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Journal of Reproductive Immunology 94 (2012) 190– 195

Contents lists available at SciVerse ScienceDirect

Journal of Reproductive Immunology

jo u r n al hom ep age : w ww.elsev ier .com/ locate / j repr imm

nalysis of monocyte subsets and toll-like receptor 4 expression ineripheral blood monocytes of women in preterm labor

imyung Kima, Younghyun Koa, Kyechul Kwona, Sunhoe Kooa, Yunee Rheeb,yunghun Kangb, Mina Leeb,∗

Department of Laboratory Medicine, Chungnam National University Hospital, Daejeon, South KoreaDepartment of Obstetrics and Gynecology, Chungnam National University Hospital, Daejeon, South Korea

r t i c l e i n f o

rticle history:eceived 17 November 2011eceived in revised form 25 January 2012ccepted 1 February 2012

eywords:low cytometryonocytes

reterm laborubsetsoll-like receptor4

a b s t r a c t

Preterm labor is associated with both localized inflammation of the uterus and elevatedproinflammatory cytokines. Recently, specific roles have been suggested for distinct mono-cyte subsets and toll-like receptor 4 (TLR4) expression in inflammation. The aim of thisstudy was to determine whether specific monocyte subsets and increased TLR4 expressionin monocyte subsets contribute to preterm labor. The study included 30 preterm labor, 40full-term labor and 20 pregnant women (not in labor). Four-color flow cytometry was usedto examine the distribution of three monocyte subsets (CD14+CD16−, CD14highCD16+, andCD14lowCD16+) and the TLR4 expression in each monocyte subset in each group of women.A larger percentage of CD14highCD16+ cells was found in the preterm labor group thanin the other groups (P = 0.08, P = 0.06). Women in preterm labor also showed significantlyhigher TLR4 expression in all monocyte subsets and increased fluorescence intensity in the

CD14+CD16− and CD14highCD16+ cells. Expression of TLR4 and mean fluorescence intensityon each monocyte subset were also significantly correlated. We conclude that women withpreterm labor have higher CD16 monocytes, with high concomitant expression of CD14and enhanced TLR4 expression in monocytes, and that monocyte TLR4 levels could be usedas a marker to predict preterm delivery.

. Introduction

Monocytes in peripheral blood are a heterogeneousopulation (Gordon and Taylor, 2005; Ziegler-Heitbrockt al., 2010). Monocytes can be divided into three sub-ets based on expression of CD14 and CD16: classicalCD14+CD16−), intermediate (CD14highCD16+), and non-

lassical (CD14lowCD16+) cells (Passlick et al., 1989;iegler-Heitbrock et al., 2010). Classical monocytes areharacterized by CD14 expression and lack of CD16

∗ Corresponding author at: Department of Obstetrics and Gynecol-gy, Chungnam National University Hospital, 282 Moonhwa-ro, Joong-gu,aejeon, South Korea. Tel.: +82 42 280 7998; fax: +82 42 257 5365.

E-mail address: solvaram@megapass.net (M. Lee).

165-0378/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jri.2012.02.002

© 2012 Elsevier Ireland Ltd. All rights reserved.

expression; they comprise approximately 90–95% of thetotal population of monocytes and have high phagocyticactivity. Nonclassical monocytes are characterized by lowexpression of CD14 and positive CD16 expression, whileintermediate monocytes exhibit high CD14 expressiontogether with CD16 expression. Compared with classi-cal monocytes, these monocyte subsets exhibit distinctchemokine-receptor expression, lower CCR2 levels, higherCX3CR1 levels, and low phagocytic capacity. In addition,classical monocytes predominantly produce the anti-inflammatory cytokine interleukin (IL)-10, whereas theCD14+CD16+ cells expand during infection and inflamma-

tion and are responsible for the release of proinflammatorycytokines such as tumor necrosis factor (TNF) and IL-1(Belge et al., 2002; Strauss-Ayali et al., 2007; Hanai et al.,2008). These findings suggest that the specific subsets

ctive Im

J. Kim et al. / Journal of Reprodu

of monocytes (CD14+CD16+ cells) may contribute to thedevelopment of inflammation.

