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Interleukin-8 level in maternal serum as a marker for screening of histological chorioamnionitis at term
International
Journal
of Gynecology
& Obstetrics
57 (1997)
1.53-159
Article
Interleukin-8 level in maternal serum as a marker for screening of histological chorioamnionitis at term K. Shimoya”y* , N. Matsuzakia, T. Taniguchib,
T. Okada”, F. Saji”, Y. Murataa
“Deparmtent of Gynecology and Obstetrics, Osaka UniversityMedical School, Osaka, Japan bDepartment of Gynecologv and Obstetrics, Osaka Rosai Hospital, Sakai City, Osaka, Japan ‘Depanment of Gynecology and Obsterrics, Fujimoto Hospital, Neyagawa City, Osaka, Japan Received
3 October
1996; revised
12 Febmaly
1997; accepted
27 February
1997
Abstract Objective: To establisha clinical method for immediatediagnosisof histologicalchorioamnionitis,by maternal blood samplingat term. Method: The seraof 22 motherswith chorioamnionitisand 81 motherswithout chorioamnionitis at term delivery were collected.The serumlevelsof cytokinesincludinginterleukin-1u (IL-l cx),interleukin-1p (IGlP), tumor necrosisfactor-a (TNF-a), interleukin-6 (IL-6) and interleukin-8 (IL-8) were titered and other conventional markers such as white blood cell and CRP were measuredsimultaneously.Chorioamnionitiswas histopathologicallyconfirmedafter delivery. Result: The seraof motherswith histologicalchorioamnionitisshoweda significantincreasein IL-8 titer, but not in those of other cytokines or conventional markers,comparedwith those without chorioamnionitis.A positive correlation was observed between maternal and cord serum IL-8 levels. Maternal IL-8 showed the highest predictive value for diagnosisof histological chorioamnionitis. Conclusion: Measurement of maternal IL8 is useful for rapid prenatal screeningof histological chorioamnionitis at term. 0 1997International Federation of Gynecology and Obstetrics Keywords:
Chorioamnionitis;Cytokine; IL-8; Maternal serum;Term pregnancy
termining factor of perinatal mortality and morbidity of both mothers and neonates despite re-
1. Introduction Intrauterine infection is a very serious concern
in perinatal
medicine,
*Corresponding 8793359; email:
author. Tel.: +81 6 8793356; [email protected]
0020-7292/97/$17.00 PI1 SOOZO-7292(97)
since the disease is a de-
0 1997 International 02891-9
fax:
Federation
+81
6
of Gynecology
cent advances in obstetric therapy and neonatal care [l]. Intra-amniotic infection is a subset of intrauterine infection and is closely related to histological chorioamnionitis, i.e. extra-amniotic infection, another subset of intrauterine infection [2]. Histological chorioamnionitis has been identiand Obstetrics
154
R Shimoya
et al. /International
Journal
fied in 20-33% of women with preterm deliveries and lo-18% of women with term deliveries [3,4]. Definitive diagnosis of histological chorioamnionitis is only possible at present by histological analysis of the placenta after delivery. The diagnosis of clinical chorioamnionitis, in contrast, is difficult owing to the lack of specific clinical signs and symptoms, and low specificity and sensitivity of diagnosis of intrauterine infection [5]. Adequate diagnostic methods to permit the precise diagnosis of histological chorioarmrionitis as early and efficiently as possible should be developed to ensure favorable outcome of pregnancy. Some cytokines such as interleukin-1 (IL-l), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6) and interleukin-8 (IL-81 have been shown to be useful for the detection and monitoring of various human inflammatory diseases. Similar analyses have been applied to diagnose intraamniotic infection by titering cytokine levels in amniotic fluid [6,7]. To examine whether cytokine levels in maternal serum are modulated by the presence or absence of intrauterine infection, we titered levels of cytokines (IL-IQ, IL-l/3, TNF-a, IL-6 and IL-S) in the sera of mothers with or without chorioamnionitis at term delivery and examined their potential for predicting histological chorioamnionitis. Since only IL-8 titers showed marked differences between both groups, we also examined whether there was any significant correlation between IL-8 levels in maternal and fetal serum in chorioamnionitis-free and chorioamnionitis-complicated states. To obtain the best prenatal diagnostic method for chorioamnionitis at term delivery, we examined the interrelationship between the presence of histological chorioamnionitis and clinical markers including maternal cytokine levels, white blood cell (WBC) count and C-reactive protein (CRP) and compared their predictive values for chorioamnionitis. 2. Materials
and methods
Between June 1992 and September 1992, 103 women in spontaneous active labor at term pregnancy were randomly selected by patient’s I.D. number and analyzed retrospectively. We selected patients if their I.D. numbers were multiples of 3. All patients were observed for signs of clinical
of Gynecology
& Obstetrics
57 (1997) 153-159
