Intrauterine infection and preterm delivery: Evidence for activation of the fetal hypothalamic-pituitary-adrenal axis

Intrauterine infection and preterm delivery: Evidence for activation of the fetal hypothalamic-pituitary-adrenal axis Michael G. Gravett, MD,a, b Jane Hitti, MD,c David L. Hess, PhD,a and David A. Eschenbach, MDc Beaverton and Portland, Oregon, and Seattle, Washington OBJECTIVE: We studied pregnant women in preterm labor with and without intrauterine infection to determine whether fetal hypothalamic-pituitary-adrenal axis activation occurs in the setting of infection-induced preterm parturition. STUDY DESIGN: Amniotic fluid collected by amniocentesis and maternal blood from patients in preterm labor with intact membranes at 24 to 34 weeks’ gestation were analyzed by radioimmunoassay for the steroid hormones estrone, estradiol, progesterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, and cortisol. Amniotic fluid was also obtained for microbial culture and for interleukin 6 measurements by enzyme immunoassay. RESULTS: Patients with intrauterine infection (n = 11) had significantly higher amniotic fluid concentrations of dehydroepiandrosterone (539 ± 79 pg/mL) and of cortisol (5.28 ± 1.0 µg/dL) than did patients with preterm labor and preterm delivery without infection (n = 11; 273 ± 82 pg/mL and 1.61 ± 1.05 µg/dL, respectively) or patients with preterm labor and subsequent term delivery (n = 11; 202 ± 79 pg/mL and 1.82 ± 1.0 µg/dL, respectively). Furthermore those patients who were delivered within 7 days after enrollment (who were also more likely to have intrauterine infection) had higher amniotic fluid concentrations than did those who were not delivered within 7 days of both estrone (586 ± 101 pg/mL vs 314 ± 98 pg/mL) and estradiol (238 ± 44 pg/mL vs 91 ± 43 pg/mL). CONCLUSION: Intrauterine infection was associated with increased fetal adrenal androgen and cortisol biosynthesis, and delivery within 7 days after the onset of preterm labor was associated with increased placental estrogen synthesis. These data are consistent with fetal hypothalamic-pituitary-adrenal axis activation in the setting of infection-associated preterm delivery. (Am J Obstet Gynecol 2000;182:1404-13.)

Key words: Fetal hypothalamic-pituitary-adrenal axis, intrauterine infection, parturition, preterm labor, steroid biosynthesis

Preterm labor that results in premature birth is the most common cause of perinatal mortality in industrialized countries, accounting for as many as 80% of perinatal deaths not attributable to congenital malformations.1 A large body of evidence suggests that intrauterine infection may be an important and potentially preventable cause of preterm labor and delivery, especially among those pregnancies that end in the second or early third trimester and result in very-low-birth-weight neonates, among whom rates of mortality and morbidity are high.2 The prevalence of histologic chorioamnionitis is inversely related to gestational age and is about 60% to 90% From the Division of Reproductive Sciences, Oregon Regional Primate Research Center,a and the Departments of Obstetrics and Gynecology, Oregon Health Sciences Universityb and the University of Washington.c Supported by National Institutes of Health grants AI42490, AI31871, RR00163, and HD18185. Presented at the Sixty-sixth Annual Meeting of the Pacific Coast Obstetrical and Gynecological Society, Cancun, Mexico, October 20-24, 1999. Reprint requests: Michael G. Gravett, MD, Department of Obstetrics and Gynecology, L458, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97201-3098. Copyright © 2000 by Mosby, Inc. 0002-9378/2000 $12.00 + 0 6/6/106180 doi:10.1067/mob.2000.106180

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among pregnancies ending between 20 and 24 weeks’ gestation3; microbial infection of the amnion and chorion occurs in 60% of patients with preterm delivery.4 There is now considerable evidence that proinflammatory cytokines play a central role in the pathogenesis of infection-associated preterm delivery.5 These inflammatory mediators include interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), and tumor necrosis factor, are produced by macrophages and decidua in response to a wide variety of bacteria or bacterial products, and stimulate the production of prostaglandins by the amnion and chorion. In contrast, activation of the fetal hypothalamic-pituitary-adrenal axis is thought to be a key step in the onset of spontaneous parturition. Spontaneous parturition in primates is characterized by fetal adrenal synthesis of C19 androgens, which, in turn, are aromatized by the placenta into estrogens, including estrone, estradiol, and estriol. Among nonhuman primates a rise in the amniotic fluid concentration of estrone precedes or coincides with increases in amniotic fluid prostaglandin concentrations, which begin to rise several days before parturition.6 In human beings placental corticotropin-

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releasing hormone (CRH),7, 8 maternal salivary estriol,9 and maternal plasma estradiol10 concentrations all increase before spontaneous preterm parturition. In contrast to infection-associated parturition, spontaneous parturition is associated with either no increases or only small increases in amniotic fluid concentrations of cytokines.11, 12 However, there is increasing evidence for important interactions between the immune system and the endocrine system both at the hypothalamic and pituitary levels and locally.13-15 IL-1, interleukin 2 (IL-2), IL-6, and tumor necrosis factor can augment the release of corticotropin, and hence adrenal steroid and glucocorticoid production, by stimulating hypothalamic CRH.14, 16 In turn, glucocorticoids exert their well-established immunosuppressant effects in part by blocking the transcription factor NF-κB, which is necessary for transcription of genes for proinflammatory cytokines.17 Such interactions are relevant in light of the important functions of both the adrenocortical system and the immune system in responding to stress. Some investigators have suggested that the response of the hypothalamicpituitary-adrenal axis to stress or infection may have evolved to suppress and modulate inflammatory responses.14 In view of the important interactions between the immune and endocrine systems, we hypothesized that normal steroid biosynthesis by the fetoplacental unit may be altered in the setting of preterm parturition associated with intrauterine infection. We therefore studied fetoplacental steroid hormone biosynthesis among a cohort of women with and without evidence of intrauterine infection who were in preterm labor with intact fetal membranes. Material and methods Study population. The study population was drawn from 309 women in premature labor with intact fetal membranes admitted to the University of Washington Medical Center or associated hospitals in Seattle between June 25, 1991, and June 30, 1997, for a separate prospective cohort study of intrauterine infection and preterm labor as previously described.18 All women provided written informed consent for the original study, and the study protocol was approved by the institutional review boards of all participating hospitals. The study population described here was drawn from participants in the original study. The participants were at 22 to 34 weeks’ gestation according to last menstrual period or the earliest available sonogram. All participants had intact fetal membranes at study enrollment. Preterm labor was defined as regular uterine contractions at a frequency ≤10 minutes with either documented cervical change or a cervical dilatation ≥1 cm or effacement ≥50%. Women with cervical dilatation >4 cm or ruptured membranes at admission were excluded. Women with multiple gestations, cervical