Preterm labor is a common cause of perinatal mortal-ity, and intrauterine infection is implicated in many casesof preterm delivery (Goldenberg et al., 2000). Elevation ofinflammatory cytokines, such as TNF, IL-1, IL-6, and IL-8, have been reported in the amniotic fluid of women inpreterm labor (Arntzen et al., 1998; Tanaka et al., 1998).It is likely that the maternal inflammatory response isrelated to the initiation of preterm labor because theseproinflammatory cytokines stimulate prostaglandin pro-duction and can induce uterine contraction. Recent reportssuggest that inflammatory responses are controlled by toll-like receptors (TLRs), which are predominantly expressedon monocytes (Sabroe et al., 2002; Kopp and Medzhitov,2003). In particular, TLR4 plays a key role in early acti-vation in response to bacterial lipopolysaccharide (LPS)(Kopp and Medzhitov, 2003). TLR4 induces expression ofgenes encoding the inflammatory cytokines elevated inpreterm labor. In one previous study (Kumazaki et al.,2004), increased expression of TLR4 in the placental villihas been observed in women with chorioamnionitis dur-ing the preterm period. Additionally, a preterm labor groupshowed elevated levels of TLR4+/CD14+ monocytes andenhanced TLR4 expression in peripheral blood monocytesin a recent study (Pawelczyk et al., 2010).

Although intrauterine infection is known to be asso-ciated with preterm labor, it is difficult to predictpreterm delivery before labor because intrauterine infec-tion is usually asymptomatic and routine amniocentesis isuncommon. Therefore, new tests are needed to predict therisk of preterm labor.

To the best of our knowledge, there are no compar-ative studies that analyze monocyte subsets in maternalperipheral blood and the levels of TLR4 expression in eachmonocyte subset in women with preterm and full-termdelivery. The purpose of this study was to investigatewhether the difference in monocyte subset distributionand TLR4 expression predict preterm labor.

2. Materials and methods

2.1. Study population

This study was approved by the Institute of ReviewBoards of Chungnam National University Hospital and writ-ten informed consent was obtained from all participants.Preterm labor was defined as the presentation of activelabor in a pregnant woman prior to 36 weeks’ gestationand was confirmed by a physician according to the clinicalcriteria for labor. Pregnant women with uterine malfor-mations, placental abruption, and placenta previa wereexcluded. Additional exclusion criteria were autoimmunedisease and use of anti-inflammatory medication. Thirtywomen in preterm labor ended up with premature birthwere included in the study. Forty women in full-term labor(delivery after 37 weeks’ gestation) were designated con-

trol subjects, and 20 pregnant women at 25–36 weeks’gestation who were not in labor were enrolled as additionalcontrol subjects. Control subjects were age-matched withpreterm subjects.

munology 94 (2012) 190– 195 191

Peripheral blood samples were obtained for all sub-jects at the time of presentation, regardless of whetherthe patients came in labor or for routine medical exami-nation. The time interval from the collection of the bloodsample to childbirth in the labor group was measured.Complete blood counts were analyzed using an auto-mated hematology analyzer XE-2100 (Sysmex Corporation,Kobe, Japan) and C-reactive protein (CRP) levels (referencerange < 0.5 mg/dL) were measured by a latex agglutinationtest using a TBA-200FR NEO system (Toshiba Medical Sys-tem Corporation, Tochigi, Japan).

2.2. Flow cytometry analysis

Blood samples were collected in EDTA tubes for flowcytometry analysis and stored at 4 ◦C until immunostain-ing. Analysis was performed within 4 h of blood collection.The following monoclonal antibodies to human antigenswere used: anti-CD16 fluorescein-isothiocyanate (FITC;Beckman Coulter, Marseille, France); anti-leukocyte anti-gen (HLA) DR phycoerythrin–cyanine 5 (PC5); anti-CD14phycoerythrin–cyanine 7 (PC7); anti-TLR4 conjugated withphycoerythrin (PE, 80 �g/mL; Santa Cruz Biotechnology,Santa Cruz, CA, USA). White blood cells were resus-pended at 5 × 103 cells/�L with phosphate buffered saline(PBS), and immunostaining was performed. Whole blood(100 �L) was stained with 3 �L anti-CD14-PC7 mAbs, 3 �Lanti-CD16-FITC mAbs, 15 �L anti-HLA-DR-PC5 mAbs, and10 �L anti-TLR4-PE mAbs. The mixture was incubated for30 min at room temperature in the dark. Isotype-matchedantibody controls were used to detect nonspecific staining.For red cell lysis, 0.5 mL OptiLyse solution was added andincubated for 15 min in the dark.