chorioamnionitis including: fever before delivery (38°C) with or without other known sites of infection; maternal and/or fetal tachycardia; uterine tenderness; leukocytosis; and foul odor of the amniotic fluid [5]. The diagnosis of clinical chorioamnionitis was made when the patient was febrile and was positive for at least two of the other criteria. Only one patient in the present study had clinical chorioamnionitis. To identify patients with histological chorioamnionitis, microscopic analysis of the placenta from each patient was performed and histopathologitally scored. The severity of chorioamnionitis was graded histologically according to Blanc’s classification [8]. The histological stages of chorioamnionitis were defined by the degree of polymorphonuclear cell (PMNC) infiltration into the subchorionic space, i.e. mild stage (stage 11, chorionic plate, i.e. moderate stage (stage 21, and amniotic membrane, i.e. severe stage (stage 3). The numbers of patients with histological chorioamnionitis were 16 (mild), four (moderate) and two (severe>. The incidence of histological chorioamnionitis was 21.4% (22/103). No significant differences were found between women without chorioamnionitis (n = 81) and those with chorioamnionitis (n = 22) in their age (27.0 f 3.5 years vs. 26.8 f 5.4 years), gestational age (39.3 + 1.5 weeks vs. 39.4 f 1.3 weeks), neonatal body weight (3040 k 431 g vs. 2909 f 489 g), gravidity (Gl: 33 vs. 9, G 2 2: 48 vs. 13) or Apgar score (I 6 at 1 min: 2 vs. 1, I 6 at 5 min: 0 vs. 0). There were no differences in mortality or morbidity of the neonates born to mothers with or without histological chorioamnionitis, and no sepsis was found in the neonates in the present study. Women with fetuses identified as having congenital malformations were excluded. Gestational age was determined from the data of their last menstrual period and ultrasonographic data of crown-rump length in the first trimester. Maternal and umbilical cord blood were obtained at delivery, and sera from both were subsequently separated and stored at -80°C until use. A portion of maternal blood was used to count WBC and determine titration of CRP. Informed consent to use maternal and cord sera was obtained from all mothers. To measure titers of IL-la, IL-l& TNF-a,
K Shimoya
et al. /International
Journal
IL-6 and IL-S in maternal and cord sera, enzyme-immunoassay (EIA) kits specific for human IL-la (R&D Systems, Minneapolis), IL-lp (R&D Systems), TNF-a (R&D Systems), IL-6 (Amersham, UK) and IL-8 (R&D Systems) were used. Serum concentration which each kit detected covered the range of over 0.2 pg/ml of IL-la, over 0.3 pg/ml of IL-l& 0.75 pg/ml of TNF-a, 3.13-300 pg/ml of IL-6, and over 18.1 pg/ml of IL-8 respectively. No mutual crossreactivity was found among these kits. Intra-assay variability of the IL-la kit was 1.2-2.1% and its inter-assay variability was 3.3-4.4%. The intraand inter-assay variabilities of the IL-l p kit were 2.88.6% and 4.1-8.6%, respectively. Intra- and inter-assay coefficients of the TNF-a kit were 5.8-9.5% and 2.9-7.8%, respectively. Intra- and inter-assay variabilities of the IL-6 kit were 2.7-4.1% and 4.1-7.8%. Intra- and inter-assay coefficients of the IL-8 kit were 5.4-9.2% and 7.3-12.2%. Statistical analysis of each cytokine level and WBC number in maternal and cord sera was conducted using the Mann-Whitney U-test. Values are expressed as the median and range. The correlation between maternal IL-8 and fetal IL-8 levels was examined by simple linear regression analysis. A value of P < 0.05 was considered statistically significant. Receiver-operator characteristic curves were constructed to describe the relationship between diagnostic indices (sensitivity and specificity) and positive and negative predictive values for cytokines, WBC count and CRP values. 3. Results
Table 1 shows the serum cytokine levels of mothers with (n = 22) or without chorioamnionitis (n = 811. A clear difference between these two groups was only observed in IL-8 level (P = O.OOOS),but not in IL-l (Y, TNF-(Y or IL-6 levels. In IL-lp titers, mothers with chorioamnionitis showed a significantly lower value than those of mothers without chorioamnionitis (P = 0.0233). In IL-8 titers, mothers with mild chorioamnionitis (n = 16; median 110 pg/ml, range O-2000 pg/ml, P = 0.0234) and those with moderate chorioamnionitis (n = 4; median 780 pg/ml, range O-6400
of Gynecology
& Obstetrics
57 (1997)
153-159
155
Table 1 Cytokine levels in the sera of mothers with or without histological chorioamnionitis Maternal cytokine
Chorioamnionitis (- 1 (w/ml)
Chorioamnionitis ( f) (pg/ml)
P-value (pg/ml)
IL-1 cx IL-l p TNF-a IL-6 (pg/ml) IL-8 (pg/ml)
0 (O-56ja 15.5 (O-70) 32 (O-340) 9.5 (O-84) 0 (o-2400)
0 (O-5) 10.2 (O-25) 0 (O-230) 6.1 (O-90) 250 (o-6400)
0.3186 0.0233 0.3186 0.5248 0.0008
‘Value represents the median and parentheses shows minimum-maximum value in the group.
pg/ml, P = 0.0253) differed significantly from those without chorioamnionitis. No significant differences in IL-8 levels were observed between the mothers with mild and moderate chorioamnionitis due to the small number of mothers with moderate chorioamnionitis. There were no significant correlations between maternal IL-8 levels, length of labor and length of rupture of membranes. WBC counts of mothers with (median 10 900 cells/mm3, range 7000-24 200 cells/mm31 or without chorioamnionitis (median 11 700 cells/mm3, range 3700-21900 cells/mm”) also did not differ significantly. The number of patients with clinical chorioamnionitis (n = 11 was obviously too small to perform similar analysis. Fig. 1 shows the distributions of cytokine levels in mothers with or without chorioamnionitis. In contrast to differences in maternal cytokine levels, chorioamnionitis affected IL-6 and IL-8 levels in cord sera. Neonates with and without chorioamnionitis showed differences in IL8 (median 225 pg/ml, range O-6500 pg/ml vs. median 0 pg/ml, range O-1100 pg/ml, P = O.OOOl> and IL-6 titers (median 0 pg/mol, range O-180 pg/ml vs. median 0 pg/ml, range O-32 pg/ml, P = 0.0008). To examine the correlation between maternal and fetal IL-8 levels, a simple linear regression analysis was performed. As shown in Fig. 2, a positive correlation between maternal and cord serum IL-8 levels was observed (Y = 0.558X + 124, r = 0.502, P < 0.0011. However, no such correlation was observed between maternal and cord serum IL-6 levels (data not shown). The clinical diagnostic value of IL-8 titer in
K Shimoya et al. /International Journal
156
of Gynecology & Obsteths
57 (1997) 153-159
B
.