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cerclage, placenta previa, abruptio placentae, diabetes, hypertension, and preeclampsia were considered eligible if they otherwise met study criteria. During the study interval 252 (23%) of the 1070 women admitted to the University of Washington Medical Center with preterm labor and intact membranes participated in the study. Review of the medical records of all women admitted with intact membranes and delivered at the University of Washington at ≤34 weeks’ gestation between October 1992 and January 1995, the majority of the study interval, revealed no significant differences in age, race, gravidity, history of preterm delivery, multiple gestation, or gestational age at delivery between study participants and nonparticipants. An additional 57 women were enrolled from two other Seattle hospitals. Women enrolled at these hospitals were older (median age, 27 years vs 24 years; P < .01) and were more likely to have multiple gestations (23% vs 10%, P < .01) than were patients enrolled at the University of Washington. There were no significant differences in maternal race, parity, history of previous preterm delivery, preterm delivery rate, or gestational age at delivery between patients enrolled at the University of Washington and those from the other hospitals. Transabdominal amniocentesis was performed under ultrasonographic guidance for all study participants, and maternal venous blood was also collected by venipuncture at the time of enrollment. Among 309 participating women 11 (4%) were unavailable for follow-up and 11 (4%) were excluded because of major fetal malformations or higher-order multiple gestations, leaving 287 women. From this study population a subset was retrospectively identified for the steroid hormone analysis reported here. This subset included 11 patients with evidence of intrauterine infection (as defined by the recovery of a microbial pathogen from amniotic fluid or an amniotic fluid IL-6 concentration ≥2000 pg/mL), a randomly selected subset of 11 patients without intrauterine infection but with preterm birth, and 11 patients without infection and with preterm labor responsive to tocolytic therapy who had subsequent term birth. These 33 patients constitute the study population for this report. Amniotic fluid studies. Amniotic fluid was placed in an anaerobic transport vial (Port-a-Cul; Becton Dickinson Microbiology Systems, Sparks, Md) and inoculated into culture media for aerobic and anaerobic bacteria and genital mycoplasmas within 12 hours after collection. Methods for the culture and identification of these organisms were designed to detect low quantities of bacteria through the use of broth enrichment, as previously described.19 The remaining amniotic fluid was stored at –70°C until assay for IL-6 and steroid hormones. Amniotic fluid IL-6 concentrations were determined by commercial enzyme immunoassay (Genzyme Corporation, Cambridge, Mass). The lower limit of detection for IL-6 was 70

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Table I. Characteristics of study population

Characteristic Maternal age (y, mean ± SD) White race (No.) Parity (mean ± SD) Nulliparity (No.) Gestational age at enrollment (wk, mean ± SD) Gestational age at delivery (wk, mean ± SD) Interval from enrollment to delivery (d, mean ± SD) Delivery ≤7 d after enrollment (No.)

Preterm delivery (<35 wk) with intrauterine infection (n = 11)

Preterm delivery (<35 wk) without intrauterine infection (n = 11)

Premature labor but with subsequent term delivery (n = 11)

Statistical significance

24.5 ± 5.4 06 (55%) 01.9 ± 1.6 03 (27%) 26.9 ± 1.1

26.6 ± 9.0 4 (36%) 01.9 ± 1.5 1 (9%)0 28.6 ± 1.1

25.6 ± 6.0 6 (55%) 03.0 ± 2.5 1 (9%)0 30.3 ± 1.1

NS NS NS NS P = .1000

27.3 ± 0.9

29.8 ± 1.0

37.0 ± 0.9

P < .0001

02.1 ± 5.6

08.4 ± 6.3

46.9 ± 5.6

P < .0001

10 (91%)

6 (55%)

0 (0%)0

P < .0010

Data were analyzed by analysis of variance for continuous data and χ2 test for categoric data. NS, Not significant.

pg/mL. An amniotic fluid IL-6 level ≥2000 pg/mL was considered elevated. This value was at the 75th percentile for the study population and reliably predicted intrauterine infection in our population.18 Quantitation of steroid hormones. Quantitation of hormone concentrations in amniotic fluid and maternal plasma were carried out at the Oregon Regional Primate Research Center by previously validated radioimmunoassay.20, 21 Progesterone, dehydroepiandrosterone (DHEA), androstenedione, estrone, and estradiol concentration were quantitated by radioimmunoassay after extraction with diethyl ether and separation by LH-20 column chromatography. Cortisol and DHEA sulfate (DHEAS) were measured by direct radioimmunoassay. Statistical analysis. Subject characteristics were analyzed with descriptive statistics. The χ2 test was used for dichotomous variables; the Student t test was used for continuous variables. Steroid hormone and IL-6 concentrations are presented as mean ± SEM. Comparisons between hormone concentrations were made by analysis of variance or Student t test as appropriate. Results Characteristics of the study population. The study population was divided into 3 groups: (1) patients with evidence of intrauterine infection according to either recovery of microorganisms from amniotic fluid or an amniotic fluid IL-6 concentration >2000 pg/mL, (2) patients with preterm labor and delivery at <35 weeks’ gestation without evidence of intrauterine infection, and (3) patients with preterm labor responsive to tocolytic therapy who were delivered at ≥35 weeks’ gestation. We chose 35 weeks’ gestation as the cutoff because the highest rates of both intrauterine infection and neonatal complications occur before this gestational age. There were no differences in maternal age, race, or parity among these three groups (Table I). However, patients with intrauterine infection were seen at a somewhat earlier gestational age at enrollment (P = .10) and were delivered at a significantly earlier