The samples were analyzed on a Cytomics FC 500 flowcytometer (Beckman Coulter). In flow cytometry analysis,mononuclear cells were first gated in a forward scatter (FS)and side scatter (SS) with 100,000 events. Monocytes wereidentified as HLA-DR positive cells using a forward scat-ter/side scatter dot plot. Monocytes were further dividedinto CD14+CD16−, CD14highCD16+, and CD14lowCD16+ sub-sets (Fig. 1). Cell surface expression of TLR4 on eachmonocyte subset was determined using scatter analysisand is shown as mean fluorescence intensity (MFI; Fig. 2).

2.3. Statistics

All statistical analyses were performed using MedCalcstatistical software 11.3.0.0 (MedCalc Software, Mariak-erke, Belgium). Continuous variables were presented aseither mean ± SD or median and range. Data comparisonsbetween two groups were performed using either a t-testor a Mann–Whitney U-test. When more than two groupswere compared, ANOVA or the Kruskal–Wallis test wasused. Linear regressions were calculated to assess correla-tions between two parameters. A P value of less than 0.05was considered significant.

3. Results

Clinical and laboratory data are summarized in Table 1.The mean maternal age and parity in the preterm labor,

192 J. Kim et al. / Journal of Reproductive Immunology 94 (2012) 190– 195

Fig. 1. Top left: mononuclear cells (A) were gated in a forward scatter (FS)/side scatter (SS) dot plot, and the indicated population (circled) was furtherprocessed. Top right: within the previously indicated population, a monocytic population (B) was defined as HLA-DR+ cells using an HLA-DR-PC5/SS dotplot. Bottom right: monocyte subsets were divided into CD14+CD16− (C), CD14highCD16+ (D), and CD14lowCD16+ (E) subsets.

Fig. 2. TLR4 expression was determined in CD14+CD16− (C), CD14highCD16+ (D), and CD14lowCD16+ (E) subsets.

J. Kim et al. / Journal of Reproductive Immunology 94 (2012) 190– 195 193

Table 1Patients characteristics.

Characteristics Preterm labor (n = 30) Full-term labor (n = 40) Pregnant control (n = 20) P value

Age (years) 31.2 ± 5.4 31.6 ± 4.7 33.3 ± 3.6 0.28Gestational age 32.7 ± 3.2 38.7 ± 1.4 32.1 ± 4.0 <0.0001Parity 0.5 ± 0.7 0.7 ± 0.7 0.7 ± 0.8 0.81Time to birth (days) 6.1 ± 8.0 1.1 ± 0.6 <0.0001Leukocyte (×103/�L) 11.0 ± 2.2 11.1 ± 3.1 9.4 ± 2.0 0.045Monocyte (/�L) 643 ± 50 668 ± 191 583 ± 188 0.32Monocyte (%) 5.8 ± 1.7 6.2 ± 1.6 6.2 ± 1.6 0.59

0.44 ±

ollection

C-reactive protein (mg/dL) 0.55 ± 0.68

Data are expressed as mean ± SD. Time to birth indicates the time from c

full-term labor, and pregnant control groups were similar.The mean time interval from blood collection to childbirthwas 6.1 ± 8.0 days in the preterm group and 1.1 ± 0.6 daysin the full-term group, and showed a significant differencebetween the two groups.

White blood cell (WBC) counts were11.0 ± 2.2 × 103/�L, 11.1 ± 3.1 × 103/�L, and9.4 ± 2.0 × 103/�L in the preterm labor, full-term labor,and pregnant control groups respectively. WBC countswere higher in the preterm and full-term labor groupsthan in the pregnant control group (P = 0.045), while nosignificant difference between the preterm labor andfull-term labor groups was shown.

The percentage of monocyte and absolute monocytecounts were 5.8 ± 1.7% and 643 ± 50/�L in the pretermlabor group, 6.2 ± 1.6% and 668 ± 191/�L in the full-termlabor group, and 6.2 ± 1.6% and 583 ± 188/�L in the preg-nant control group. The percentage of monocyte andabsolute monocyte counts in the preterm labor group didnot differ from those in the full-term labor and pregnantcontrol groups (P = 0.59, P = 0.32 respectively). There was alinear correlation between the percentage monocyte countmeasured by the automated analyzer and that measured byflow cytometry (r = 0.34, P = 0.0009).