. 6CI
. . .
. .
: .
.i
E .
m 9b
600(
I $ 4oof 5
Fig. 1. Maternal serum IL-la (A), IGlp (B), TNF-a (C), IL6 (D) and IL8 (E) levels of patients with and without histological chorioamnionitis. Y-axis represents cytokine titers (pg/ml). Bars represent the median value for each group.
maternal serum for chorioamnionitis was compared with other serum markers such as CRP, WBC count, IL-la, IGlP, TNF-a and IG6. As evident from Table 2, the clinical detection of IL8 levels in maternal sera was quite sensitive (64%) and specific (81%) for evaluating chorioamnionitis, compared with conventional clinical markers and other cytokines. Moreover, cord IL-8 was also more sensitive (73% vs. 50%) and specific (81% vs. 53%) than cord IL-6 for diagnosis of chorioadionitis. 4. Discussion Since intrauterine infection is still one of the most important causes of maternal and neonatal morbidity and mortality [1,2], the prenatal diagnosis of intrauterine infection is a prerequisite for
the proper clinical management of both mother and infant. There have been a number of reports demonstrating different methods for detection of intra-amniotic or extra-amniotic infection. Culture of placental blocks or amniotic fluid and identification of pathological bacteria are indispensable for diagnosis of intrauterine infection, and provide important information for the medical treatment of mothers and neonates [2]. However, it takes several days to obtain final results and shows low sensitivity and specificity. Romero et al. reported the usefulness of amniotic fluid analysis for rapid detection of intra-amniotic infection by demonstrating Gram staining of bacteria in the amniotic fluid [9], by determining the levels of cytokines such as IL-l (Y,IL-1 j3, IL-6 and IL8 [6,7], and titering glucose concentration in the amniotic fluid [lo]. In contrast, the titration of
K Shimoya et al. / Intemutional Journal
of Gynecology
&
Obstetrics 57 (1997) 153-159
157
Table 2 Sensitivity and specificity of markers used for diagnosis of histological chorioamnionitis
(4 6000-
o&y:,
..
1000
,
2000
Maternal
.
,
3000
I
4000
Serum
IL-8
I
5000
I
6000
(pg/mL)
Markers (cut off value)
Sensitivity Specificity PPV’ NPVb (%o) (%o) (%/o) (%o)
Maternal blood WBC (12 OOO/mm3) CRP (0.3 mg/dl) IL1 a (4.0 pg/ml) IL1 p (12 pg/ml) TNF-a (32 pg/ml) IL6 (11 pg/ml) IL-8 (90 pg/ml)
45 50 23 50 36 43 64
52 67 43 51 54 81
20 22 16 19 16 18 48
77 80 76 76 75 76 89
Cord blood IL-8 (100 pg/ml)
73
81
52
92
49
aPPV represents ‘positive predictive value’. bNPV represents ‘negative predictive value’.
0
::. $2: 0 ‘... .’
I 100
Maternal
1
I
400
500
I
200
300 Serum
IL-8
bdmL)
Fig. 2. Correlation between maternal serum IL-8 and cord serum IL8. Maternal serum IL-8 titers are indicated on the X-axis and cord serum IL8 titers on the Y-axis (A), while the data in the range of O-500 pg/ml has been replotted (Bl.
IL-8 level in fetal sera obtained at preterm delivery has been found to be potentially useful for immediate detection of extra-amniotic infection, i.e. chorioamnionitis, and shows a close relation to the stage of histological chorioamnionitis [ll]. Counting the number of WBC and titration of CRP in maternal sera have been used as conventional markers for early detection of histological chorioamnionitis, but these are of limited value [12,13]. The present study, however, showed that maternal blood sampling could be used as a more reliable method for screening histological chorioamnionitis than conventional markers, which would be useful for and applicable to the man-
agement or screening of chorioamnionitis with premature rupture of membranes (PROM) at preterm or term delivery. Indeed, Len&i et al. also reported the usefulness of maternal blood sampling for detection of clinical chorioamnionitis at preterm pregnancy [14]. Romero et al. [7] showed the effects of labor and chorioamnionitis on elevation of amniotic IL-8 level. However, maternal IL-8 level was only modulated by chorioamnionitis and not by labor. The discrepancy in dynamic changes of IL-8 levels between amniotic fluid and maternal serum remains unknown at present. The titration of maternal IL-8 level was found here to be modulated only by the presence or absence of chorioamnionitis. Such an increase in IL-8 level in maternal sera was observed even in the early stage of chorioamnionitis, suggesting that maternal IL-8 might be useful for prenatal diagnosis of chorioamnionitis. However, further studies are required to examine the relationship between maternal IL-8 and intra-amniotic infection. The accurate diagnosis of chorioamnionitis by titrating maternal serum IL-8 would make optimal management of the fetus possible in utero, thus assuring the most favorable outcome of pregnancy. Our previous report showed elevation and usefulness of titration of cord IL-8 level at preterm delivery for prenatal detection of chorioamnioni-
158
IC Shimoya et al. /International Journal of Gynecology & Obstetrics 57 (1997) 153-159
tis [ll]. The prediction of histological chorioamnionitis by cord IL-8 level at term delivery is also possible because of its higher predictive value than cord IL-6 level. Thus, maternal and neonatal IL8 were shown to be the best indicator available for diagnosis of chorioanmionitis at term pregnancy. Although our results revealed a good positive correlation in IL-8 levels between mother and baby irrespective of the presence or absence of chorioamnionitis, the levels did not necessarily show identical titers. The differences in the main source of IL-8 may explain this; cord IL-8 might be mainly derived from immunocompetent cells such as macrophages [15], trophoblasts, endothelial cells and fibroblasts in the placenta [16]. In contrast, maternal IL-8 might be mainly derived from these cells in the decidua. Each IL-8 level was elevated by IL-S-producing cells activated by inflammatory stimuli in choriodeciduitis, i.e. histological chorioamnionitis. It is also possible that the differences might be induced by the clearance of IL8 