gestational age than were patients with preterm delivery without infection or those with term delivery (27.3 ± 0.9 weeks’ gestation vs 29.8 ± 1.0 and 37.0 ± 0.9 weeks’ gestation, respectively; P < .0001). In addition, those with intrauterine infection had a significantly shorter interval from enrollment to delivery (2.1 ± 5.6 days, vs 8.4 ± 6.3 and 46.9 ± 5.6 days for the other two groups; P < .0001). Ninety-one percent of those with intrauterine infection were delivered within 7 days after enrollment. Among the 11 patients with infection microorganisms were recovered in 4 cases (2 patients had Escherichia coli, 1 had Candida albicans, and 1 had mixed anaerobes); all of these patients were delivered within 7 days. Seven other patients were included in the infection group because of amniotic fluid IL-6 concentrations >2000 pg/mL. The mean amniotic fluid concentration of IL-6 among these patients was 27.7 ± 7.8 ng/mL, compared with 0.68 ± 0.20 ng/mL among those with preterm delivery without infection and 0.25 ± 0.13 ng/mL among those with preterm labor and term delivery (P < .01). Fetoplacental steroid biosynthesis in intrauterine infection. Patients with intrauterine infection had significantly elevated mean amniotic fluid concentrations of DHEA and cortisol with respect to those of patients with preterm delivery without infection or those of patients with preterm labor and term delivery (Table II and Fig 1). The amniotic fluid DHEA concentration among patients with intrauterine infection was more than twice as high as the concentrations among women with preterm delivery without infection and among women with subsequent term delivery (539 ± 79 pg/mL vs 273 ± 82 pg/mL and 202 ± 79 pg/mL, respectively; P = .01). Amniotic fluid concentrations of both androstenedione and DHEAS were also increased among the patients with intrauterine infection, but these increases did not achieve statistical significance. There were no significant differences in amniotic fluid estrogen or progesterone concentrations among the 3 groups of patients (Table II).

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A

B

Fig 1. Amniotic fluid concentrations of cortisol (A) and DHEA (B) among patients with infection-associated preterm delivery (IUI), spontaneous preterm delivery (PMD), and preterm labor without delivery (PML). Data are presented as individual values (points), means (lines), and 95% confidence intervals (brackets). Dashed line, Mean concentration of all 3 groups. Patients with intrauterine infection had higher amniotic fluid concentrations of cortisol and DHEA than did patients with spontaneous preterm delivery or preterm labor without delivery (P < .05 by analysis of variance).

Table II. Mean amniotic fluid steroid hormone concentrations among women in preterm labor with and without intrauterine infection

Estrogens Estrone (pg/mL) Estradiol (pg/mL) Androgens Androstenedione (pg/mL) DHEA (pg/mL) DHEAS (ng/mL) Glucocorticoids Cortisol (µg/dL) Progesterone (ng/mL)

Preterm delivery (<35 wk) with intrauterine infection (n = 11)

Preterm delivery (<35 wk) without intrauterine infection (n = 11)

Premature labor but with subsequent term delivery (n = 11)

460 ± 125 161 ± 55

570 ± 131 241 ± 57

319 ± 125 92 ± 55

NS NS

717 ± 94 539 ± 79 277 ± 65

519 ± 98 273 ± 82 171 ± 68

499 ± 94 202 ± 79 211 ± 65

NS P = .01 NS

5.28 ± 1.0 24.8 ± 5.8

1.61 ± 1.0 35.5 ± 6.1

1.82 ± 1.0 28.0 ± 5.8

P = .02 NS

Statistical significance

Data are presented as mean ± SEM and were analyzed by analysis of variance. NS, Not significant.

The mean amniotic fluid concentration of cortisol was also significantly higher in the setting of intrauterine infection. Cortisol concentrations were 5.28 ± 1.0 µg/dL among patients with intrauterine infection, 1.61 ± 1.05 µg/dL among those with preterm delivery without infection, and 1.82 ± 1.0 µg/dL among those with preterm labor with subsequent term delivery. The increases in fetal adrenal androgen and cortisol concentrations seen in the setting of infection were not reflected by similar differences in maternal plasma concentrations. Maternal plasma differences were observed only in progesterone concentrations. Maternal plasma concentrations of progesterone were lower among patients with infection than among patients in the other two groups. The mean maternal plasma concentrations of progesterone were 82.7 ng/mL among patients with infection, 176.5 ng/mL among those with preterm delivery without infection, and 215.6 ng/mL among those with

subsequent term delivery (SEM, 34.86 ng/mL; P = .03). These differences reflected the earlier gestational age at which patients with intrauterine infection were enrolled and were no longer present after adjustment for gestational age. Relationship to interval from enrollment to delivery ≤7 days. To investigate the role of estrogens in imminent delivery in the setting of infection we analyzed steroid hormone concentrations with and without intrauterine infection in relationship to an interval from enrollment to delivery ≤1 week. Sixteen of the 33 patients (48%), including 10 of the 11 patients with intrauterine infection (91%), were delivered within 7 days after enrollment. Analysis of amniotic fluid obtained at the time of initial enrollment revealed significantly higher concentrations of fetal androgens androstenedione and DHEA and of estradiol (Table III). Differences in estrogen concentrations but not androgen concentrations persisted in a fur-

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Table III. Mean amniotic fluid steroid hormone concentrations among women with and without preterm delivery ≤7 days after admission Delivery ≤7 d Delivery >7 d after admission after admission Statistical (n = 16) (n = 17) significance Estrogens Estrone (pg/mL) 586 ± 101 Estradiol (pg/mL) 238 ± 44 Androgens Androstenedione 698 ± 76 (pg/mL) DHEA (pg/mL) 466 ± 68 DHEAS (ng/mL) 219 ± 55 Glucocorticoids Cortisol (µg/dL) 3.89 ± 0.9 Progesterone 30.4 ± 4.9 (ng/mL)