The mean CRP was 0.55 ± 0.68, 0.44 ± 0.37, and0.28 ± 0.23 in the preterm labor, full-term labor, and preg-nant control groups respectively. The CRP levels werehigher in the preterm labor group than in the other twogroups, but did not reach statistical significance (P = 0.09,P = 0.07). The frequency of elevated CRP levels was 33.3%(10/30) in the preterm labor, 22.5% (9/40) in the full-termlabor, and 10% (2/20) in the pregnant control groups. Thefrequency of elevated CRP levels in the preterm labor groupwas not significantly different from that in the full-termlabor and pregnant control groups (P = 0.42, P = 0.09 respec-tively).

Within the population of monocytes, CD14+CD16−

monocytes constituted the largest proportion and rep-resented 69.2% (16.5%–81.4%), 71.7% (26.5%–84.5%), and72.7% (58.8%–82.8%) in the preterm labor, full-term labor,and pregnant control groups respectively. CD14highCD16+

monocytes were present in the following proportions:preterm, 17.1% (4.2–44.9%); full-term, 15.3% (2.3–35.4%);and pregnant controls, 14.6% (2.2–29.2%). CD14lowCD16+

monocytes were present in the following proportions:preterm, 6.0% (1.5–15.5%); full-term, 5.9% (0.8–13.3%); andpregnant controls, 5.5% (0.8–8.9%). There were no signifi-cant differences in the percentage of each monocyte subset

0.37 0.28 ± 0.23 0.14

of the study specimen to childbirth.

among the groups, but the preterm labor group demon-strated a higher percentage of CD14highCD16+ monocytesthan did the full-term (P = 0.08) and pregnant control(P = 0.06) groups.

In the preterm labor group, the percentage ofCD14+CD16−, CD14highCD16+, and CD14lowCD16+ mono-cytes with TLR4 expression was 69.8% (16.5–99.6%),80.0% (33.1–99.4%), and 42.3% (11.2–95.2%) respectively.In the full-term labor group, 39.4% (10.1–92.9%), 49.1%(13.5–97.4%), and 28.1% (7.2–92.9%) of each monocytesubset (CD14+CD16−, CD14highCD16+, and CD14lowCD16+,respectively) was TLR4-positive. Finally, 22.5% (3.5–65.4%),31.8% (5.8–73.2%), and 20.1% (3.1–63.8%) of each monocytesubset (CD14+CD16−, CD14highCD16+, and CD14lowCD16+

respectively) was TLR4-positive in the pregnant controlgroup. Compared with other groups, TLR4 expression ineach monocyte subset was significantly higher in thepreterm labor group (P < 0.0001, P < 0.0001, P = 0.02). Incontrast, TLR4 expression in each monocyte subset did notdiffer between the full-term labor and pregnant controlgroups (P = 0.11, P = 0.12, P = 0.30).

The TLR4 MFI values for CD14+CD16−, CD14highCD16+,and CD14lowCD16+ monocytes were 1.54 (0.26–6.36),1.98 (0.59–5.81), and 1.03 (0.43–3.18) in the pretermlabor group; 0.80 (0.16–3.54), 1.12 (0.17–3.98), and0.83 (0.32–2.38) in the full-term labor group; and 0.54(0.10–1.69), 0.78 (0.10–1.85), and 0.80 (0.37–1.42) in thepregnant control group respectively. In the preterm laborgroup, the TLR4 MFI values of CD14+CD16− (P = 0.0015,P < 0.0001) and CD14highCD16+ monocytes (P = 0.0004,P < 0.0001) were significantly higher than the values inother groups. However, the TLR4 MFI of CD14lowCD16+

monocytes did not differ significantly between groups(P = 0.14, P = 0.10). Finally, the TLR4 MFI for each monocytesubset did not differ between the full-term labor and thepregnant control groups (P = 0.10, P = 0.12, P = 0.69; Table 2).

In the 70 women in the preterm and full-term laborgroups, leukocyte count, monocyte percentage, and abso-lute monocyte counts were not related to CRP. In addition,the percentage of each monocyte subset, TLR4 expression,and MFI of each monocyte subset did not correlate withCRP. However, TLR4 expression and MFI on each mono-cyte subset was significantly correlated (r = 0.86, P < 0.0001,r = 0.87, P < 0.0001, r = 0.84, P < 0.0001).