molecules from the circulation and circulatory volume between mother and fetus. Increased IL-8 production by the placenta and the decidua under inflammatory conditions might flow between the maternal and fetal circulation, thus causing a difference in maternal and fetal IL-8 levels. The present findings suggest that IL-8 molecules do not simply diffuse out into the maternal and fetal circulation, but rather that IL8 levels in their circulations are strictly regulated. A similar correlation was also observed in soluble IL-2 receptor concentrations in maternal and fetal serum [14]. Recent studies have demonstrated high IL-8 levels in the plasma of patients with clinical disease or those in whom inflammation was induced experimentally [17,181. The increase in IL-8 coincides with the resolution of early granulocytopenia and subsequent granulocytosis in endotoxemia and IL-1 administration into humans [17]. Since IL-8 in maternal serum is enhanced by the chorioamnionitis, the elevated IL-8 may induce changes in the number of granulocytes in the maternal circulation. Such IL-8 may also express chemotactic and activating activity toward neu-
trophils, causing them to accumulate at sites of inflammation, thus preventing the bacteria from entering into the maternal blood stream from the intrauterine space. IL-6 is another cytokine used as a marker to monitor the activity of inflammatory disease and is essential for the host defense mechanism against infection and tissue damage. Increases in serum IL-6 have been observed in patients with various types of inflammatory disease such as SLE [19] and AIDS [20]. High IL-6 titer in amniotic fluid indicates active intra-amniotic infection [21]. Moreover, high IL-6 levels in babies with chorioamnionitis at term pregnancy have been observed to revert to normal levels following proper medical treatment, thus indicating that IL6 is useful as a marker to monitor infected infants [22]. We expected that maternal IL-6 would also be a good marker for prenatal diagnosis of chorioamnionitis at term, but found it to be inferior to IL-8 for this purpose. The precise reason why maternal IL-6 levels showed no difference between mothers with or without chorioamnionitis at term, while the placenta constitutively produces IL-6 and enhances its production in chorioamnionitis is unclear at present [23]. Moreover, human amniotic fluid even under chorioamnionitis-free conditions contains a significant amount of IL6 during pregnancy [21], while that with chorioamnionitis contains a significantly higher amount of IL-6 [93. However, it is noteworthy that differences in IL-6 serum levels were observed between fetuses with and without chorioamnionitis (present study, [ 141). Thus, IL-6 as well as other cytokines such as IL-la, IL-lp and TNF-a in maternal sera are clearly not as sensitive and specific for prenatal detection of histological chorioamnionitis as IL-8 at term. Recently we have reported that maternal IL-6 levels are more sensitive and specific for prenatal detection of histological chorioamnionitis in preterm delivery [24], while this study showed IL-8 titers in maternal sera was most sensitive and specific at term. The reason for the differences between cytokine levels of maternal sera in preterm delivery and at term is unknown. Further investigation would be needed to clarify this.
K Shimoya
et al. /International
Journal
Acknowledgments
This study was supported by Grants-in-Aids for Scientific Research (Nos. 07457387, 07671780, 07671781,08671888 and 08671889) from the Ministry of Education, Science and Culture, Tokyo, Japan. References
ill
Seo K, McGregor JA, French JI. Preterm birth is associated with increased risk of maternal and neonatal infection. Obstet Gynecol 1992; 79: 75-80. Dl Hillier SL, Martius J, Krohn M, Kiviat N, Holmes KK, Eschenbach DA. A case-control study of chorioamnionic infection and histologic chorioamnionitis in prematurity. New Engl J Med 1988; 319: 972-978. [31 Guzick DS, Winn K. The association of chorioamnionitis with preterm delivery. Obstet Gynecol 1985; 65: 11-16. [41 Mueller-Heubach E, Rubinstein DN, Schwarz SS. Histologic chorioamnionitis and preterm delivery in different patient populations. Obstet Gynecol 1990; 75: 622-626. [Sl Hollander D. Diagnosis of chorioamninitis. Clin Obstet Gynecol 1986; 25: 816-825. [61 Romero R, Avila C, Santhanam U, Sehgal PB. Amniotic fluid interleukin-6 in preterm laborAssociation with infection. J Clin Invest 1990; 85: 1392-1400. [71 Romero R, Ceska M, Avila C, Mazor M, Behnke E, Lindley I. Neutrophil attractant/activating peptide/interleukin-8 in term and preterm parturition. Am J Obstet Gynecol 1991; 165: 8133820. 181 Blanc WA. Amniotic infection syndrome: pathogenesis, morphology and significance in circumnatal mortality. Clin Obstet Gynecol 1959; 2: 715-734. [91 Romero R, Sirtori M, Oyarzun E, Avila C, Mazor M, Callahan R et al. Infection and labor V. Prevalence, microbiology, and clinical significance of intraamniotic infection in women with preterm labor and intact membranes. Am J Obstet Gynecol 1989; 161: 817-824. DO1 Kirshon B, Rosenfeld B, Mari G, Belfort M. Amniotic fluid glucose and intrauterine infection. Am J Obstet Gynecol 1991; 164: 818-820. [111 Shimoya K, Matsuzaki N, Taniguchi T, Jo T, Saji F. Kitajima H et al. Interleukin-8 in cord sera: A sensitive and specific marker for the detection of preterm chorioamnionitis. J Infect Dis 1992; 165: 957-960. [I21 Ismail MA, Zinaman MJ, Lowensohn RI, Moawad AH. The significance of C-reactive protein levels in women with premature rupture of membranes. Am J Obstet Gynecol 1985; 151: 541-544. [131 Ernest JM, Swain M, Block SM, Nelson LH, Hatjis CG, Meis PJ. C-reactive protein: A limited test for managing
