regulator1 (NUPR1) and S100 calcium binding protein A8 (S100A8)were also altered and several genes with unknown function


ICAM-1

GAPDH

Control group ZDY groupuNK group

(a)

002040608

112141618

2


Prot

ein

leve

l of I

CAM

-1

a

b

(b)

Figure 2 (a) Expression of endometrial ICAM-1 protein inendometrial epithelial cells The GAPDH band was used as theinternal loading control in each lane (b) Comparison of thenormalized expression intensity of endometrial ICAM-1 protein inthe control uNK and ZDY groupsThe data are expressed as meansplusmn SEM a

119875 lt 001 for the comparison between the uNK and controlgroups b

119875 lt 001 for the comparison between the ZDY and controlgroups

were found to be upregulatedThe downregulated transcriptsincluded only two genes of known function that were down-regulated by more than 4fold lamin B1 (LMNB1 a struc-tural factor) and tankyrase TRF1-interacting ankyrin-relatedADP-ribose polymerase 2 (TNKS2 an enzyme activator)Thegenes that were downregulated by more than 2fold includedthe genes encoding the cytokines chemokine (C-X-C motif)ligand 3 (CXCL3) and caprin family member 2 (CAPRIN2)the transporter component of the oligomeric golgi complex5 (COG5) and various genes associated with transcriptionkinase ion binding and nucleotide metabolism

32 Quantitative Real-Time RT-PCR (qRT-PCR) To verifythese changes the transcript levels of the following genesweremeasured by qRT-PCR CXCL-10 IL-15 ICAM-1 NLRC-5and IRF-1 (Figure 1) The qRT-PCR results confirmed thesubstantial and significant changes in expression observed inthe microarray analysis Epithelial cells treated with the uNKcell-secretion medium showed increased expression levelsof CXCL-10 (184-fold) IL-15 (117-fold) ICAM-1 (125-fold)NLRC-5 (315-fold) and IRF-1 (156-fold) (all 119875 lt 0001)compared with the control group

We also confirmed that epithelial cells pretreated withZDY and then treated with the uNK cell-secretion mediumexhibited downregulated expression levels of CXCL-10 (09-fold 001 lt 119875 lt 005) and NLRC-5 (10-fold001 lt 119875 lt005) and upregulated levels of IL-15 (11-fold 001 lt 119875 lt005) ICAM-1(11-fold 119875 gt 005) and IRF-1 (11-fold 119875 gt005) compared with epithelial cells treated with the uNK

cell-secretion medium However none of these changes weresignificant

33 Western Blot To confirm these effects at the proteinlevel the amount of ICAM-1 was analyzed by western blot(Figure 2) Epithelial cells treated with the uNK cell-secretionmedium showed a 21-fold (119875 lt 001) increase in theexpression of ICAM-1 However the epithelial cells treatedwith ZDY and the uNK cell-secretion medium showed a 14-fold increase (001 lt 119875 lt 005) in the expression of ICAM-1compared with the epithelial cells treated with the uNK cell-secretion medium Thus there was no significant differencebetween the uNK and ZDY groups

4 Discussion

Progesterone plays a central role in reproduction and isinvolved in implantation and pregnancy When pregnancyoccurs high progesterone levels are critical not only forfacilitating implantation but also for maintaining pregnancyby stimulating uterine growth [20 21] Several observa-tions suggest that uNK cells also play an important rolein reproduction (a) these cells are hormonally regulatedbecause they increase in number during the luteal phasewhen implantation occurs [7] (b) these cells are present inearly gestation at the time placental cells invade the maternalarteries [22] and (c) these cells are particularly abundantaround trophoblast cells [23] The proliferation of uNK cellsis the foundation of their reproductive function One of themechanisms underlying the increase in uNK cell numbersis the stimulation of the proliferation of existing uNK cellsby progesterone In mouse uterine tissues the significantupregulation in the decidual NK population is regulatedby progesterone [24] Because there are no progesteronereceptors on uNK cells it is hypothesized that the paracrinecommunication between uNKand epithelial cells participatesin this process In this study we used the uNK cell-secretionmedium to stimulate epithelial cells in an attempt to find theinteraction andor paracrine effects between these cells Thedata obtained in the present study demonstrate that uNK cellshave a significant effect on the gene expression in epithelialcells in the nonpregnant state and these paracrine effects alsohave a profound effect on uNK proliferation

