Amniotic fluid interleukin-6 and preterm delivery: a review

23.

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25.

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27.

28.

29. 30.

31.

32.

for Meconium Project (CRAMP): 1. South Africa. Br J Obstet Gynaecol 1998;105:304 – 8. Mahomed K, Mulambo T, Woelk G, Hofmeyr GJ, Gulmezoglu AM. The Collaborative Randomised Amnioinfusion for Meconium Project (CRAMP): 2. Zimbabwe. Br J Obstet Gynaecol 1998;105: 309 –13. Khosla AH, Sangwan K, Ahuja SD. Prophylactic amnioinfusion during labor complicated by meconium. Aust N Z J Obstet Gynaecol 1997;37:294 – 6. Moodley J, Matchaba P, Payne AJ. Intrapartum amnioinfusion for meconium-stained liquor in developing countries. Trop Doct 1998; 28:31– 4. Chalmers I, Hetherington J, Elbourne D, Keirse MJNC, Enkin M. Materials and methods used for synthesizing evidence to evaluate the effects of care during pregnancy and childbirth. In: Chalmers I, Enkin M, Keirse MJNC, eds. A guide to effective care in pregnancy and childbirth. Oxford: Oxford University Press, 1989:39 – 65. Mantel N, Haenszel W. Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst 1959;22: 719 – 48. Breslow NE, Day NE. Statistical methods in cancer research: The analysis of case control studies. Lyons, France: IARC Scientific Publications, 1980. Egger M, Davey Smith G, Minder C. Bias in meta-analysis detected by a simple, graphical test. BMJ 1997;315:629 –34. Wu BT, Sun LJ, Tang LY. Intrapartum amnioinfusion for replacement of meconium-stained-amniotic-fluid to prevent meconium aspiration syndrome. Chin Med J 1991;104:221– 4. Peng TC, Gutcher GR, Van Dorsten JP. A selective aggressive approach to the neonate exposed to meconium-stained amniotic fluid. Am J Obstet Gynecol 1996;175:296 –303. Hofmeyr GJ, Gulmezoglu AM, Nikodem VC, De Jager M. Amnioinfusion. Eur J Obstet Gynecol Reprod Biol 1996;64:159 – 65.

Amniotic fluid interleukin-6 and preterm delivery: A review Amira Y. El-Bastawissi, MBCHB, PhD, Michelle A. Williams, ScD, Donald E. Riley, PhD, Jane Hitti, MD, MPH, and John N. Krieger, MD Objective: To evaluate the potential role of amniotic fluid (AF) interleukin (IL)-6 as a predictor of preterm delivery and to consider possible explanations for the proportion of women with elevated AF IL-6 who deliver preterm yet lack microbiologically detectable intra-amniotic infection. Data Sources: We searched the English language human literature in MEDLINE, 1966 through September 1999, using the keywords “labor/infant,” “premature,” “cytokines/ interleukin-6,” and “AF.” We also examined abstracts from From the Departments of Epidemiology, Urology, Pathobiology, and Obstetrics and Gynecology, University of Washington; and the Center for Perinatal Studies, Swedish Medical Center, Seattle, Washington. This work was supported by grant R40 MC 00113 from the Maternal and Child Health Bureau (Title V, Social Security Act), Health Resources and Services Administration, Department of Health and Human Services.

1056 0029-7844/00/$20.00 PII S0029-7844(00)00875-9

33. Hofmeyr GJ. Amnioinfusion for meconium-stained liquor in labor. In: Neilson JP, Crowther CA, Hodnett ED, Hofmeyr GJ, Keirse MJNC, eds. Pregnancy and childbirth module of the Cochrane database of systematic reviews. The Cochrane Library, Vol 2. Oxford: Update Software, 1999. 34. Thompson SG. Why sources of heterogeneity in meta-analysis should be investigated. BMJ 1994;309:1351–5. 35. Posner MD, Ballagh SA, Paul RH. The effect of amnioinfusion on uterine pressure and activity: A preliminary report. Am J Obstet Gynecol 1990;163:813– 8. 36. Wenstrom K, Andrews WW, Maher JE. Amnioinfusion survey: Prevalence, protocols, and complications. Obstet Gynecol 1995;86: 572– 6.