314 ± 98 91 ± 43

P = .06 P = .02

469 ± 74

P = .04

220 ± 66 223 ± 53

P = .01 NS

2.05 ± 0.88 28.2 ± 4.8

NS NS

Data are presented as mean ± SEM and were analyzed by Student t test. NS, Not significant.

ther analysis in which patients with intrauterine infection were excluded (Table IV). In this secondary analysis of patients in preterm labor without infection both estrone and estradiol concentrations remained significantly higher among those who were delivered within 7 days. Amniotic fluid estrone concentrations were 694 ± 148 pg/mL among those with delivery within 7 days and 327 ± 98 pg/mL among those without delivery within 7 days (P = .05). Estradiol concentrations were 309 ± 74 pg/mL among those with infection and 100 ± 49 pg/mL among those without infection (P = .03). There was no correlation between mean amniotic fluid or maternal plasma cortisol concentration and an interval from enrollment to delivery ≤7 days. Comment We found significantly higher amniotic fluid concentrations of cortisol and DHEA among women with preterm delivery associated with intrauterine infection than among either women with idiopathic preterm delivery or women with preterm labor without delivery. In addition, women who were delivered within 7 days after the onset of preterm labor, a group that included 10 of the 11 patients with intrauterine infection, had higher amniotic fluid concentrations of fetal adrenal androgens (androstenedione and DHEA) and estrogens (estrone and estradiol). Although we did not measure corticotropin or catechol concentrations in our study, the endocrine profiles that we observed during intrauterine infection are consistent with fetal stress and subsequent activation of the fetal hypothalamic-pituitary-adrenal axis. We used amniotic fluid steroid hormone concentrations to reflect fetal rather than maternal production of androgens and cortisol for the following reasons:

Table IV. Mean amniotic fluid steroid hormone concentrations among women with and without preterm delivery ≤7 days after admission without intrauterine infection Delivery ≤7 d Delivery >7 d after admission after admission Statistical (n = 6) (n = 16) significance Estrogens Estrone (pg/mL) 694 ± 148 Estradiol (pg/mL) 309 ± 74 Androgens Androstenedione 519 ± 82 (pg/mL) DHEA (pg/mL) 293 ± 69 DHEAS (ng/mL) 196 ± 70 Glucocorticoids Cortisol (µg/mL) 1.58 ± 0.3 Progesterone 34.4 ± 7.0 (ng/mL)

327 ± 98 100 ± 49

P = .05 P = .03

504 ± 55

NS

211 ± 46 190 ± 47

NS NS

1.78 ± 0.2 30.4 ± 4.6

NS NS

Data are presented as mean ± SEM and were analyzed by Student t test. NS, Not significant.

(1) The presence of normal concentrations of 17-ketosteroids in amniotic fluid of women after adrenalectomy suggests a fetal origin of amniotic fluid androgens22; (2) as gestation progresses, both fetal and amniotic fluid cortisol concentrations increase but maternal cortisol concentration does not23; (3) experimental intra-amniotic infection in nonhuman primates is associated with increases in fetal but not maternal DHEAS and cortisol concentrations and correlates with amniotic fluid concentrations.24 The human placenta contains steroid aromatase but not 17α-hydroxylase. Thus estrogen production during pregnancy is dependent on an intact fetoplacental unit, with fetal adrenal androgens as precursors to placentally derived estrogens. Normally spontaneous parturition is preceded by increased synthesis of fetal androgens, which are converted by the placenta into estrogens. Among nonhuman primates a rise in amniotic fluid estrone concentration precedes or coincides with increases in amniotic fluid prostaglandin concentrations, which begin several days before parturition.6 Among human beings increases in maternal plasma estradiol and salivary estriol concentrations have been noted to precede the onset preterm parturition.9, 10 The mechanism for these increases is likely to be through activation of the fetal hypothalamic-pituitary-adrenal axis by increases in placentally derived CRH, which also occurs before preterm parturition.7, 8 In contrast, proinflammatory cytokines are thought to play a central role in infection-associated parturition. These inflammatory mediators include IL-1, IL-6, IL-8, and tumor necrosis factor, are produced by macrophages and decidual cells in response to a wide variety of bacteria or bacterial products, and have been demonstrated to

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stimulate production of prostaglandins by the amnion and decidua.25-27 Further, administration of recombinant IL-1 induces preterm labor in mice28 and in nonhuman primates.29 Thus in the setting of infection the interaction between proinflammatory cytokines and prostaglandins occurring at the level of the amnion and chorion and decidua is fundamentally important, and labor may proceed by alternative mechanisms to spontaneous parturition. As alluded to earlier, however, complex interactions between the immune system and the endocrine system may result in activation and participation of the fetal hypothalamic-pituitary-adrenal axis in the setting of intrauterine infection and preterm delivery, as we found in this study. Similar findings have also been noted by others. Yoon et al30 reported increases in fetal plasma cortisol and IL-6 concentrations but not in DHEAS concentration among women with pregnancies complicated by premature rupture of membranes and evidence of intrauterine infection. In contrast to our study, in which all women had intact fetal membranes, no differences were found in amniotic fluid concentrations of cortisol or DHEAS. However, the amniotic fluid cortisol concentration among their patients without infection was much higher (median, 3.3 µg/dL) than that among our patients without infection (mean, 1.6 µg/dL). It is possible that activation of the fetal hypothalamic-pituitaryadrenal axis had already occurred in their population after rupture of membranes. Amniotic fluid concentrations of cortisol in the setting of infection were similar in the two studies (4.4 µg/dL and 5.28 µg/dL). Amniotic fluid DHEAS concentration was not elevated in the study by Yoon et al,30 but other fetal adrenal androgen concentrations were not reported. In our study we found significant increases in the concentration of androstenedione, a fetal androgen not measured in the study by Yoon et al.30 Nonetheless, the two studies provide complementary evidence for fetal hypothalamic-pituitaryadrenal activation in preterm delivery. It remains unknown whether the elevated proinflammatory cytokine amniotic fluid concentrations reported in the setting of infection-induced preterm labor act primarily or in synergy with corticotropin in stimulating the fetal hypothalamic-pituitary-adrenal axis. We also observed increased concentrations within amniotic fluid of adrenal androgens (androstenedione and DHEA) and placental estrogens (estrone and estradiol) when delivery occurred within 7 days after the onset of preterm labor, which also suggests activation of the fetal hypothalamic-pituitary-adrenal axis. Increases in amniotic fluid estrone concentration among nonhuman primates6 and in salivary estriol9 and maternal plasma estradiol10 concentrations among human beings all occur before spontaneous parturition without infection. It has previously been demonstrated that elevated