The largest group of upregulated genes induced in epithe-lial cells by the soluble factors derived fromuNKcells inducedcytokines and immunological factors including IL-15 andIL-15RA IL-15 is critical for NK cell differentiation in miceand human lymphoid tissue [25 26] IL-15 acts on precursorand immature NK cells In humans IL-15 transcription ismore abundant during the secretory compared with the pro-liferative cycle phase is maintained in early pregnancy andlocalizes to the endothelium and decidual spiral arteries [27]The correlative time strongly suggests that IL-15 participatesin uNK cell proliferation and that IL-15 may also contributeto the localization of uNK cells Our results show that uNKcells exhibit upregulated levels of both IL-15 and IL-15RAThis may activate a powerful proliferation loop because IL-15 is presented by IL-15RA on epithelial cells to promote uNKproliferation


Endometrial epithelial cells

IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation

120573 EGF HB-EGFIGFs

LIF IL-6 IL-11 TGF

Prol

ifera

tion

Figure 3Model of the proliferation and function of uNK cells During implantation and early pregnancy the uNKparacrine signals stimulateendometrial epithelial cells to produce IL-15 VEGF and other unidentified factors that may regulate uNK cell proliferation The immuneparacrine communication between uNK and endometrial epithelial cells may support implantation and trophoblast development

There are temporal and quantitative features associatedwith the activities of uNK cells and trophoblast cells inuterine spiral artery maturation There is evidence that IL-15upregulates the level of VEGF-C mRNA expression whichsuggests that uNK cells may play an important role inendometrial angiogenesis and regeneration [28] In additionCXCL-10 is involved in uNK cell production directs themigration and invasion of CXCR1+ CXCR3+ CXCR4+ andCCR3+ trophoblast [12] and promotes angiogenesis in theplacental bed [29] Furthermore in the absence of uNKcells spiral artery extension is delayed and the tunica mediaremain intact [30] and these studies are consistent withan angiogenicvascular remodeling role for uNK cells [412 31] We found some evidence that uNK cells promotechanges in endometrial epithelial cells in the nonpregnantstate The level of VEGF-C which is upregulated in epithelialcells by the uNK cell-secretion medium increases markedlyduring implantation and early pregnancy [32] Our datasuggest that uNK may prime some of the decidualizationchanges that occur in epithelial cells prior to the onset of fulldecidualization

The exact function of uNK cells has not yet been unequiv-ocally determined but it is known that these cells expressa different cytokine profile compared with peripheral NKcells For example these cells express colony-stimulating fac-tor (CSF) macrophage colony-stimulating factor (M-CSF)granulocyte-macrophage colony-stimulating factor (GM-CSF) tumor necrosis factor alpha (TNF-120572) interferon-120574(IFN-120574) and leukemia inhibitory (LIF) [33 34] Duringhuman pregnancy these cells are found in close proximity tothe implantation site and in close contact with the infiltratingextravillous trophoblasts Furthermore uNK cells increasetheir numbers in early pregnancy due to hormonal depen-dence and exhibit close proximity to trophoblasts Takentogether these data support the conclusion that uNK cells

play an important role in the regulation for the maternalimmune response for the control of implantation and tro-phoblast invasion during human pregnancy The summaryof the paracrine effects of uNK cells on the epithelial cells isshown in Figure 3

In addition this study provides the first demonstration ofthe paracrine communication between uNK and endometrialepithelial cells Because our previous study showed thatZDY can improve the morphology of the endometriumin mice we attempted to identify a relationship betweenthe paracrine communication (uNKepithelial cells) and themechanism of action of ZDY However the majority of theup- and downregulated genes induced in the ZDY pretreatedepithelial cells were of unknown function To the best ofour knowledge there are no published studies on the effectsof traditional Chinese medicine on the human endometrialparacrine communication Our results suggest that ZDYmaynot regulate this process in vitro ZDY may improve themorphology of the uterus through another mechanism