Address reprint requests to:

Francisco L. Gaudier, MD University of Florida Health Science Center Department of Obstetrics and Gynecology 653-1 West 8th Street Jacksonville, FL 32209

Received July 1, 1999. Received in revised form November 1, 1999. Accepted November 15, 1999.

Copyright © 2000 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.

the 1999 meetings of the Society for Maternal-Fetal Medicine and the Society for Epidemiologic Research. We identified other studies by reviewing the reference lists of published articles. Methods of Study Selection: The MEDLINE search yielded 55 citations. We focused on studies that reported on the association between AF IL-6 and preterm delivery. Tabulation, Integration, and Results: There is consensus in the literature that elevated AF IL-6 is a stronger predictor of preterm delivery than intra-amniotic infection detected by either microbiologic culture or polymerase chain reaction (PCR). Among women with elevated AF IL-6, PCR could detect a higher proportion of intra-amniotic infection than culture. A number of women with elevated AF IL-6 (33–70%) deliver preterm and do not have evidence of intra-amniotic infection by either culture or PCR. Possible explanations for this observation are considered. Conclusion: Elevated AF IL-6 is strongly associated with preterm delivery and merits future consideration in clinical settings to predict preterm delivery and guide patient care. Development of improved polymerase chain reaction-based clinical methods to detect intra-amniotic infection is necessary to better understand the relationship between elevated AF IL-6, intra-amniotic infection, and preterm delivery. (Obstet Gynecol 2000;95:1056– 64. © 2000 by The American College of Obstetricians and Gynecologists.)

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Preterm delivery, defined as birth before 37 weeks’ gestation, accounts for approximately 75% of perinatal morbidity and mortality.1 An important public health concern is the increase in the preterm delivery rate from 8% in 19821 to 11.4% in 1997.2 Therefore, one of the most urgent problems in perinatology is to identify women at high risk for delivering preterm and develop appropriate preventive measures for preterm delivery.3 Further reductions of the perinatal mortality rate, the cost of neonatal care, and of subsequent physical and neurologic impairment among preterm infant survivors depend on reducing the preterm delivery rate.4 The association between lower and upper genital tract infection and preterm delivery has been well established. Investigators have used a variety of study designs in diverse populations and have published many studies supporting the importance of infections in the etiology of preterm delivery. Methodologic approaches taken to demonstrate this association included attempts to isolate microorganisms from amniotic fluid (AF), placenta, and the lower genital tract,5–11 to identify bacterial byproducts in AF,10 and to administer prophylactic antibiotic treatment in clinical trials of antimicrobial therapy.12,13 Subclinical and histologic chorioamnionitis, untreated pyelonephritis, and asymptomatic bacteriuria also have been associated with preterm delivery.14 –19 These studies support the view that the association between genital tract infection and preterm delivery is most likely causal; however, microbial invasion after onset of labor should not be precluded. The prevalence of microbial invasion of the amniotic cavity, as detected by AF culture, has been reported to range from 4.2% (one of 24) to 21.6% (24 of 111) among women with preterm labor and intact membranes who subsequently delivered a preterm neonate.20 –22 Such intra-amniotic infection is often subclinical and requires extensive microbiologic studies for accurate diagnosis. Most clinical laboratories do not have adequate facilities for such extensive studies, which to date, require evaluation in research laboratories. The early identification of subclinical intra-amniotic infection is a desirable clinical goal because neonates born to mothers with intra-amniotic infection have a higher risk of both infectious and noninfectious complications than infants born to mothers with negative AF cultures.10,20,22 Moreover, women with intra-amniotic infection are more likely to develop clinical chorioamnionitis, spontaneous rupture of the fetal membranes, and to not respond to tocolysis because of the underlying inflammation.10,16,20,22–27 However, early diagnosis of intraamniotic infection is difficult because the classic clinical signs of infection (fever, uterine tenderness, foulsmelling vaginal discharge, fetal tachycardia, and maternal leukocytosis) occur late and are present in only a