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patients with preterm labor or preterm rupture of membranes. Am J Obstet Gynecol 1987; 156: 449-454. 1141 Len&i SG, Maciulla MB, Eglinton GS. Maternal and umbilical cord serum interleukin levels in preterm labor with clinical chorioamnionitis. Am J Obstet Gynecol 1994; 170: 1345-1351. [151 Taniguchi T, Matsuzaki N, Shimoya K, Neki R, Okada T, Kitajima H et al. Fetal mononuclear cells show a comparable capacity with maternal mononuclear cells to produce IL-8 in response to lipopolysaccharide in chorioamnionitis. J Reprod Immunol 1993; 23: 1-12. 1161 Shimoya K, Matsuzaki N, Taniguchi T, Kameda T, Koyama M, Neki R et al. Human placenta constitutively produces interleukin-8 during pregnancy and enhance its production in intrauterine infection. Biol Reprod 1992; 47: 220-226. [171 Van-Zee KJ, DeForge LE, Fischer E, Marano MA, Kenney JS, Remick DG et al. IL-8 in septic shock, endotoxemia, and after IL-1 administration. J Immunol 1991; 146: 347883482. [181 Martich GD, Danner RL, Ceska M, Suffredini AF. Detection of interleukin-8 and tumor necrosis factor in normal humans after intravenous endotoxin: the effect of anti-inflammatory agents. J Exp Med 1991; 173: 1021-1024. 1191 Linker-Israeli M, Deans RJ, Wallace DJ, Prehn J, Ozeri-Chen T, Klinenberg JR. Elevated levels of endogenous IL-6 in systemic lupus erythematodes. A putative role in pathogenesis. J Immunol 1991; 147: 117-123. m Nakajima K, Martinez-Maza 0, Hirano T, Breen EC. Nishanian PG, Salazar-Gonzalez JF et al. Induction of IL-6 (B cell stimulatory factor-Z/IFN- beta 2) production by HIV. J Immunol 1989; 142: 531-536. ml Greig PC, Ernest JM, Teot L, Erickson M, Talley R. Amniotic fluid interleukin-6 levels correlate with histologic chorioamnionitis and amniotic fluid cultures in patients with premature labor with intact membranes. Am J Obstet Gynecol 1993; 169: 1035-1044. WI Matsuzaki N, Saji F, Kameda T, Yoshizaki K, Okada T, Sawai K et al. In vivo and in vitro production of interleukin-6 by fetal mononuclear cells, Clin Immunol Immunopathol 1990; 55: 305-314. WI Matsuzaki N, Taniguchi T, Shimoya K, Neki R, Okada T, Saji F et al. Placental interleukin-6 production is enhanced in intrauterine infection, but not in labor. Am J Obstet Gynecol 1993; 168: 94-97. 1241 Maeda K, Matsuzaki N, Fuke S, Mitsuda N, Shimoya K, Nakayama M et al. Diagnostic value of maternal interleukin-level for determination of histologic chorioamnionitis in preterm delivery. Gynecol Obstet Invest 1997: in press.
Journal
of Gynecology
& Obstetrics
57 (1997)
1.53-159
Article
Interleukin-8 level in maternal serum as a marker for screening of histological chorioamnionitis at term K. Shimoya”y* , N. Matsuzakia, T. Taniguchib,
T. Okada”, F. Saji”, Y. Murataa
“Deparmtent of Gynecology and Obstetrics, Osaka UniversityMedical School, Osaka, Japan bDepartment of Gynecologv and Obstetrics, Osaka Rosai Hospital, Sakai City, Osaka, Japan ‘Depanment of Gynecology and Obsterrics, Fujimoto Hospital, Neyagawa City, Osaka, Japan Received
3 October
1996; revised
12 Febmaly
1997; accepted
27 February
1997
Abstract Objective: To establisha clinical method for immediatediagnosisof histologicalchorioamnionitis,by maternal blood samplingat term. Method: The seraof 22 motherswith chorioamnionitisand 81 motherswithout chorioamnionitis at term delivery were collected.The serumlevelsof cytokinesincludinginterleukin-1u (IL-l cx),interleukin-1p (IGlP), tumor necrosisfactor-a (TNF-a), interleukin-6 (IL-6) and interleukin-8 (IL-8) were titered and other conventional markers such as white blood cell and CRP were measuredsimultaneously.Chorioamnionitiswas histopathologicallyconfirmedafter delivery. Result: The seraof motherswith histologicalchorioamnionitisshoweda significantincreasein IL-8 titer, but not in those of other cytokines or conventional markers,comparedwith those without chorioamnionitis.A positive correlation was observed between maternal and cord serum IL-8 levels. Maternal IL-8 showed the highest predictive value for diagnosisof histological chorioamnionitis. Conclusion: Measurement of maternal IL8 is useful for rapid prenatal screeningof histological chorioamnionitis at term. 0 1997International Federation of Gynecology and Obstetrics Keywords:
Chorioamnionitis;Cytokine; IL-8; Maternal serum;Term pregnancy
termining factor of perinatal mortality and morbidity of both mothers and neonates despite re-
1. Introduction Intrauterine infection is a very serious concern
in perinatal
medicine,
*Corresponding 8793359; email:
author. Tel.: +81 6 8793356; [email protected]
0020-7292/97/$17.00 PI1 SOOZO-7292(97)
since the disease is a de-
0 1997 International 02891-9
fax:
Federation
+81
6
of Gynecology
cent advances in obstetric therapy and neonatal care [l]. Intra-amniotic infection is a subset of intrauterine infection and is closely related to histological chorioamnionitis, i.e. extra-amniotic infection, another subset of intrauterine infection [2]. Histological chorioamnionitis has been identiand Obstetrics
154
R Shimoya
et al. /International
Journal
fied in 20-33% of women with preterm deliveries and lo-18% of women with term deliveries [3,4]. Definitive diagnosis of histological chorioamnionitis is only possible at present by histological analysis of the placenta after delivery. The diagnosis of clinical chorioamnionitis, in contrast, is difficult owing to the lack of specific clinical signs and symptoms, and low specificity and sensitivity of diagnosis of intrauterine infection [5]. Adequate diagnostic methods to permit the precise diagnosis of histological chorioarmrionitis as early and efficiently as possible should be developed to ensure favorable outcome of pregnancy. Some cytokines such as interleukin-1 (IL-l), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6) and interleukin-8 (IL-81 have been shown to be useful for the detection and monitoring of various human inflammatory diseases. Similar analyses have been applied to diagnose intraamniotic infection by titering cytokine levels in amniotic fluid [6,7]. To examine whether cytokine levels in maternal serum are modulated by the presence or absence of intrauterine infection, we titered levels of cytokines (IL-IQ, IL-l/3, TNF-a, IL-6 and IL-S) in the sera of mothers with or without chorioamnionitis at term delivery and examined their potential for predicting histological chorioamnionitis. Since only IL-8 titers showed marked differences between both groups, we also examined whether there was any significant correlation between IL-8 levels in maternal and fetal serum in chorioamnionitis-free and chorioamnionitis-complicated states. To obtain the best prenatal diagnostic method for chorioamnionitis at term delivery, we examined the interrelationship between the presence of histological chorioamnionitis and clinical markers including maternal cytokine levels, white blood cell (WBC) count and C-reactive protein (CRP) and compared their predictive values for chorioamnionitis. 2. Materials