5 Conclusions

Overall this study provides the first detailed demonstrationof the paracrine interaction between immune cells andepithelial cells in the endometrial environment The findingsindicate that these paracrine signals may contribute to uNKcell proliferation during implantation and early pregnancy Inaddition we failed to find evidence indicating that ZDY reg-ulates the paracrine effects between uNK cells and epithelialcells in vitro

Conflict of Interests

The authors declare that they have no conflict of interests


Acknowledgments

This project was supported by the Natural Science Founda-tion of China The project number is 81173292

References

[1] J Zhang Z Chen G N Smith and B A Croy ldquoNatural killercell-triggered vascular transformation maternal care beforebirthrdquo Cellular amp Molecular Immunology vol 8 no 1 pp 1ndash112010

[2] S Quenby H Nik B Innes et al ldquoUterine natural killer cellsand angiogenesis in recurrent reproductive failurerdquo HumanReproduction vol 24 no 1 pp 45ndash54 2009

[3] M-J Guimond J A Luross B Wang C Terhorst S Danialand B A Croy ldquoAbsence of natural killer cells during murinepregnancy is associated with reproductive compromise inTgE26micerdquo Biology of Reproduction vol 56 no 1 pp 169ndash1791997

[4] G E Lash S C Robson and J N Bulmer ldquoReview functionalrole of uterine natural killer (uNK) cells in human earlypregnancy deciduardquo Placenta vol 31 pp S87ndashS92 2010

[5] J N Bulmer L Morrison M Longfellow A Ritson and DPace ldquoGranulated lymphocytes in human endometrium his-tochemical and immunohistochemical studiesrdquo Human Repro-duction vol 6 no 6 pp 791ndash798 1991

[6] H Okada T Nakajima M Sanezumi A Ikuta K Yasuda andH Kanzaki ldquoProgesterone enhances interleukin-15 productionin human endometrial stromal cells in vitrordquo Journal of ClinicalEndocrinology amp Metabolism vol 85 no 12 pp 4765ndash47702000

[7] A King V Wellings L Gardner and Y W Loke ldquoImmunocy-tochemical characterization of the unusual large granular lym-phocytes in human endometrium throughout the menstrualcyclerdquo Human Immunology vol 24 no 3 pp 195ndash205 1989

[8] S Verma S E Hiby YW Loke and A King ldquoHuman decidualnatural killer cells express the receptor for and respond to thecytokine interleukin 15rdquo Biology of Reproduction vol 62 no 4pp 959ndash968 2000

[9] T A Henderson P T K Saunders A Moffett-King N PGroome and H O D Critchley ldquoSteroid receptor expressionin uterine natural killer cellsrdquo Journal of Clinical Endocrinologyand Metabolism vol 88 no 1 pp 440ndash449 2003

[10] O Gubbay H O D Critchley J M Bowen A King and H NJabbour ldquoProlactin induces ERK phosphorylation in epithelialand CD56+ natural killer cells of the human endometriumrdquoJournal of Clinical Endocrinology amp Metabolism vol 87 no 5pp 2329ndash2335 2002

[11] A Germeyer A M Sharkey M Prasadajudio et al ldquoParacrineeffects of uterine leucocytes on gene expression of humanuterine stromal fibroblastsrdquo Molecular Human Reproductionvol 15 no 1 pp 39ndash48 2009

[12] J Hanna D Goldman-Wohl Y Hamani et al ldquoDecidual NKcells regulate key developmental processes at the human fetal-maternal interfacerdquo Nature Medicine vol 12 no 9 pp 1065ndash1074 2006

[13] A S Crimmel C S Conner and M Monga ldquoWithered Yanga review of traditional Chinese medical treatment of maleinfertility and erectile dysfunctionrdquo Journal of Andrology vol22 no 2 pp 173ndash182 2001