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small proportion of women with microbiologic evidence of intra-amniotic infection. For example, in a study of 264 women with preterm labor and intact membranes, Romero et al22 noted that only 12.5% of women with positive AF cultures (n ⫽ 24) had clinical signs of infection. The need to treat patients with subclinical intraamniotic infection promptly has led to considerable interest in developing rapid, yet highly sensitive and specific, assays for the identification of microbial invasion of the amniotic cavity. Examples of relatively rapid assays used to diagnose intra-amniotic infection include Gram stain,27 limulus amebocyte assay,28 acridine orange tests,29 leukocyte esterase,30 white blood cell count,31 glucose concentration,32,33 gas-liquid chromatography,34 and leukoattractants.35 The limited sensitivity and specificity of some of these tests and their inability to identify the specific organism that caused the infection limits their clinical usefulness. Sensitive tests are necessary to detect the presence or absence of intra-amniotic infection to avoid failing to treat infected women, and specific tests are necessary to avoid overuse of antibiotics in noninfected women. It is also crucial to identify the specific organisms that caused the infection to facilitate selection of appropriate antimicrobial agents. Infection-induced stimulation of immune cells to produce cytokines with cell-mediated immunity leads to cytokine production and the subsequent synthesis and release of prostaglandin E2 and prostaglandin F2␣,36 –39 which have been shown to induce cervical ripening, uterine contractions, and labor at term.40 Cytokines have also been implicated in the pathophysiologic mechanisms of preterm labor.36,41 The association between several AF cytokines and preterm delivery has been evaluated including: interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor (TNF-␣), and granulocyte-colony stimulating factor.39,41– 45 In the setting of infection it has been shown that preterm delivery is associated with dramatic alterations in the AF concentration of several cytokines.39,43,46 Clinical studies have shown that elevated maternal serum C-reactive protein precedes the development of clinical chorioamnionitis and onset of preterm labor in women with preterm premature rupture of membranes (PROM).47– 49 Additionally, women in preterm labor with elevated C-reactive protein are less likely to respond to tocolysis compared with women who have nondetectable C-reactive protein.17,49,50 Because IL-6 plays a critical role in the induction of C-reactive protein synthesis,51,52 it has been hypothesized that this cytokine, regarded as a major mediator of the host response to infection and tissue damage, is also impor-

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tant in the host response to intrauterine infection. Indeed, AF IL-6 concentration alterations consistently exceeded the alterations in levels of the other cytokines tested.39,41,46 These findings suggest that AF IL-6 is the cytokine most likely involved in the pathophysiology of preterm labor associated with infection. Amniotic fluid IL-6 concentrations above 1.5 ng/mL were detected in 88% (28 of 32) of women who delivered at or before 34 weeks’ gestation compared with 12% (two of 17) of women who delivered after 34 weeks’ gestation (P ⬍ .001).39 These observations are important because the highest rate of intra-amniotic infection among women in preterm labor occurs earlier than 34 weeks’ gestation.21 The proportions of women who delivered at or before 34 weeks’ gestation and who had elevated AF IL-1␣, IL-1␤, and TNF-␣ were only 50%, 42%, and 44%, respectively.39 These findings suggest that, of cytokines studied, IL-6 has the strongest association with preterm labor in a setting of intra-amniotic infection. Moreover, bacteria were present in 100% (nine of nine), 88% (seven of eight), 63% (five of eight), and 89% (eight of nine) of women with preterm labor and intact membranes, and elevated AF IL-6, IL-1␣, IL-1␤, and TNF-␣, respectively.39 Similarly, elevated AF IL-6 was more likely than the other cytokines to be associated with histologic chorioamnionitis and presence of bacteria in the chorioamnion. Histologic chorioamnionitis was present in 100% (15 of 15), 64% (nine of 14), 64% (nine of 14), and 67% (ten of 15) of women who had elevated AF IL-6, IL-1␣, IL-1␤, and TNF-␣, respectively.39 Presence of bacteria in the chorioamnion was documented in 100% (nine of nine), 38% (three of eight), 50% (four of eight), and 67% (six of nine) of women who had elevated AF IL-6, IL-1␣, IL-1␤, and TNF-␣, respectively.39 These findings provide further support for the conclusion that AF IL-6 is the cytokine more likely than other cytokines to be associated with intra-amniotic infection, histologic chorioamnionitis, and the presence of bacteria in chorioamnion. The recent development of the highly sensitive enzyme-linked immunosorbent assay (ELISA) for measuring cytokines has resulted in a substantial increase in cytokine research, particularly AF IL-6. Furthermore, high levels of AF IL-6 in preterm delivery with infection makes AF IL-6 readily detectable in minute aliquots of AF.43 Because AF IL-6 is a biomarker of infection and inflammation, we expected that concentrations of this cytokine would be associated with detection of intraamniotic infection. However, many women (63–70%) with elevated AF IL-6 levels delivered preterm but lacked positive AF cultures. We reviewed the literature to determine the association between AF IL-6 and preterm labor or preterm delivery and to provide possible explanations for why many women with ele-