and methods
Between June 1992 and September 1992, 103 women in spontaneous active labor at term pregnancy were randomly selected by patient’s I.D. number and analyzed retrospectively. We selected patients if their I.D. numbers were multiples of 3. All patients were observed for signs of clinical
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chorioamnionitis including: fever before delivery (38°C) with or without other known sites of infection; maternal and/or fetal tachycardia; uterine tenderness; leukocytosis; and foul odor of the amniotic fluid [5]. The diagnosis of clinical chorioamnionitis was made when the patient was febrile and was positive for at least two of the other criteria. Only one patient in the present study had clinical chorioamnionitis. To identify patients with histological chorioamnionitis, microscopic analysis of the placenta from each patient was performed and histopathologitally scored. The severity of chorioamnionitis was graded histologically according to Blanc’s classification [8]. The histological stages of chorioamnionitis were defined by the degree of polymorphonuclear cell (PMNC) infiltration into the subchorionic space, i.e. mild stage (stage 11, chorionic plate, i.e. moderate stage (stage 21, and amniotic membrane, i.e. severe stage (stage 3). The numbers of patients with histological chorioamnionitis were 16 (mild), four (moderate) and two (severe>. The incidence of histological chorioamnionitis was 21.4% (22/103). No significant differences were found between women without chorioamnionitis (n = 81) and those with chorioamnionitis (n = 22) in their age (27.0 f 3.5 years vs. 26.8 f 5.4 years), gestational age (39.3 + 1.5 weeks vs. 39.4 f 1.3 weeks), neonatal body weight (3040 k 431 g vs. 2909 f 489 g), gravidity (Gl: 33 vs. 9, G 2 2: 48 vs. 13) or Apgar score (I 6 at 1 min: 2 vs. 1, I 6 at 5 min: 0 vs. 0). There were no differences in mortality or morbidity of the neonates born to mothers with or without histological chorioamnionitis, and no sepsis was found in the neonates in the present study. Women with fetuses identified as having congenital malformations were excluded. Gestational age was determined from the data of their last menstrual period and ultrasonographic data of crown-rump length in the first trimester. Maternal and umbilical cord blood were obtained at delivery, and sera from both were subsequently separated and stored at -80°C until use. A portion of maternal blood was used to count WBC and determine titration of CRP. Informed consent to use maternal and cord sera was obtained from all mothers. To measure titers of IL-la, IL-l& TNF-a,
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IL-6 and IL-S in maternal and cord sera, enzyme-immunoassay (EIA) kits specific for human IL-la (R&D Systems, Minneapolis), IL-lp (R&D Systems), TNF-a (R&D Systems), IL-6 (Amersham, UK) and IL-8 (R&D Systems) were used. Serum concentration which each kit detected covered the range of over 0.2 pg/ml of IL-la, over 0.3 pg/ml of IL-l& 0.75 pg/ml of TNF-a, 3.13-300 pg/ml of IL-6, and over 18.1 pg/ml of IL-8 respectively. No mutual crossreactivity was found among these kits. Intra-assay variability of the IL-la kit was 1.2-2.1% and its inter-assay variability was 3.3-4.4%. The intraand inter-assay variabilities of the IL-l p kit were 2.88.6% and 4.1-8.6%, respectively. Intra- and inter-assay coefficients of the TNF-a kit were 5.8-9.5% and 2.9-7.8%, respectively. Intra- and inter-assay variabilities of the IL-6 kit were 2.7-4.1% and 4.1-7.8%. Intra- and inter-assay coefficients of the IL-8 kit were 5.4-9.2% and 7.3-12.2%. Statistical analysis of each cytokine level and WBC number in maternal and cord sera was conducted using the Mann-Whitney U-test. Values are expressed as the median and range. The correlation between maternal IL-8 and fetal IL-8 levels was examined by simple linear regression analysis. A value of P < 0.05 was considered statistically significant. Receiver-operator characteristic curves were constructed to describe the relationship between diagnostic indices (sensitivity and specificity) and positive and negative predictive values for cytokines, WBC count and CRP values. 3. Results
Table 1 shows the serum cytokine levels of mothers with (n = 22) or without chorioamnionitis (n = 811. A clear difference between these two groups was only observed in IL-8 level (P = O.OOOS),but not in IL-l (Y, TNF-(Y or IL-6 levels. In IL-lp titers, mothers with chorioamnionitis showed a significantly lower value than those of mothers without chorioamnionitis (P = 0.0233). In IL-8 titers, mothers with mild chorioamnionitis (n = 16; median 110 pg/ml, range O-2000 pg/ml, P = 0.0234) and those with moderate chorioamnionitis (n = 4; median 780 pg/ml, range O-6400
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Table 1 Cytokine levels in the sera of mothers with or without histological chorioamnionitis Maternal cytokine
Chorioamnionitis (- 1 (w/ml)
Chorioamnionitis ( f) (pg/ml)
P-value (pg/ml)
IL-1 cx IL-l p TNF-a IL-6 (pg/ml) IL-8 (pg/ml)
0 (O-56ja 15.5 (O-70) 32 (O-340) 9.5 (O-84) 0 (o-2400)
0 (O-5) 10.2 (O-25) 0 (O-230) 6.1 (O-90) 250 (o-6400)
0.3186 0.0233 0.3186 0.5248 0.0008
‘Value represents the median and parentheses shows minimum-maximum value in the group.