[14] C Qian J Zhe L Qiu-dan C Ying and L Nan ldquoAffection ofZidan decoction containing serum on endometrial receptivity

in uterine immunity environmentrdquo Guo Ji Zhong Yi Zhong YaoZa Zhi vol 2012 no 3 pp 220ndash223 2012

[15] D L Carlone and V Rider ldquoEmbryonic modulation of basicfibroblast growth factor in the rat uterusrdquo Biology of Reproduc-tion vol 49 no 4 pp 653ndash665 1993

[16] P Vigano B Gaffuri E Somigliana M Infantino M Vignaliand A M Di Blasio ldquoInterleukin-10 is produced by humanuterine natural killer cells but does not affect their productionof interferon-120574rdquo Molecular Human Reproduction vol 7 no 10pp 971ndash977 2001


[18] M S Diamond D E Staunton A R De Fougerolles et alldquoICAM-1 (CD54) a counter-receptor forMac-1 (CD11bCD18)rdquoJournal of Cell Biology vol 111 no 6 pp 3129ndash3139 1990

[19] S D Marlin and T A Springer ldquoPurified intercellular adhesionmolecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1)rdquoCell vol 51 no 5 pp 813ndash819 1987

[20] C L Clarke and R L Sutherland ldquoProgestin regulation ofcellular proliferationrdquo Endocrine Reviews vol 11 no 2 pp 266ndash301 1990

[21] J Dinny Graham and C L Clarke ldquoPhysiological action ofprogesterone in target tissuesrdquo Endocrine Reviews vol 18 no4 pp 502ndash519 1997

[22] J Bulmer P Johnson andD Bulmer ldquoLeukocyte populations inhuman decidua and endometriumrdquo in Immunoregulation andFetal Survival T C Gill and T Wegmann Eds pp 111ndash134Oxford University Press New York NY USA 1987

[23] Y Loke Human Implantation Cell Biology and ImmunologyCambridge University Press Cambridge UK 1995

[24] Y S Choe C Shim D Choi C S Lee K K Lee and K KimldquoExpression of galectin-1 mRNA in the mouse uterus is underthe control of ovarian steroids during blastocyst implantationrdquoMolecular Reproduction and Development vol 48 no 2 pp261ndash266 1997

[25] M K Kennedy M Glaccum S N Brown et al ldquoReversibledefects in natural killer and memory CD8 T cell lineagesin interleukin 15-deficient micerdquo Journal of ExperimentalMedicine vol 191 no 5 pp 771ndash780 2000

[26] W E Carson J G Giri M J Lindemann et al ldquoInterleukin (IL)15 is a novel cytokine that activates human natural killer cellsvia components of the IL-2 receptorrdquo Journal of ExperimentalMedicine vol 180 no 4 pp 1395ndash1403 1994

[27] K Kitaya J Yasuda I Yagi Y Tada S Fushiki and H HonjoldquoIL-15 expression at human endometrium and deciduardquo Biologyof Reproduction vol 63 no 3 pp 683ndash687 2000

[28] X F Li D S Charnock-Jones E Zhang et al ldquoAngiogenicgrowth factor messenger ribonucleic acids in uterine naturalkiller cellsrdquo Journal of Clinical Endocrinology ampMetabolism vol86 no 4 pp 1823ndash1834 2001

[29] A Moffett-King ldquoNatural killer cells and pregnancyrdquo NatureReviews Immunology vol 2 no 9 pp 656ndash663 2002

[30] D Chakraborty M A Karim Rumi T Konno and M JSoares ldquoNatural killer cells direct hemochorial placentationby regulating hypoxia-inducible factor dependent trophoblastlineage decisionsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 39 pp16295ndash16300 2011


[31] S D Smith C E Dunk J D Aplin L K Harris and R LJones ldquoEvidence for immune cell involvement in decidual spiralarteriole remodeling in early human pregnancyrdquoThe AmericanJournal of Pathology vol 174 no 5 pp 1959ndash1971 2009