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vated AF IL-6 lack detectable intra-amniotic infection in preterm labor or preterm delivery.

Data Sources For the main exposure (AF IL-6) and outcome (preterm labor or preterm delivery) of interest in this review article, we searched the English-language human literature in MEDLINE (1966 through September 1999), using the keywords “labor or infant,” “premature,” and “cytokines or interleukin-6,” and “amniotic fluid.” We also examined abstracts from the 19th Annual Meeting of the Society for Maternal-Fetal Medicine, San Francisco, California, 1999 and from the 32nd Annual Meeting of the Society for Epidemiologic Research, Baltimore, Maryland, 1999. We identified other studies by reviewing the reference lists of published articles. We also conducted other MEDLINE searches and accumulated literature that pertained to our review article objectives, such as laboratory and microbiologic assays performed to detect intra-amniotic infection in preterm labor or preterm delivery (eg, polymerase chain reaction [PCR], AF antibacterial activity, and chorioamnionitis). The MEDLINE search on the specific exposure (AF IL-6) and outcome (preterm labor or preterm delivery) of interest yielded 55 citations. We focused on studies that reported on the association between AF IL-6 and preterm delivery and its subtypes, such as preterm delivery less than 34 weeks’ gestation, spontaneous preterm labor, preterm PROM, or medically indicated preterm delivery, and that had pertinent information with respect to our objectives. We also used relevant references from the other MEDLINE searches we conducted and from the reference lists of published articles.

Tabulation, Integration, and Results Table 1 and Table 2 summarize results from studies that examined the association between AF IL-6 in intraamniotic infection and preterm labor or preterm delivery and that we could fit into the paradigm presented.39,41,43,46,53– 61 Other studies are referred to in the text when appropriate. Because of the importance of appropriate specimen collection and storage in laboratory studies of AF, we summarized this information in Table 1. Storage and collection are particularly important because of the multicellular nature of AF, which can lead to further cytokine production or degradation that would yield erroneous results if AF is inappropriately collected, handled, processed, or stored. We did not attempt to combine the studies’ quantitative results because of their considerable methodologic heterogeneity.

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Table 1. Amniotic Fluid Interleukin-6 Research: Study Methods AF collection

AF storage (°C)

Greig et al, 1993

TA

Romero et al, 199043

Study

IL-6 determination method

Population studied

Study design

⫺70

ELISA (Predicta Interleukin-6 kit, Genzyme, Cambridge, MA)

Cross-sectional

TA

⫺70

Hepatocyte-stimulating factor assay for IL-6 biologic activity

Romero et al, 199354

TA

Unclear

Romero et al, 199355

TA

⫺70

Two-site ELISA; sensitivity ⫽ 0.3 ng/mL Same as Romero et al54; sensitivity ⫽ 0.1 ng/mL

Coultrip et al, 199456

TA

⫺40 or ⫺70

Liechty et al, 199157

TA

⫺20

Hillier et al, 199339

TA

⫺70

ELISA (Genzyme, Boston, MA)