pg/ml, P = 0.0253) differed significantly from those without chorioamnionitis. No significant differences in IL-8 levels were observed between the mothers with mild and moderate chorioamnionitis due to the small number of mothers with moderate chorioamnionitis. There were no significant correlations between maternal IL-8 levels, length of labor and length of rupture of membranes. WBC counts of mothers with (median 10 900 cells/mm3, range 7000-24 200 cells/mm31 or without chorioamnionitis (median 11 700 cells/mm3, range 3700-21900 cells/mm”) also did not differ significantly. The number of patients with clinical chorioamnionitis (n = 11 was obviously too small to perform similar analysis. Fig. 1 shows the distributions of cytokine levels in mothers with or without chorioamnionitis. In contrast to differences in maternal cytokine levels, chorioamnionitis affected IL-6 and IL-8 levels in cord sera. Neonates with and without chorioamnionitis showed differences in IL8 (median 225 pg/ml, range O-6500 pg/ml vs. median 0 pg/ml, range O-1100 pg/ml, P = O.OOOl> and IL-6 titers (median 0 pg/mol, range O-180 pg/ml vs. median 0 pg/ml, range O-32 pg/ml, P = 0.0008). To examine the correlation between maternal and fetal IL-8 levels, a simple linear regression analysis was performed. As shown in Fig. 2, a positive correlation between maternal and cord serum IL-8 levels was observed (Y = 0.558X + 124, r = 0.502, P < 0.0011. However, no such correlation was observed between maternal and cord serum IL-6 levels (data not shown). The clinical diagnostic value of IL-8 titer in
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B
.
. 6CI
. . .
. .
: .
.i
E .
m 9b
600(
I $ 4oof 5
Fig. 1. Maternal serum IL-la (A), IGlp (B), TNF-a (C), IL6 (D) and IL8 (E) levels of patients with and without histological chorioamnionitis. Y-axis represents cytokine titers (pg/ml). Bars represent the median value for each group.
maternal serum for chorioamnionitis was compared with other serum markers such as CRP, WBC count, IL-la, IGlP, TNF-a and IG6. As evident from Table 2, the clinical detection of IL8 levels in maternal sera was quite sensitive (64%) and specific (81%) for evaluating chorioamnionitis, compared with conventional clinical markers and other cytokines. Moreover, cord IL-8 was also more sensitive (73% vs. 50%) and specific (81% vs. 53%) than cord IL-6 for diagnosis of chorioadionitis. 4. Discussion Since intrauterine infection is still one of the most important causes of maternal and neonatal morbidity and mortality [1,2], the prenatal diagnosis of intrauterine infection is a prerequisite for
the proper clinical management of both mother and infant. There have been a number of reports demonstrating different methods for detection of intra-amniotic or extra-amniotic infection. Culture of placental blocks or amniotic fluid and identification of pathological bacteria are indispensable for diagnosis of intrauterine infection, and provide important information for the medical treatment of mothers and neonates [2]. However, it takes several days to obtain final results and shows low sensitivity and specificity. Romero et al. reported the usefulness of amniotic fluid analysis for rapid detection of intra-amniotic infection by demonstrating Gram staining of bacteria in the amniotic fluid [9], by determining the levels of cytokines such as IL-l (Y,IL-1 j3, IL-6 and IL8 [6,7], and titering glucose concentration in the amniotic fluid [lo]. In contrast, the titration of
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Table 2 Sensitivity and specificity of markers used for diagnosis of histological chorioamnionitis
(4 6000-
o&y:,
..
1000
,
2000
Maternal
.
,
3000
I
4000
Serum
IL-8
I
5000
I
6000
(pg/mL)
Markers (cut off value)
Sensitivity Specificity PPV’ NPVb (%o) (%o) (%/o) (%o)
Maternal blood WBC (12 OOO/mm3) CRP (0.3 mg/dl) IL1 a (4.0 pg/ml) IL1 p (12 pg/ml) TNF-a (32 pg/ml) IL6 (11 pg/ml) IL-8 (90 pg/ml)
45 50 23 50 36 43 64
52 67 43 51 54 81
20 22 16 19 16 18 48
77 80 76 76 75 76 89
Cord blood IL-8 (100 pg/ml)
73
81
52
92
49
aPPV represents ‘positive predictive value’. bNPV represents ‘negative predictive value’.
0
::. $2: 0 ‘... .’
I 100
Maternal
1
I
400
500
I
200
300 Serum
IL-8
bdmL)
Fig. 2. Correlation between maternal serum IL-8 and cord serum IL8. Maternal serum IL-8 titers are indicated on the X-axis and cord serum IL8 titers on the Y-axis (A), while the data in the range of O-500 pg/ml has been replotted (Bl.