In 19 subjects with elevated CRP levels out of the 70women in the preterm and full-term labor groups, therates of TLR4-positive monocytes were 46.4%, 52.4%, and40.6% in CD14+CD16−, CD14highCD16+, and CD14lowCD16+

194 J. Kim et al. / Journal of Reproductive Immunology 94 (2012) 190– 195

Table 2Distribution of monocyte subsets and TLR4 expression in pregnant women in preterm labor, full-term labor, and pregnant controls.

Preterm labor (n = 30) Full-term labor (n = 40) Pregnant control (n = 20) P value

CD14+CD16− (%) 69.2 (16.5–81.4) 71.7 (26.5–84.5) 72.7 (58.8–82.8) 0.46TLR4 positivity (%) 69.8 (16.5–99.6) 39.4 (10.1–92.9) 22.5 (3.5–65.4) <0.0001TLR4 MFI 1.54 (0.26–6.36) 0.80 (0.16–3.54) 0.54 (0.10–1.69) 0.0002

CD14highCD16+ (%) 17.1 (4.2–44.9) 15.3 (2.3–35.4) 14.6 (2.2–29.2) 0.11TLR4 positivity (%) 80.0 (33.1–99.4) 49.1 (13.5–97.4) 31.8 (5.8–73.2) <0.0001TLR4 MFI 1.98 (0.59–5.81) 1.12 (0.17–3.98) 0.78 (0.10–1.85) <0.0001

CD14lowCD16+ (%) 6.0 (1.5–15.5) 5.9 (0.8–13.3) 5.5 (0.8–8.9) 0.94TLR4 positivity (%) 42.3 (11.2–95.2) 28.1 (7.2–92.9) 20.1 (3.1–63.8) 0.02

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onocyte subset. These values were higher than thoseetected in the remaining 51 subjects with normal CRP

evels (40.7%, 44.4%, and 27.1% in the CD14+CD16−,D14highCD16+, and CD14lowCD16+ monocyte subset).owever, there was no significant difference between the

wo groups (P = 0.19, P = 0.21, P = 0.10 respectively). TheFIs of CD14+CD16−, CD14highCD16+, and CD14lowCD16+

onocytes in the group with elevated CRP levels were 1.20.20–6.36), 1.7 (0.36–5.81), and 1.1 (0.32–3.18) respec-ively. These values were higher than the MFI values of.9 (0.16–4.13), 1.2 (0.17–3.24), and 0.8 (0.20–2.24) in theroup with normal CRP levels, although the differencesere not significant (P = 0.14, P = 0.17, P = 0.09 respectively).

. Discussion

Preterm labor has been strongly associated withntrauterine infection (Romero et al., 2002). Although local-zed intrauterine infection is often subclinical, it can causeecruitment and activation of monocytes and result inroduction of proinflammatory cytokines. Activation ofonocytes has been implicated in the pathogenesis of

reterm labor (Norman et al., 2007).This is the first report that characterizes monocyte

ubsets in pregnant women who are or are not be inabor. We observed that pregnant women showed a higher

ean percentage of CD14highCD16+ monocytes and a lowerercentage of CD14+CD16− monocytes compared with arevious report in normal subjects (Tallone et al., 2011),hereas the mean percentage of CD14lowCD16+ cells in the

urrent report matches closely with the values from therevious study.

Populations of CD16-positive monocytes in humansave been shown to expand in inflammatory diseasesNockher and Scherberich, 1998; Strauss-Ayali et al., 2007;iegler-Heitbrock et al., 2010), and several studies sug-est that CD16+ monocytes play an important role innflammation (Belge et al., 2002; Strauss-Ayali et al., 2007;anai et al., 2008; Heron et al., 2008). CD16+ monocytesave endothelial affinity owing to the high expression ofhemokine-receptor CX3CR1 and these cells can rapidlyxtravasate in response to inflammatory stimuli (Auffrayt al., 2007; Gautier et al., 2009).