Saito et al, 199341

Unclear*

⫺80

Yoon et al, 199558

TA

⫺70

IL-6-dependent cell line MH60.BSF2 mouse hybridoma cells Same as Romero et al55

n ⫽ 258; 57 PTL/intact membranes; 75 term; and 126 at ⬍ 37 WG (no labor/intact membranes), Winston-Salem, NC n ⫽ 205; 109 PTL/intact membranes; 71 term (31 term, no labor, 40 term, active labor); and 25 secondtrimester AF, New Haven, CT n ⫽ 146 PTL/intact membranes; location unknown n ⫽ 120 PTL/intact membranes, New Haven, CT and Detroit, MI n ⫽ 89 PTL/intact membranes, Washington, DC n ⫽ 25; 20 PTL at ⱕ 34 WG; 5 term, Salt Lake City, UT, and New Haven, CT n ⫽ 50 PTL/intact membranes at 23–34 WG, Seattle, WA n ⫽ 104; 51 PTD; 53 term, Japan

Hsu et al, 199859

TA

⫺80

Santhanam et al, 199160

TA

⫺70

Yoon et al, 199846

TA

⫺70

ELISA (R&D Systems, Minneapolis, MN)

Romero et al, 199361

TA

⫺70

Same as Romero et al55

53

ELISA (Endogen, Inc., Boston, MA) ELISA (R&D Systems, Minneapolis, MN)

ELISA (R&D Systems, Minneapolis, MN) IL-6-dependent murine hybridoma cell line B9

n ⫽ 50 PTL/intact membranes (n ⫽ 29) or preterm PROM (n ⫽ 21); all had PTD at 24 –35 WG, Seoul, Korea n ⫽ 41 PTL/preterm PROM, New Haven, CT n ⫽ 227; 96 preterm PROM; 88 term; and 43 secondtrimester, New Haven, CT n ⫽ 120 preterm PROM all delivered preterm, Seoul, Korea n ⫽ 110 preterm PROM, New Haven, CT and Detroit, MI

Cross-sectional

Cross-sectional Cross-sectional

Cross-sectional Cross-sectional

Cross-sectional Prospective

Cross-sectional

Cross-sectional Cross-sectional

Cross-sectional

Cross-sectional

AF ⫽ amniotic fluid; IL ⫽ interleukin; TA ⫽ transabdominal amniocentesis; ELISA ⫽ enzyme-linked immunosorbent assay; PTL ⫽ preterm labor; WG ⫽ weeks gestation; PTD ⫽ preterm delivery; PROM ⫽ premature rupture of membranes. * Presumably transcervical.

Several important issues with respect to AF IL-6 merit discussion. First, there is consensus that elevated AF IL-6 levels are associated with preterm labor or preterm delivery, including subgroups of these outcomes, such as preterm PROM,46,59 – 61 preterm delivery at 34 weeks’ gestation or less,39,57,62,63 and spontaneous preterm delivery,62,63 but not in medically indicated preterm delivery.62,63 Furthermore, AF IL-6 levels have been found to be elevated in women with positive AF cultures (median range of AF IL-6 for women in preterm labor or preterm delivery who have intra-amniotic infection is 2.6 –375 ng/mL compared with 0.2–1.8 for women in preterm labor or preterm delivery who do not have

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intra-amniotic infection) (Table 2), in women with positive PCR for AF infection,64 in women with histologic chorioamnionitis,41,53,63,65 and in women with clinical chorioamnionitis.55 Notably, high levels of AF IL-6 were associated with certain types of organisms, such as Ureaplasma urealyticum,46,66 whereas low levels were associated with Staphylococcus epidermidis and grampositive Bacillus.66 It should be noted that culturepositive AF with normal or low AF IL-6 could be from skin contamination rather than true intra-amniotic infection. This finding suggests that levels of AF IL-6 can help identify sample contamination or false-negative culture results. For example, a low level of AF IL-6 and

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Table 2. Amniotic Fluid Interleukin-6 Research: Study Outcomes Outcome measured

Study 53

Median AF IL-6 (range) ng/mL preterm/IAI ‡

Median AF IL-6 (range) ng/mL preterm/no IAI

Median AF IL-6 (range) ng/mL term/no IAI §

P†

Comments

⬍ .001

⬍ .001

...