IL-8 level in fetal sera obtained at preterm delivery has been found to be potentially useful for immediate detection of extra-amniotic infection, i.e. chorioamnionitis, and shows a close relation to the stage of histological chorioamnionitis [ll]. Counting the number of WBC and titration of CRP in maternal sera have been used as conventional markers for early detection of histological chorioamnionitis, but these are of limited value [12,13]. The present study, however, showed that maternal blood sampling could be used as a more reliable method for screening histological chorioamnionitis than conventional markers, which would be useful for and applicable to the man-
agement or screening of chorioamnionitis with premature rupture of membranes (PROM) at preterm or term delivery. Indeed, Len&i et al. also reported the usefulness of maternal blood sampling for detection of clinical chorioamnionitis at preterm pregnancy [14]. Romero et al. [7] showed the effects of labor and chorioamnionitis on elevation of amniotic IL-8 level. However, maternal IL-8 level was only modulated by chorioamnionitis and not by labor. The discrepancy in dynamic changes of IL-8 levels between amniotic fluid and maternal serum remains unknown at present. The titration of maternal IL-8 level was found here to be modulated only by the presence or absence of chorioamnionitis. Such an increase in IL-8 level in maternal sera was observed even in the early stage of chorioamnionitis, suggesting that maternal IL-8 might be useful for prenatal diagnosis of chorioamnionitis. However, further studies are required to examine the relationship between maternal IL-8 and intra-amniotic infection. The accurate diagnosis of chorioamnionitis by titrating maternal serum IL-8 would make optimal management of the fetus possible in utero, thus assuring the most favorable outcome of pregnancy. Our previous report showed elevation and usefulness of titration of cord IL-8 level at preterm delivery for prenatal detection of chorioamnioni-
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tis [ll]. The prediction of histological chorioamnionitis by cord IL-8 level at term delivery is also possible because of its higher predictive value than cord IL-6 level. Thus, maternal and neonatal IL8 were shown to be the best indicator available for diagnosis of chorioanmionitis at term pregnancy. Although our results revealed a good positive correlation in IL-8 levels between mother and baby irrespective of the presence or absence of chorioamnionitis, the levels did not necessarily show identical titers. The differences in the main source of IL-8 may explain this; cord IL-8 might be mainly derived from immunocompetent cells such as macrophages [15], trophoblasts, endothelial cells and fibroblasts in the placenta [16]. In contrast, maternal IL-8 might be mainly derived from these cells in the decidua. Each IL-8 level was elevated by IL-S-producing cells activated by inflammatory stimuli in choriodeciduitis, i.e. histological chorioamnionitis. It is also possible that the differences might be induced by the clearance of IL8 molecules from the circulation and circulatory volume between mother and fetus. Increased IL-8 production by the placenta and the decidua under inflammatory conditions might flow between the maternal and fetal circulation, thus causing a difference in maternal and fetal IL-8 levels. The present findings suggest that IL-8 molecules do not simply diffuse out into the maternal and fetal circulation, but rather that IL8 levels in their circulations are strictly regulated. A similar correlation was also observed in soluble IL-2 receptor concentrations in maternal and fetal serum [14]. Recent studies have demonstrated high IL-8 levels in the plasma of patients with clinical disease or those in whom inflammation was induced experimentally [17,181. The increase in IL-8 coincides with the resolution of early granulocytopenia and subsequent granulocytosis in endotoxemia and IL-1 administration into humans [17]. Since IL-8 in maternal serum is enhanced by the chorioamnionitis, the elevated IL-8 may induce changes in the number of granulocytes in the maternal circulation. Such IL-8 may also express chemotactic and activating activity toward neu-
trophils, causing them to accumulate at sites of inflammation, thus preventing the bacteria from entering into the maternal blood stream from the intrauterine space. IL-6 is another cytokine used as a marker to monitor the activity of inflammatory disease and is essential for the host defense mechanism against infection and tissue damage. Increases in serum IL-6 have been observed in patients with various types of inflammatory disease such as SLE [19] and AIDS [20]. High IL-6 titer in amniotic fluid indicates active intra-amniotic infection [21]. Moreover, high IL-6 levels in babies with chorioamnionitis at term pregnancy have been observed to revert to normal levels following proper medical treatment, thus indicating that IL6 is useful as a marker to monitor infected infants [22]. We expected that maternal IL-6 would also be a good marker for prenatal diagnosis of chorioamnionitis at term, but found it to be inferior to IL-8 for this purpose. The precise reason why maternal IL-6 levels showed no difference between mothers with or without chorioamnionitis at term, while the placenta constitutively produces IL-6 and enhances its production in chorioamnionitis is unclear at present [23]. Moreover, human amniotic fluid even under chorioamnionitis-free conditions contains a significant amount of IL6 during pregnancy [21], while that with chorioamnionitis contains a significantly higher amount of IL-6 [93. However, it is noteworthy that differences in IL-6 serum levels were observed between fetuses with and without chorioamnionitis (present study, [ 141). Thus, IL-6 as well as other cytokines such as IL-la, IL-lp and TNF-a in maternal sera are clearly not as sensitive and specific for prenatal detection of histological chorioamnionitis as IL-8 at term. Recently we have reported that maternal IL-6 levels are more sensitive and specific for prenatal detection of histological chorioamnionitis in preterm delivery [24], while this study showed IL-8 titers in maternal sera was most sensitive and specific at term. The reason for the differences between cytokine levels of maternal sera in preterm delivery and at term is unknown. Further investigation would be needed to clarify this.
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Acknowledgments
This study was supported by Grants-in-Aids for Scientific Research (Nos. 07457387, 07671780, 07671781,08671888 and 08671889) from the Ministry of Education, Science and Culture, Tokyo, Japan. References
ill
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