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estrogen levels result from an augmented supply of fetal androgens.31 Thus the increases in amniotic fluid estrogen concentrations observed in this study are not surprising. However, previous studies from our laboratory with nonhuman primates in preterm labor with experimental intrauterine infection did not demonstrate increases in estrogen concentrations, despite dramatic increases in fetal adrenal androgen production.24 This suggested a selective placental dysfunction in steroid aromatase related to the infection (direct cytotoxic effects of the infection, diminished placental blood flow, relative hypoxemia, or impairments of the steroid aromatase cytochrome P-450 enzyme system).32 Such an effect was not observed in this study; estrogen concentrations rose as expected. It is likely that the experimental model for intrauterine infection represented a more advanced infection than was observed in this study. This supposition is supported by the observation that IL-6 amniotic fluid concentrations were higher after experimental infection (49 ng/mL) than among the infected women in this study (27.7 ng/mL). It is possible that estrogen biosynthesis is enhanced by an augmented supply of androgens early in the course of preterm labor associated with intrauterine infection but is impaired in a more advanced infection. Although maternal estriol concentration was not measured in this study, our data provide additional support for monitoring concentrations of estrogens or other hormones in the evaluation of preterm labor. We present evidence for activation of the fetal hypothalamic-pituitary-adrenal axis in the setting of infectioninduced preterm labor. However, many important questions remain. Are the adrenal effects the result of stress-induced CRH-corticotropin stimulation, are they the result of direct action of proinflammatory cytokines on the fetal adrenal, or are they a combination of both? What are the effects of increased fetal cortisol concentration? Although increased glucocorticoid concentrations may downwardly regulate the inflammatory response, they may also serve to stimulate placental CRH production.33 Do increased glucocorticoid concentrations lead to a functional progesterone blockade (by competing with progesterone for the glucocorticoid receptor) and thus to labor? Finally, do increases in estrogen concentrations prepare the uterus for parturition, or in advanced infections does labor proceed by alternative mechanisms involving the proinflammatory primarily cytokine–prostaglandin cascade? Undoubtedly, in the near future other endocrine, paracrine, and immune interactions will be elucidated that will contribute to our understanding of the multifactorial etiologies and differing pathophysiologic mechanisms leading to preterm labor. An understanding of these interactions will have profound implications for the diagnosis and management of preterm labor.

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REFERENCES

1. Rush RW, Keirse MJ, Howat P, Baum JD, Anderson AB, Turnbull AC. Contribution of preterm delivery to perinatal mortality. BMJ 1976;2:965-8. 2. Romero R, Avila C, Brekus CA, Morotti R. The role of systemic and intrauterine infection in preterm parturition. Ann N Y Acad Sci 1991;622:355-75. 3. Russell P. Inflammatory lesions of the human placenta. I. Clinical significance of acute chorioamnionitis. Am J Diagn Gynecol Obstet 1979;1:127-37. 4. Hillier SL, Martius J, Krohn M, Kiviat N, Holmes KK, Eschenbach DA. A case-control study of chorioamnionitis in prematurity. N Engl J Med 1988;319:972-8. 5. Dudley DJ, Trautman MS. Infection, inflammation, and contractions: the role of cytokines in the pathophysiology of preterm labor. Semin Reprod Endocrinol 1994;12:263-72. 6. Walsh SW, Stanczyk FZ, Novy MJ. Daily hormonal changes in the maternal, fetal and amniotic fluid compartments before parturition in a primate species. J Clin Endocrinol Metab 1984;58: 629-39. 7. McLean M, Bisits A, Davies J, Woods R, Lowry P, Smith R. A placental clock controlling the length of human pregnancy. Nat Med 1995;1:460-3. 8. Korebrits C, Ramirez MM, Watson L, Brinkman E, Bocking AD, Challis JR. Maternal corticotropin-releasing hormone is increased with impending preterm birth. J Clin Endocrinol Metab 1998;83:1585-91. 9. McGregor JA, Jackson GM, Lachelin G. Salivary estriol as risk assessment for preterm labor: a prospective trial. Am J Obstet Gynecol 1995;173:1337-42. 10. Germain AM, Kato S, Villarroel LA, Valenzuela GJ, Serron-Ferre M. Human term and preterm delivery is preceded by a rise in maternal plasma 17β-estradiol. Prenat Neonat Med 1996;1:57-63. 11. Opsjon SL, Wathen NC, Tingulstad S, Wiedswang G, Sundon A, Waage A, et al. Tumor necrosis factor, interleukin-1, and interleukin-6 in normal human pregnancy. Am J Obstet Gynecol 1993;169:397-404. 12. Gravett MG, Witkin SS, Haluska GJ, Edwards JL, Cook MJ, Novy MJ. An experimental model for intraamniotic infection and preterm labor in rhesus monkeys. Am J Obstet Gynecol 1994;171:1660-7. 13. Aoki N, Ohno Y, Omamura M. Physiological interactions between the immune and endocrine system: are cytokines hormones? Med Sci Res 1990;18:195-201. 14. Riechlin S. Neuroendocrine-immune interactions. N Engl J Med 1993;329:1246-53. 15. Besedovsky HO, del Rey A. Immune-neuro-endocrine interactions: facts and hypotheses. Endocr Rev 1996;17:64-102. 16. Sweep F, Rijnkels C, Hermus A. Activation of the hypothalamuspituitary-adrenal axis by cytokines. Acta Endocrinol (Copenh) 1991;125 Suppl 1:84-91. 17. Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M. Immunosuppression by glucocorticoids: inhibition of NF-κB activity through induction of Iκ B synthesis. Science 1995;270:286-90. 18. Hitti J, Krohn MA, Patton DL, Tarczy-Hornoch P, Hillier SL, Cassen EM, et al. Amniotic fluid tumor necrosis factor-α and the risk of respiratory distress syndrome among preterm infants. Am J Obstet Gynecol 1997;177:50-6. 19. Watts DH, Krohn MA , Hillier SL, Eschenbach DA. The association of occult amniotic fluid infection with gestational age and neonatal outcome among women in preterm labor. Obstet Gynecol 1992;79:351-7. 20. Resko JA, Pleom JG, Stadelman HL. Estrogens in fetal and maternal plasma of the rhesus monkey. Endocrinology 1975;97: 425-30. 21. Resko JA, Ellinwood WE, Pasztor LM, Huhl AE. Sex steroids in the umbilical circulation of fetal rhesus monkeys from the time of gonadal differentiation. J Clin Endocrinol Metab 1980; 50:900-5. 22. Wade AP, Abramovich DR. The distribution of 17-oxosteroids and 17-hydroxycorticoids in amniotic fluid. Steroids 1967;10: 669-86.