⬍ .001

...

Greig et al, 1993

PTL

2.6 (0.6 –3.6)

0.2 (0.0 –1.5)

Romero et al, 199043

PTL

375 (30 –5000)

1.5 (0.0 –500)

Romero et al, 199354

PTL

91.2 (0.9 – 437)

0.4 (⬍ 0.3–195)

...

⬍ .001

...

Romero et al, 199355 Coultrip et al, 199456

PTL PTL

35.9 (32.8 – 68.8) 241.8 (0.3–241.8)¶

1.7 (0.1– 69.1) 0.3 (0.0 –241.8)¶

... ...

⬍ .001 ⬍ .001

... ...

Liechty et al, 199157 Hillier et al, 199339

PTL at ⱕ 34 WG PTL at ⱕ 34 WG

0.5 (0.015– 6.5)** 1.8 (0.0 – 8.0)**

0.03 (ⱕ 0.005– 0.8)** ...

... ⬍ .05

... ...

Saito et al, 199341 Yoon et al, 199558 Hsu et al, 199859

PTD at ⱕ 36 WG 69.8 (3.2–230.4)**††‡‡ 6.2 (0.8 –15.4)**†† PTD at ⱕ 35 WG 70.8 (0.7– 499.2)‡‡ 2.9 (0.8 –16.0) PTL/preterm 2005 (27– 4071)㛳 㛳 990 (7.5–3409)㛳 㛳 PROM Preterm PROM 14.4 (0.2–28.8) 0.8 (0.2–26.8)

3.1 (0.1–14.8)**††§§ ... ...

⬍ .001 ⬍ .001 .005

... ...

No cultures on AF of women at term in labor since they had transvaginal amniotomy (median AF IL-6 ⫽ 19 ng/mL; range 4.0 –500). All women with AF IL-6 ⱖ 11.3 ng/mL and a negative AF culture delivered preterm. ... All women with AF IL-6 level ⬎ 6.2 ng/mL delivered preterm. ... IAI was present in all women who had AF IL-6 level of ⬎ 1.5 ng/mL. ... ... ...

0.2 (0.2– 0.8)§

⬍ .001

...

...

⬍ .01 ⬍ .001

... ...

... ...

Santhanam et al, 199160 Yoon et al, 199846 Romero et al, 199361

Preterm PROM Preterm PROM

8.0 (5.1–14.4)** 9.5 (7.5–10.0)**

13.4 (0.7–115.2) 26.8 (0.8 – 47.2)

0.9 (0.001–137.2) 3.8 (0.2– 46.7)

0.0 (0.0 – 0.5) ; 0.1 (0.0 –1.9)㛳 13 (3.5– 60)§

P*

... ...

IAI ⫽ intra-amniotic infection. Other abbreviations as in Table 1. * P for preterm/IAI compared with preterm/no IAI. † P for preterm/IAI compared with term/no IAI. ‡ AF IL-6 values are for patients with either histologic chorioamnionitis or IAI. § Term no labor. 㛳 Term in labor. ¶ Ranges are measured from figures. ** Medians are measured from figures. †† AF IL-6 levels are expressed in U/mL. ‡‡ AF IL-6 values are for histologic chorioamnionitis. §§ IAI was determined by measuring AF endotoxin with limulus endoxin-specific test. 㛳㛳 AF IL-6 levels are expressed in ng/mg creatinine and were normalized by AF creatinine levels.

a positive AF culture might indicate skin contamination rather than true intra-amniotic infection, and a high level of AF IL-6 and a negative culture might indicate failure of the culture to detect intra-amniotic infection. Second, AF IL-6 has been associated with preterm delivery in the absence of apparent clinical infection.54 Therefore, it might be worthwhile to investigate the possibility of using AF IL-6 as a sensitive biomarker to identify women with subclinical infections who might be at risk for preterm delivery. Third, higher levels of AF IL-6 appear to predict