[32] G E Lash B Schiessl M Kirkley et al ldquoExpression ofangiogenic growth factors by uterine natural killer cells duringearly pregnancyrdquo Journal of Leukocyte Biology vol 80 no 3 pp572ndash580 2006

[33] P P Jokhi A King A M Sharkey S K Smith and YW Loke ldquoScreening for cytokine messenger ribonucleic acidsin purified human decidual lymphocyte populations by thereverse-transcriptase polymerase chain reactionrdquo Journal ofImmunology vol 153 no 10 pp 4427ndash4435 1994

[34] S Saito K Nishikawa T Morii et al ldquoCytokine productionby CD16-CD56(bright) natural killer cells in the human earlypregnancy deciduardquo International Immunology vol 5 no 5 pp559ndash563 1993

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Composition of Zi Dan Yin (ZDY)

Components Ratio(1) Sheng Di (Rehmannia glutinosa (Gaertn) Libosch root) 15(2) Dan Shen (Salviae Miltiorrhizae Bge root) 10(3) Dang gui (Angelica sinensis (Oliv) Diels root) 12(4) Chuan Duan (Dipsacus asperoides C Y Cheng et TMAi root) 15

(5) Du Zhong (Eucommia ulmoides Oliv cortex) 12(6) Shan Yao (Dioscorea oppositaThunb rhizome) 15(7) Mei Gui-hua (Rosa rugosaThunb flower) 6(8) Chuan Xiong (Ligusticum Chuanxiong Hort rhizome) 6(9) Yi Yi-ren (Coix lacryma-jobi L var ma-yuen (Roman)Stapf seed) 12

pregnancy thus there is no doubt that the preparation ofepithelial cells is directed towards these process We hypoth-esize that there similar paracrine communications occurbetween uNK cells and uterine epithelial cells Our resultsrevealed that soluble factors from uNK cells have substantialeffects on endometrial epithelial gene expression uNK cellswere found to upregulate transcript levels in epithelial cellsthat are known to promote uNK cell proliferation and theseupregulated transcripts may contribute to the preparation forimplantation and trophoblast migration and invasion

In China oriental herbal medicines have been used totreat infertility since ancient times [13] Zi Dan Yin (ZDY)a compound of nine herbs (Table 1) was found to improvethe morphology of the endometrium in mice [14] Althoughthe treatment effects of ZDY on mice have been confirmedit is unclear whether ZDY contributes to the local immuneresponses However our results do not support the hypothe-sis that ZDY can improve the paracrine effects between uNKand endometrial epithelial cells

2 Materials and Methods

21 Ethics Statement All of the subjects understood andsigned the informed consent form before participation Theexperimental protocolswere approved by the Ethics Commit-tee of the Dongfang Hospital Human Ethics Committee (no2011090201)

22 Collection of Material Decidual tissues were obtainedfrom healthy women undergoing elective termination ofa normal pregnancy between 7 and 8 weeks of gestationThe endometrial tissues were collected from endometrialbiopsies taken during the proliferative phase of the cyclefrom women undergoing laparoscopy (Dongfang Hospital ofBeijing University of Chinese Medicine China) for benigndisease The average age was 30 plusmn 13 years and the bodymass index (BMI) was 221 plusmn 08 kgm2 All of the ninewomen were Chinese of Han ethnicity The exclusion criteriawere hormonal stimulation cancerous lesions and irregularmenstrual bleeding