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23. Belisle S, Tulchinsky D. Amniotic fluid hormones. In: Tulchinsky D, Ryan KJ, editors. Maternal-fetal endocrinology. Philadelphia: WB Saunders; 1980. p. 169-95. 24. Gravett MG, Haluska GJ, Cook MJ, Novy MJ. Fetal and maternal endocrine responses to experimental intrauterine infection in rhesus monkeys. Am J Obstet Gynecol 1996;174:1725-33. 25. Romero R, Avila C, Santhanam U, Sehgal PB. Amniotic fluid interleukin-6 in preterm labor: association with infection. J Clin Invest 1990;85:1392-400. 26. Mitchell MD, Dudley DJ, Edwin SS. Interleukin-6 stimulates prostaglandin production by human amnion and decidual cells. Eur J Pharmacol 1991;192:189-91. 27. Romero R, Durum S, Dinarello CA, Oyarzun E, Hobbins JC, Mitchell MD. Interleukin-1 stimulates prostaglandin biosynthesis by human amnion. Prostaglandins 1989;37:13-22. 28. Romero R, Mazur M, Tartakovsky B. Systemic administration of interleukin-1 induces preterm parturition in mice. Am J Obstet Gynecol 1991;165:969-71. 29. Baggia S, Gravett MG, Witkin SS, Haluska GJ, Novy MJ. Interleukin-1β intraamniotic infusion induces tumor necrosis factor-α, prostaglandin production, and preterm contractions in pregnant rhesus monkeys. J Soc Gynecol Investig 1996;3:121-6. 30. Yoon BH, Romero R, Jun JK, Maymon E, Gomez R, Mazor M, et al. An increase in fetal plasma cortisol but not dehydroepiandrosterone sulfate is followed by the onset of preterm labor in patients with preterm premature rupture of the membranes. Am J Obstet Gynecol 1998;179:1107-14. 31. Fritz MA, Stanczyk FZ, Novy MJ. Relationship of uteroplacental blood flow to the placental clearance of maternal dehydroepiandrosterone through estradiol formation in the pregnant baboon. J Clin Endocrinol Metab 1985;61:1023-30. 32. Masuda M, Kubota T, Kamada S, Aso T. Nitric oxide inhibits steroidogenesis in cultured porcine granulosa cells. Mol Hum Reprod 1997;3:4:285-92. 33. Riley SC, Challis JR. Corticotrophin-releasing hormone production by the placenta and fetal membranes. Placenta 1991;12: 105-19.

Editors’ note: This manuscript was revised after these discussions were presented. Discussion DR MICHAEL T. MEDCHILL, Phoenix, Arizona. Preterm labor is the common final pathway of several uterine pathologic processes, including infections, ischemia, overdistention, and hormonal disturbances.1 Because of our lack of understanding of the pathophysiology of the inciting event, we currently are forced to treat the woman with preterm labor with tocolytic agents or deliver her of a premature infant. Gravett et al have provided us with new data about the steroid milieu of the amniotic fluid of patients with intact membranes and evidence of intrauterine infection. Midpregnancy amniotic fluid steroid concentrations have been extensively analyzed and found to be highly accurate for the diagnosis of congenital adrenal hyperplasia.2 However, relatively little information is available about the steroid concentration changes associated with infection-induced preterm labor. Yoon et al3 recently studied the hormonal milieu of the fetus in pregnancies with preterm premature rupture of membranes. They found fetal plasma cortisol and IL-6 levels elevated among patients with positive culture results. However, amniotic fluid cortisol and DHEAS concentrations were not statistically different from those among patients with negative culture results. DHEA lev-