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failure of tocolysis,43,54 which might prove useful for clinical management of spontaneous preterm labor. Future research is needed to investigate this possibility. Finally, it should be noted that most studies of AF IL-6 and preterm delivery were cross-sectional (ie, specimens collected at the time of preterm labor or PROM diagnosis) (Table 1). Hence for most of them, it was not possible to determine the temporal relationship between intra-amniotic infection, increased AF IL-6 levels, and onset of labor. This remains an important goal to better understand the pathophysiology of pre-

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term labor. Although it is beyond the scope of this review article to discuss animal models, it is worthwhile to mention that Gravett et al67 clearly showed that in the rhesus monkey, after intra-amniotic infection, there was an increase in AF IL-6 and other cytokines, followed by increases in prostaglandin E2 and prostaglandin F2␣. These increases all occurred before uterine contractility increased and before clinical signs of infection. It has been reported that only 30% (nine of 30) to 37% (11 of 30) of pregnancies with preterm labor and intact membranes with elevated AF IL-6 had positive AF cultures, although all women with elevated AF IL-6 delivered preterm.39,55 The cutoff for elevated AF IL-6 used by Hillier et al39 was 1.5 ng/mL, and that used by Romero et al55 was 11.3 ng/mL. Until recently, AF cultures were considered the gold standard for diagnosing microbial invasion of AF, but AF cultures are limited by their inability to isolate the infectious agent because of low levels of infectious inoculum, previous antibiotic treatment, presence of unculturable microorganisms,68,69 or difficulty in isolating fastidious microorganisms.21 Investigators have shown positive Gram stain for AF bacteria, but this occurred in patients whose AF grew Corynebacterium species, which are considered a skin contaminant.43 Researchers also have failed to recover bacteria from patients who have clinical as well as histopathologic evidence of chorioamnionitis.43 Because of culture limitations and the persistent need to identify causative microorganisms in intraamniotic infection, researchers have resorted to PCR amplification of bacterial 16S ribosomal RNA encoding DNA (rDNA), which is universal among bacteria. This approach has recently become an accepted technique for detecting unculturable microorganisms.70 Deoxyribonucleic acid sequencing of the PCR products can lead to identification of infecting bacteria. Although only a few investigators have used those molecular methods to detect intra-amniotic infection to date, the early preliminary results are promising.64,71 (Oyarzun E, Yamamoto M, Kato S, Lizama L, Moenne A. PCR gene amplification of bacterial 16S rRNA gene in the detection of intra-amniotic infection and prediction of preterm delivery. Am J Obstet Gynecol 1998;178: 518 [abstract].) Hitti et al71 detected bacterial 16S rDNA by PCR in five of 14 (36%) afebrile patients in preterm labor with negative cultures but elevated AF IL-6 (over 2.0 ng/mL). The authors reported a sensitivity of 95% for PCR compared with 76% for culture for detecting intra-amniotic infection, where true-positive results were considered to be patients with either culturepositive or PCR-positive AF and an AF IL-6 level of over 2.0 ng/mL. Similarly, Oyarzun et al (Oyarzun E et al, Am J Obstet Gynecol 1998;178:518 [abstract].) iden-