23 Isolation of the Uterine NK (uNK) Cells The uNK cellswere isolated as previously described [15 16] Briefly thedecidual tissues were extensively washed with Ca2+- andMg2+-free Hankrsquos balanced salt solution (HBSS) containingantibiotics minced thoroughly between two scalpels intofragments of 1-2mm3 and digested for 1 h at 37∘Cwith gentleagitation in HBSS with 01 (wv) collagenase I (GibcoUSA)The cell suspensions were layered over Ficoll-Hypaquemedium (General Electric USA) and centrifuged at 800timesgand room temperature for 25min The cells at the interfacewere washed twice with Roswell Park Memorial Institute(RPMI)-1640 media with 10 fetal calf serum (FCS) andantibiotics After incubation for 20min at 4∘C with anti-CD56 microbeads (Miltenyi Biotec Ltd Germany) thecells were washed with washing buffer (PBS 2mM EDTAand 05BSA (wv)) and then loaded onto a MiniMACSSeparator (MS) column in a MiniMACS magnet (MiltenyiBiotec Ltd Germany) After flushing the MS column threetimes the CD56+ cells were flushed as indicated by the man-ufacturerThe purity of the uNK cells was gt90CD56+CD3minusaccording to the flow cytometric analysis The uNK cellswere cultured in RPMI 1640 media with 1 FCS and IL-15(10 ngmL) (RampD systems USA) in which to maintain theviability of the purified uNK cells [17]

24 Production of uNKCell-SecretionMedium TheuNKcell-secretion medium was prepared using 200120583L of RPMI 1640media with 1 FCS and IL-15 (10 ngmL) containing 5times105 ofthe purified uNK cells The cell suspension was placed in theupper chamber of a 04 120583m pore hanging cell culture insert(Millipore USA) in a 24-well tissue plate and 1300 120583L of thesamemediawithout cells was placed in the lower chamber Asa result only the soluble molecules from the uNK cells canpass through the filter into the lower chamber The controlmedium was 1500120583L of the RPMI 1640 media with 1 FCSand IL-15 (10 ngmL) and this control medium was alsoused in the subsequent experiments After incubation for24 h at 37∘C the uNK cell-secretion medium (in the lowerchamber) and the control medium were collected and frozenat minus80∘C The cells in the upper chamber were collected andthe cell viability was measured using a livedead viability kit(Invitrogen USA) Only the conditional medium from uNKpopulations with less than 35 dead cells after overnightincubation was used in the subsequent experiments

25 Human Endometrial Epithelial Cell Culture Humanendometrial tissue was cut using scissors and dissociated intosingle-cell suspensions using 01 (wv) collagenase I for 50ndash60min Every 15min the digests were pipetted and their dis-sociation was monitored by microscopyThe cell suspensionswere filtered using a 40-mesh screen to separate the singlecells from undigested tissue To remove the erythrocytes thecells were resuspended in 4mL ofDulbeccorsquosmodified Eaglersquosmedium and Hamrsquos nutrient F12 (DMEMF12) with 1 FCSlayered over Ficoll-Paque PLUS and centrifuged for 25minat 800timesg The leukocytes were removed with CD45-coatedDynabeads (Dynal Biotech USA)The purified epithelial cellsuspensions were then obtained through a further round of















GeneID Description

















Table 3 Continued


GeneID Description














Transcription




Table 3 Continued


GeneID Description












Table 3 Continued


GeneID Description














Table 3 Continued


GeneID Description










3 Results
















Others







Transcription




Table 4 Continued





Kinase


Enzyme activity







02468

101214161820


mRN

A le

vel o

f CXC

L-10

a

b

(a)

0

2

4

6

8

10

12

14


mRN

A le

vel o

f IL-

15

ab

(b)

0

2

4

6

8

10

12

14

16


mRN

A le

vel o

f ICA

M-1

a

b

(c)

0

5

10

15

20

25

30

35


mRN

A le

vel o

f NLR

C-5

a b

(d)

0

10

20


mRN

A le

vel o

f IRF

-1

ab

18

16

14

12

8

6

4

2

(e)







ICAM-1

GAPDH


(a)

002040608

112141618

2


Prot

ein

leve

l of I

CAM

-1

a

b

(b)









4 Discussion





IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation


LIF IL-6 IL-11 TGF

Prol

ifera

tion






5 Conclusions





Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























GeneID Description

















Table 3 Continued


GeneID Description














Transcription




Table 3 Continued


GeneID Description












Table 3 Continued


GeneID Description














Table 3 Continued


GeneID Description










3 Results
















Others







Transcription




Table 4 Continued





Kinase


Enzyme activity







02468

101214161820


mRN

A le

vel o

f CXC

L-10

a

b

(a)