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els were not reported.3 In contrast, Gravett et al found an elevated amniotic fluid concentration of cortisol. They also found elevated DHEA levels but equivalent DHEAS concentrations in their infected population with respect to control patients. In this study only 4 of the 11 patients considered to be infected had positive culture results. The other seven patients were considered to be infected because of high levels of IL-6. Unfortunately, even though the infection may have cleared in these seven patients the preterm labor continued unabated, and delivery within 1 week was the rule. According to Dudley1 the maternal immune response, rather than the infectious agent itself, probably mediates the preterm labor. These data suggest that once the cytokine cascade has been initiated, the body lacks the ability to halt the process. This may explain why antibiotics may stop the infection but fail to stop preterm labor. CRH has recently been postulated to play a central role in human labor.4, 5 Several studies have demonstrated a 20- to 100-fold increase in CRH concentration before labor.4-6 Infections also stimulate CRH production through mediators such as catecholamines and such inflammatory cytokines as IL-1β, IL-6, and tumor necrosis factor α.1-7 CRH and cytokines mediate many immunologic and pathologic effects in the brain.7, 8 Cytokines have been implicated in the development of periventricular leukomalacia and cerebral palsy,9 learning disabilities,10 and anxiety and depression.11 It is interesting that children born to women with fever and bacteriuria have a higher incidence of neurologic defects.12, 13 One might speculatively theorize a pathophysiologic relationship between CRH or cytokines and the observation of O’Callaghan et al14 of a link between prenatal influenza A2 infection and schizophrenia. CRH receptor antagonists are currently being vigorously researched and touted as possibly the next generation of antidepressants.15 Perhaps CRH receptor antagonists will be useful for the prevention not only of preterm labor but also some of the serious sequelae of intrauterine infections. Dr Gravett, I have the following questions for you: 1. Amniocentesis was performed on 287 patients, but only 33 were evaluated in this study. Why were not all patients included in the study? How were the patients selected? 2. Patients with multiple gestations and preeclampsia were included in the study. Other studies have suggested that levels of steroids, cytokines, and CRH are altered in these conditions.2 Why did you consider it valid to include such patients in the study? 3. Can you explain why you found elevated concentrations of androgens (DHEA) and cortisol in the amniotic fluid of infected patients, whereas Yoon et al3 did not? 4. In light of the probable central role of CRH in labor as well as infections, were CRH levels considered for evaluation in this study? REFERENCES

1. Dudley DJ. Immunoendocrinology of preterm labor: the link be-

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12. 13.

14.

15.

tween corticotropin-releasing hormone and inflammation. Am J Obstet Gynecol 1999;180(1 Pt 3):S251-6. Wudy SA, Dorr HG, Solleder C, Djalali M, Homoki J. Profiling steroid hormones in amniotic fluid of midpregnancy by routine stable isotope dilution/gas chromatography-mass spectrometry: reference values and concentrations in fetuses at risk for 21-hydroxylase deficiency. J Clin Endocrinol Metab 1999;84:2724-8. Yoon BH, Romero R, Jun JK, Maymon E, Gomez R, Mazor M, et al. An increase in fetal plasma cortisol but not dehydroepiandrosterone sulfate is followed by the onset of preterm labor in patients with preterm premature rupture of the membranes. Am J Obstet Gynecol 1998;179:1107-14. Majzoub JA, McGregor JA, Lockwood CJ, Smith R, Taggart MS, Schulkin J. A central theory of preterm and term labor: putative role for corticotropin-releasing hormone. Am J Obstet Gynecol 1999;180(1 Pt 3):S232-41. Majzoub JA, Karalis DP. Placental corticotropin-releasing hormone: function and regulation. Am J Obstet Gynecol 1999; 180(1 Pt 3):S242-6. Frim DM, Emanuel RL, Robinson BG, Smas CM, Adler GK, Majzoub JA. Characterization and gestational regulation of corticotropin-releasing hormone messenger RNA in human placenta. J Clin Invest 1988;82:287-92. Raber J, Sorg O, Horn TF, Yu N, Koob GF, Campbell IL, et al. Inflammatory cytokines: putative regulators of neuronal and neuro-endocrine function. Brain Res Brain Res Rev 1998;26:3206. Reul JM, Stec I, Wiegers GJ, Labeur MS, Linthorst AC, Arzt E. Prenatal immune challenge alters the hypothalamic-pituitaryadrenocortical axis in adult rats. J Clin Invest 1994;93:2600-7. Yoon BH, Romero R, Yang SH, Jun JK, Kim IO, Choi JH, et al. Interleukin-6 concentrations in umbilical cord plasma are elevated in neonates with white matter lesions associated with periventricular leukomalacia. Am J Obstet Gynecol 1996;174:1433-40. Shimizu I, Adachi N, Liu K. Lei B, Nagaro T, Arai T. Sepsis facilitates brain serotonin activity and impairs learning ability in rats. Brain Res 1999;830:94-100. Arborelius L, Owens MJ, Plotsky PM, Nemeroff CB. The role of CRF in depression and anxiety disorders. J Endocrinol 1999; 160:1-12. Patrick MJ. Influence of maternal renal infection on the foetus and infant. Arch Dis Child 1967;42:208-13. Niswander KR, Gordon M. The women and their pregnancies. In: The collaborative perinatal study of the National Institute of Neurological Diseases and Stroke. Philadelphia: WB Saunders; 1992. p. 252-6. O’Callaghan E, Sham P, Takei N, Glover G, Murray RM. Schizophrenia after prenatal exposure to 1957 A2 influenza epidemic. Lancet 1991;337:1248-50. Holsboer F. The rationale for corticotropin-releasing hormone receptor (CRH-R) antagonists to treat depression and anxiety. J Psychiatr Res 1999;33:181-214.

DR ROBERT P. PRINS, Anacortes, Washington. My question concerns the amniotic fluid concentrations of cortisol and DHEA. I noticed that there were three or four outliers that seemed to basically produce your high average value, whereas the majority of your values were in a more normal range. I am curious as to whether you looked at those three or four outliers and whether there is something that was peculiar to them as opposed to the others, which were in a more normal range? DR JOHN A. ENBOM, Corvallis, Oregon. When you looked at the definition of infection, you used both patients with positive culture results and those with high IL-6 levels. Were there instances in which you did not have an elevated cytokine level in a patient in whom bacteria were detected, or did you always have a cytokine elevation regardless of whether bacteria were found?