In the present study, the three groups of women showedo differences in monocyte numbers, as represented byhe percentage of monocytes and the absolute monocyteounts. However, a tendency toward elevation was seen for

–2.38) 0.80 (0.37–1.42) 0.17

the CD14highCD16+ monocyte subset in women in pretermlabor compared with women in full-term labor or nolabor, while the other monocyte subsets (CD14+CD16− orCD14lowCD16+) showed no differences. These data suggestthat populations of CD16+ monocytes with high con-comitant expression of CD14 are the primary populationsinvolved in immunological activation in preterm labor.

Well-known inflammatory markers such as leukocytecounts and CRP showed no significant differences betweenthe full-term and preterm labor groups in our presentstudy. The mean WBC counts in both of these labor groupswere similar, but were higher than in the pregnant (nolabor) control group. Leukocyte counts did not correlatewith the value of CRP.

These results showed that the process of labor leads toan elevation of WBCs. We presumed that elevated WBCcount at the time of labor has a limited capacity to indi-cate the existence of inflammation or infection. A recentstudy reported that CRP level is a valuable marker for pre-dicting spontaneous preterm delivery in a preterm laborgroup (Kim et al., 2011). In the current study, the mean CRPlevel and the frequency of elevated CRP in the preterm laborgroup were higher than in the other groups, but the dif-ference was not statistically significant. The percentage ofTLR4-positive monocytes and expression intensity did notdirectly correlate with the level of CRP. However, the TLR4expression and the MFI of each monocyte subset in womenwith elevated CRP levels were higher than in women withnormal CRP levels. The inability to define the influence ofCRP in preterm labor in this study was likely related tolimitations in sample size.

The activation pathways for monocytes have not beenfully elucidated, but monocytes bearing TLR4 seem to playa critical role in immune activation (Sabroe et al., 2002;Kopp and Medzhitov, 2003). This study demonstrated thatwomen in preterm labor had a higher percentage of TLR4-positive monocytes in each monocyte subset than womenin full-term labor or no labor. In addition, TLR4 expressionintensity in CD14+CD16− and CD14highCD16+ monocyteswas higher in women in preterm labor than in those in full-term labor or no labor. These findings are consistent withthose of a previous study (Pawelczyk et al., 2010) in whichwomen undergoing preterm labor showed a significant

increase in TLR4 mRNA expression and in the percent-age of TLR4-positive monocytes. Because of limitationsand potential complications associated with amniocente-sis, evaluation of the presence of intrauterine infection was

ctive Im

1998. Interleukin-1� and interleukin-8 in cervicovaginal fluid during

J. Kim et al. / Journal of Reprodu

not possible in the women in preterm labor in the presentstudy. However, increased TLR4 expression in monocytesin the preterm labor group most likely represents a hostresponse that occurs when maternal immune cells arestimulated by infection.

Previous studies have shown that TLR4 expression inmaternal neutrophils changes minimally during normalpregnancy (Nitsche et al., 2010), and in a murine model,treatment with LPS resulted in preterm delivery throughthe interaction with TLR4 (Lin et al., 2006). A recent studyalso showed that blockage of TLR4 significantly decreasedLPS-induced preterm delivery (Li et al., 2010). Therefore,the increased TLR4 expression in monocytes in womenwith preterm delivery may indicate the initiation of aninflammatory response secondary to the possible infec-tious stimuli and may be related to the subsequent pretermlabor.

Our study is limited by its small data set, but the resultssupport the contention that elevated TLR4 expression onperipheral monocytes may be used as a marker for pretermlabor. This elevation of TLR4 expression would have tooccur prior to the development of preterm labor for itto be an effective marker. However, we were unable toinvestigate the status of TLR4-positive monocytes prior topreterm labor because all of our enrolled patients werealready affected by preterm labor. Future clinical use of thisparameter will require monitoring of TLR4 expression inmonocyte subsets throughout pregnancy and confirmationthat changes in TLR4 expression in monocyte subsets cor-relate with pregnancy outcomes. Therefore, the diagnosticpotential of TLR4 expression as a predictor of preterm laborshould be confirmed with a larger prospective cohort study.

In conclusion, women in preterm labor have higherlevels of CD16 monocytes with high concomitant expres-sion of CD14 and enhanced TLR4 expression in monocytes.These findings suggest that preterm labor is immunologi-cally activated. The expression of TLR4 in specific monocytesubsets could be used as a parameter for predicting the riskof preterm labor.

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