0

2

4

6

8

10

12

14


mRN

A le

vel o

f IL-

15

ab

(b)

0

2

4

6

8

10

12

14

16


mRN

A le

vel o

f ICA

M-1

a

b

(c)

0

5

10

15

20

25

30

35


mRN

A le

vel o

f NLR

C-5

a b

(d)

0

10

20


mRN

A le

vel o

f IRF

-1

ab

18

16

14

12

8

6

4

2

(e)







ICAM-1

GAPDH


(a)

002040608

112141618

2


Prot

ein

leve

l of I

CAM

-1

a

b

(b)









4 Discussion





IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation


LIF IL-6 IL-11 TGF

Prol

ifera

tion






5 Conclusions





Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 3 Continued


GeneID Description














Transcription




Table 3 Continued


GeneID Description












Table 3 Continued


GeneID Description














Table 3 Continued


GeneID Description










3 Results
















Others







Transcription




Table 4 Continued





Kinase


Enzyme activity







02468

101214161820


mRN

A le

vel o

f CXC

L-10

a

b

(a)

0

2

4

6

8

10

12

14


mRN

A le

vel o

f IL-

15

ab

(b)

0

2

4

6

8

10

12

14

16


mRN

A le

vel o

f ICA

M-1

a

b

(c)

0

5

10

15

20

25

30

35


mRN

A le

vel o

f NLR

C-5

a b

(d)

0

10

20


mRN

A le

vel o

f IRF

-1

ab

18

16

14

12

8

6

4

2

(e)







ICAM-1

GAPDH


(a)

002040608

112141618

2


Prot

ein

leve

l of I

CAM

-1

a

b

(b)









4 Discussion





IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation


LIF IL-6 IL-11 TGF

Prol

ifera

tion






5 Conclusions





Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 3 Continued


GeneID Description












Table 3 Continued


GeneID Description














Table 3 Continued


GeneID Description










3 Results
















Others







Transcription




Table 4 Continued





Kinase


Enzyme activity







02468

101214161820


mRN

A le

vel o

f CXC

L-10

a

b

(a)

0

2

4

6

8

10

12

14


mRN

A le

vel o

f IL-

15

ab

(b)

0

2

4

6

8

10

12

14

16


mRN

A le

vel o

f ICA

M-1

a

b

(c)

0

5

10

15

20

25

30

35


mRN

A le

vel o

f NLR

C-5

a b

(d)

0

10

20


mRN

A le

vel o

f IRF

-1

ab

18

16

14

12

8

6

4

2

(e)







ICAM-1

GAPDH


(a)

002040608

112141618

2


Prot

ein

leve

l of I

CAM

-1

a

b

(b)









4 Discussion





IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation


LIF IL-6 IL-11 TGF

Prol

ifera

tion






5 Conclusions





Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 3 Continued


GeneID Description














Table 3 Continued


GeneID Description










3 Results
















Others







Transcription




Table 4 Continued





Kinase


Enzyme activity







02468

101214161820


mRN

A le

vel o

f CXC

L-10

a

b

(a)

0

2

4

6

8

10

12

14


mRN

A le

vel o

f IL-

15

ab

(b)

0

2

4

6

8

10

12

14

16


mRN

A le

vel o

f ICA

M-1

a

b

(c)

0

5

10

15

20

25

30

35


mRN

A le

vel o

f NLR

C-5

a b

(d)

0

10

20


mRN

A le

vel o

f IRF

-1

ab

18

16

14

12

8

6

4

2

(e)







ICAM-1

GAPDH


(a)

002040608

112141618

2


Prot

ein

leve

l of I

CAM

-1

a

b

(b)









4 Discussion





IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation


LIF IL-6 IL-11 TGF

Prol

ifera

tion






5 Conclusions





Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 3 Continued


GeneID Description










3 Results
















Others







Transcription




Table 4 Continued





Kinase


Enzyme activity







02468

101214161820


mRN

A le

vel o

f CXC

L-10

a

b

(a)