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DR CALVIN HOBEL, Los Angeles, California. This article adds new information to try to sort out the differences between women with preterm labor with infection and those without infection. When we treat patients in preterm labor, we administer glucocorticoids, which have a dramatic effect on CRH concentration. They can stimulate CRH production within the placenta, and they can cross the placenta and inhibit the fetal pituitary-adrenal axis. Glucocorticoid treatment is a confounding variable that may explain some of the variation in some of the downstream hormone concentrations that you were measuring. Did all your patients receive glucocorticoids? If some did not, what were the differences observed? DR MARK NICHOLS, Portland, Oregon. My question is similar to Dr Enbom’s concerning the definition of infection. Was there a correlation with the diagnosis of infection by evaluation of amniotic membranes? Were stains performed to show that chorioamnionitis was present in the patients in the intrauterine infection group? DR GRAVETT (Closing). I would like to consolidate several of the questions regarding the role of IL-6 in the diagnosis of intrauterine infection, particularly with respect to those seven patients with elevated amniotic fluid IL-6 concentrations and negative microbial culture results. Dr Medchill speculated that in those patients a preexisting infection might have spontaneously cleared yet already have stimulated preterm delivery. I would suggest alternatively that these patients had very early intrauterine infections at the time of enrollment and that the infection progressed after sampling. My coinvestigators have detected bacterial ribosomal ribonucleic acid– encoding deoxyribonucleic acid within amniotic fluid in a large proportion of these patients by polymerase chain reaction.1 It is also possible that chorionic-decidual infection leads to increases in amniotic fluid IL-6 concentration before transgression of the membranes by bacteria and subsequent amniotic fluid colonization. Thus to answer the questions of Drs Nichols and Enbom, I suggest that IL-6 is an early and specific marker for intrauterine infection, and it has been strongly associated with histologic chorioamnionitis. Further, although a wide range of steroid hormone concentrations was observed in the setting of infection, as correctly pointed out by Dr Prins, there were no distinguishing characteristics of these outliers with respect to IL-6 concentrations or pathogen recovery. To directly address Dr Medchill’s questions, patients in this study were retrospectively identified. We first found the cases of all patients with evidence of intrauterine infection for whom maternal blood samples were also available for assay; samples were not available in all case. We then defined our two comparison groups and randomly selected equal numbers of patients from the total population for each comparison group. We did not intentionally exclude patients with preeclampsia or multiple gestation. In our selected subset of patients, however, none had preeclampsia. There was one case of multiple gestation in the group with infection and one in the group without infection and term delivery. Each of these was counted as a single event for statistical analysis.

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Placental CRH concentration was not measured in this study. Placental CRH was first identified in 1981, but it was not until 1995, more than halfway through the enrollment for our study, that its role as a placental clock regulating the length of gestation was first proposed.2 There was insufficient maternal blood from the early samples to measure CRH concentration, although your question regarding the relationship of intrauterine infection to placental CRH production suggests an important avenue for future research. The role of CRH in steroid hormone biosynthesis is relevant because of the frequent use of antepartum glucocorticoids to stimulate fetal lung maturity. Dr Hobel, in this study glucocorticoids were not routinely administered until after enrollment and amniocentesis, and thus glucocorticoid treatment would not have affected the steroid profiles observed in this study. Dr Hobel’s question is intriguing, however, because, in contrast to hypothalamic CRH production, placental CRH production is stimulated by glucocorticoids. Thus we are left with the conundrum that, although glucocorticoids may down-regulate the proinflammatory response, they may up-regulate placental CRH production and lead to fetal hypothalamic-pituitary-adrenal activation and possibly labor. Finally, Dr Medchill asked the important question of how our results compare with those previously reported by Yoon et al.3 First, we reported on different populations. We studied women with intact membranes in preterm labor. In contrast, Yoon et al3 studied women with premature rupture of membranes who were not in labor at the time of enrollment. I think that this difference in populations may explain some of the differences in results. After rupture of membranes there are rapid changes in the amniotic fluid volume, which may lead to perturbations in the concentrations of steroid hormones within amniotic fluid. In addition, Yoon et al3 had higher baseline concentrations of cortisol; their baseline concentrations of cortisol in the noninfected group were more than twice as high as those observed in our study. It is possible that stress associated with premature rupture of the membranes had already resulted in increased amniotic fluid cortisol concentrations, as has been previously reported.4 It is of interest that the peak amniotic fluid concentrations seen in the setting of infection in the two studies were similar. Our patients were also enrolled at a mean of 28.6 weeks’ gestation, earlier than the approximately 33 weeks’ gestation of the population of Yoon et al.3 It is possible that at the more advanced gestational age the fetal adrenal adult zone, which preferentially makes cortisol, may have been more active than the fetal zone, which preferentially makes androgens. If so, the adrenal glands were simply responding differentially by gestational age to the same stimulus by the hypothalamic-pituitary-adrenal axis. Instead of focusing on these differences, however, I would rather emphasize the similarities between our study and that of Yoon et al.3 Although we studied different populations at different gestational ages, we both found evidence of fetal hypothalamic-pituitary-adrenal axis activation. Yoon et al3 reported increases in fetal

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plasma cortisol concentration, whereas our study reported increases in amniotic fluid cortisol and adrenal androgen concentrations. These studies are therefore complementary. Both support the hypothesis that, regardless of the underlying etiology of preterm birth, activation of the fetal hypothalamic-pituitary-adrenal axis represents an important common pathway that may contribute to labor. REFERENCES

1. Hitti J, Riley DE, Krohn MA, Hillier SL, Agnew KJ, Krieger JN,

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et al. Broad-spectrum bacterial rDNA polymerase chain reaction assay for detecting amniotic fluid infection among women in premature labor. Clin Infect Dis 1997;24:1228-32. 2. McLean M, Bisits A, Davies J, Woods R, Lowry P, Smith R. A placental clock controlling the length of human pregnancy. Nat Med 1995;1:460-3. 3. Yoon BH, Romero R, Jun JK, Maymon E, Gomez R, Mazor M, et al. An increase in fetal plasma cortisol but not dehydroepiandrosterone sulfate is followed by the onset of preterm labor in patients with preterm premature rupture of the membranes. Am J Obstet Gynecol 1998;179:1107-14. 4. Cohen W, Fend MM, Tulchinsky D. Amniotic fluid cortisol after premature rupture of membranes. J Pediatr 1976;88:1007-9.

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