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tified by PCR micro-organisms in 23 of 50 (46%) patients with preterm labor and intact membranes, whereas cultures identified only six (12%). The authors also reported that the sensitivity of PCR and cultures for the identification of patients delivering before 34 weeks’ gestation was 64% and 18%, respectively. Markenson et al64 showed that 67% (six of nine) women in preterm labor who had no clinical evidence of intra-amniotic infection with elevated AF IL-6 (over 0.6 ng/mL) had positive PCR results, whereas only one (11%) had a positive culture. All women with elevated AF IL-6 had preterm delivery. Taken together, these findings suggest that, among women with elevated AF IL-6, 16S rDNA PCRs are more sensitive than microbiologic cultures in detecting intra-amniotic infection (67% in the study of Markenson et al64 compared with 30 –37% in the studies of Hillier et al39 and Romero et al55). These findings also suggest that an even higher proportion of women with elevated AF IL-6 could have intra-amniotic infection. There remain some women (33–70%) with elevated AF IL-6 who deliver preterm but who do not have evidence of intra-amniotic infection by either culture or PCR. However, inferences from those studies are limited because AF samples were obtained at the time of onset of preterm labor and so it cannot be determined whether the detected infection initiated the onset of preterm labor or whether the infection started during or after labor onset. In theory, PCR is very rapid technology that can provide substantial clinical advantages, especially compared with microbiologic cultures for slowgrowing or fastidious organisms that require prolonged cultivation in research laboratories. It should be noted that general use of PCR is currently limited by extreme measures needed to prevent contamination, by labor intensiveness, and by high cost. Despite these limitations, diagnostic 16S rDNA PCR methods appear promising, because the organism does not need to be alive or fully intact for PCR-based detection. This might contribute to the higher proportion of intra-amniotic infection that PCR detects among women with elevated AF IL-6 who delivered preterm64 compared with the proportion that culture methods could detect.39,55 Although 16S rDNA is essentially universal for bacteria, more research is needed to develop broad-spectrum PCRs for nonbacterial agents, such as viruses. Development of extended PCR approaches might facilitate further refinement of the association between detectable intra-amniotic infection and elevated AF IL-6 levels. Even with highly sensitive and specific methods to detect intra-amniotic infection in preterm labor or preterm delivery, there might remain a proportion of women with elevated AF IL-6 who have undetectable intra-amniotic infection, which might be due to inflam-

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matory conditions other than bacterial intra-amniotic infection, such as histologic chorioamnionitis.39,53,55 Histologic chorioamnionitis could trigger the production of a large enough amount of IL-6 in AF to stimulate onset of preterm labor before microorganisms can actually invade the amniotic cavity. Nonbacterial intraamniotic infection, ie, viral intra-amniotic infection might increase AF IL-6.72 These viral infections cannot be detected by routine microbiologic cultures. Another biologically plausible explanation is the increased antibacterial activity of AF in certain women, which might hinder the growth of microorganisms73 that could be colonizing the chorioamnion. Antibiotic treatment before AF specimens are obtained might also hamper recovery of microorganisms.43 High levels of AF IL-6 have also been associated with the physiologic process of labor.43,53,60 Finally, there may be genetic variation in the degree of inflammatory response to certain stimuli. A genetic predisposition to an exaggerated inflammatory response has been documented among individuals with meningococcemia74 and periodontitis.75

Conclusion We conclude that the literature has reached consensus that elevated AF IL-6 is consistently associated with intra-amniotic infection and preterm delivery. Future research should focus on clinical application of ELISA assays to detect AF IL-6 as it might indicate intraamniotic infection. It should be noted that finding elevated AF IL-6 does not identify the specific organisms involved in intra-amniotic infection. Identification remains an important scientific as well as clinical goal. Polymerase chain reaction-based approaches seem to be more promising methods than microbiologic culture to diagnose intra-amniotic infection and to better explain elevated AF IL-6 level. Important research issues include development of improved PCR assays for detection of specific organisms associated with intraamniotic infection and further refinement of the relationships between intra-amniotic infection, elevated AF IL-6, and preterm delivery. This might narrow the search for causes other than infection to explain elevated levels of AF IL-6. It is crucial for clinicians to understand the underlying cause of elevated AF IL-6 to optimize obstetric treatment. Future studies should be large and prospectively seek potential causal relationships between intra-amniotic infection, elevated AF IL-6, and preterm delivery. These promising approaches offer the potential to improve obstetrical outcomes substantially.

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Amira Y. El-Bastawissi, PhD Swedish Medical Center Center for Perinatal Studies 747 Broadway Seattle, WA 98122-4307 E-mail: [email protected]

Received September 20, 1999. Received in revised form January 18, 2000. Accepted February 10, 2000. Copyright © 2000 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.

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