0

2

4

6

8

10

12

14


mRN

A le

vel o

f IL-

15

ab

(b)

0

2

4

6

8

10

12

14

16


mRN

A le

vel o

f ICA

M-1

a

b

(c)

0

5

10

15

20

25

30

35


mRN

A le

vel o

f NLR

C-5

a b

(d)

0

10

20


mRN

A le

vel o

f IRF

-1

ab

18

16

14

12

8

6

4

2

(e)







ICAM-1

GAPDH


(a)

002040608

112141618

2


Prot

ein

leve

l of I

CAM

-1

a

b

(b)









4 Discussion





IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation


LIF IL-6 IL-11 TGF

Prol

ifera

tion






5 Conclusions





Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Others







Transcription




Table 4 Continued





Kinase


Enzyme activity







02468

101214161820


mRN

A le

vel o

f CXC

L-10

a

b

(a)

0

2

4

6

8

10

12

14


mRN

A le

vel o

f IL-

15

ab

(b)

0

2

4

6

8

10

12

14

16


mRN

A le

vel o

f ICA

M-1

a

b

(c)

0

5

10

15

20

25

30

35


mRN

A le

vel o

f NLR

C-5

a b

(d)

0

10

20


mRN

A le

vel o

f IRF

-1

ab

18

16

14

12

8

6

4

2

(e)







ICAM-1

GAPDH


(a)

002040608

112141618

2


Prot

ein

leve

l of I

CAM

-1

a

b

(b)









4 Discussion





IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation


LIF IL-6 IL-11 TGF

Prol

ifera

tion






5 Conclusions





Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 4 Continued





Kinase


Enzyme activity







02468

101214161820


mRN

A le

vel o

f CXC

L-10

a

b

(a)

0

2

4

6

8

10

12

14


mRN

A le

vel o

f IL-

15

ab

(b)

0

2

4

6

8

10

12

14

16


mRN

A le

vel o

f ICA

M-1

a

b

(c)

0

5

10

15

20

25

30

35


mRN

A le

vel o

f NLR

C-5

a b

(d)

0

10

20


mRN

A le

vel o

f IRF

-1

ab

18

16

14

12

8

6

4

2

(e)







ICAM-1

GAPDH


(a)

002040608

112141618

2


Prot

ein

leve

l of I

CAM

-1

a

b

(b)









4 Discussion





IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation


LIF IL-6 IL-11 TGF

Prol

ifera

tion






5 Conclusions





Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















02468

101214161820


mRN

A le

vel o

f CXC

L-10

a

b

(a)

0

2

4

6

8

10

12

14


mRN

A le

vel o

f IL-

15

ab

(b)

0

2

4

6

8

10

12

14

16


mRN

A le

vel o

f ICA

M-1

a

b

(c)

0

5

10

15

20

25

30

35


mRN

A le

vel o

f NLR

C-5

a b

(d)

0

10

20


mRN

A le

vel o

f IRF

-1

ab

18

16

14

12

8

6

4

2

(e)







ICAM-1

GAPDH


(a)

002040608

112141618

2


Prot

ein

leve

l of I

CAM

-1

a

b

(b)









4 Discussion





IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation


LIF IL-6 IL-11 TGF

Prol

ifera

tion






5 Conclusions





Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ICAM-1

GAPDH


(a)

002040608

112141618

2


Prot

ein

leve

l of I

CAM

-1

a

b

(b)









4 Discussion





IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation


LIF IL-6 IL-11 TGF

Prol

ifera

tion






5 Conclusions





Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















IL-15

VEGF

Other factors

uNK

uNK uNK

uNK

uNK

uNK

uNKuNK

uNK

uNK uNK

uNK uNK

uNKImplantation

and pregnancy

Differentiation


LIF IL-6 IL-11 TGF

Prol

ifera

tion






5 Conclusions





Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article gene expression profiling of the...

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