, Offer Erez , Bo H. Yoon 1,5 Clinical chorioamnionitis at

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2018. JÚNIUS NŐGYÓGYÁSZATI ÉS SZÜLÉSZETI TOVÁBBKÉPZŐ SZEMLE 103

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Clinical chorioamnionitis at term: New insights into the etiology, microbiology, and the fetal, maternal and amniotic cavity inflammatory responses

Roberto Romero1-4, Nardhy Gomez-Lopez1,5,6, Juan Pedro Kusanovic1,7, Percy Pacora1,5,

Bogdan Panaitescu1,5, Offer Erez1,5, Bo H. Yoon1,8

1 Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U S Department of Health and Human Services, Bethesda, Maryland, and Detroit, Michigan, USA

2 Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Michigan, USA3 Department of Epidemiology and Biostatistics, Michigan State University, East Lansing, Michigan, USA4 Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA5 Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan, USA6 Department of Immunology, Microbiology and Biochemistry, Wayne State University School of Medicine, Detroit, Michigan, USA7 Division of Obstetrics and Gynecology, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile8 Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Republic of Korea

Roberto Romero, M.D., D.Med.Sci.

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104 NŐGYÓGYÁSZATI ÉS SZÜLÉSZETI TOVÁBBKÉPZŐ SZEMLE 2018. JÚNIUS

Abstract

Clinical chorioamnionitis is the most common infection related diagnosis made in labor and delivery units worldwide. It is traditionally believed to be due to microbial invasion of the amniotic cavity, which elicits a maternal inflammatory response characterized by maternal fever, uterine tenderness, maternal tachycardia and leukocytosis. The condition is often as-sociated with fetal tachycardia and a foul smelling amniotic fluid. Recent studies in which amniocentesis has been used to characterize the microbiologic state of the amniotic cavity and the inflammatory response show that only 60% of patients with the diagnosis of clinical chorioamnionitis have proven infection using culture or molecular microbiologic techniques. The remainder of the patients have intra-amniotic inflammation without demonstrable microorganisms or a maternal systemic inflammatory response (fever) in the absence of in-tra-amniotic inflammation. The latter cases often represent a systemic inflammatory response after epidural anesthesia/analgesia has been administered. The most common microorganisms are Ureaplasma species and Gardnerella vaginalis. In the presence of ruptured membranes, the frequency of infection is 70%, which is substantially higher than patients who have intact membranes (25%). The amniotic fluid inflammatory response is characterized by an infiltra-tion of neutrophils and monocytes. Both cell types are activated in the presence of infection and can produce inflammatory cytokines. The white blood cells in the amniotic fluid can be of fetal or maternal origin. The maternal inflammatory response is characterized by an elevation in the concentration of pyrogenic cytokines. The cytokine plasma concentrations in the fetal circulation are elevated even if there is no evidence of an intra-amniotic inflam-matory response suggesting that maternal plasma cytokines may cross the placental barrier and induce a mild fetal inflammatory response. Placental pathology is of limited value in the diagnosis of proven intra-amniotic infection. The clinical criteria traditionally used in clinical medicine have accuracy around 50% and therefore, they cannot distinguish between patients with a proven intra-amniotic infection and those with intra-amniotic inflammation alone. Analysis of amniotic fluid with a bedside test for MMP-8 can allow the rapid identification of the patient at risk for infection and may decrease the need for antibiotic administration to neonates. An important consideration is whether antibiotics effective against Ureaplasma species should be administered to patients with clinical chorioamnionitis, given that these genital mycoplasmas are the most common organisms found in the amniotic fluid. The emer-gent picture is that clinical chorioamnionitis is a heterogeneous syndrome, which requires further study to optimize maternal and neonatal outcomes.

Keywords

Intra-amniotic infection, maternal fever, fetal tachycardia, amniotic fluid, cytokines, neutrophils, monocytes, MMP-8, biomarkers, rapid diagnosis, treatment, antibiotics,

maternal morbidity, neonatal morbidity

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IntroductionClinical chorioamnionitis is the most common infection-re-lated diagnosis made in labor and delivery units worldwide [1-4]. The standard clinical definition refers to the presence of maternal fever associated with clinical signs (i.e. foul smelling discharge, uterine tenderness, maternal and fetal tachycardia) as well as laboratory abnormalities (i.e. leukocytosis) [5, 6]. These signs are thought to be manifestations of both local and systemic maternal-fetal inflammatory processes initiat-ed in response to microbial invasion of the amniotic cavity (MIAC) [7-13].

Clinical chorioamnionitis at term is associated with a 2- to 4-fold increase in endometritis [14], wound infection [14], septic pelvic thrombophlebitis [3, 14], pelvic abscess [3, 14], maternal admission to the intensive care unit [3, 15], and postpartum hemorrhage [3, 16]. Importantly, neonates born to mothers with clinical signs of chorioamnionitis have a high risk of neonatal mortality [1], short- and long-term complications such as neonatal sepsis [17-19], meconium aspiration syndrome [20, 21], stillbirth [22, 23], and neurodevelopmental disorders including cerebral palsy [24-35].

Although this condition is common, affecting thousands of women daily and has been known for centuries as a precursor of puerperal (or childbed) fever [36, 37], new information has emerged which has characterized, for the first time, the mi-crobiology and inflammatory responses in the mother, fetus, and amniotic cavity. This article will review new insight gained from studies published in the last three years.

The syndrome of clinical chorioamnionitis: evidence for microbiologic and inflammatory heterogeneityAmong patients with clinical chorioamnionitis, we have iden ti-fied three distinct groups [38]: 1) patients who have intra-amni-otic infection, defined by the presence of an elevated concentra-tion of interleukin (IL)-6 [a marker of inflammation [39]] and the presence of microorganisms; 2) patients who have sterile intra-amniotic inflammation, in which no organisms can be detected using standard cultivation techniques or 16S RNA broad assays, although they have an elevated amniotic fluid IL-6; and 3) patients who have neither intra-amniotic inflammation or microorganisms in the amniotic cavity. The frequency with which these conditions are present varies according to whether the membranes are ruptured at the time of amniocentesis to assess the microbial state of the amniotic cavity [38]. When the membranes are intact, the frequency of intra-amniotic infection

is 25%, while it is 70% when the membranes are ruptured [38]. The relative contribution of the different entities presenting as clinical chorioamnionitis are illustrated in Figure 1. A small group of patients has microbial invasion of the amniotic cavity without inflammation.

The mechanism responsible for sterile intra-amniotic inflam-mation have not been defined [38, 40-42] and could represent inflammation induced by danger signals (or alarmins) [43-47], or alternatively, infections which have escaped detection with current cultivation and molecular microbiologic techniques [48-54]. For example, it is possible that deep DNA and RNA sequencing may identify organisms which are present below the level of detection of culture and 16S PCR; however, this question remains to be addressed.

What are the microorganisms found in the amniotic cavity in clinical chorioamnionitis at term?The original study describing the microbiology of clinical chorioamnionitis at term was performed using cultivation techniques of amniotic fluid obtained with transcervical catheters placed in the amniotic cavity [5]. This approach may not have provided an accurate description of the microbiology of clinical chorioamnionitis, since the placement of a transcervical catheter is frequently associated with contamination of the vaginal ecosystem. Conventional cultivation techniques cannot identify the entire microbial diversity of an ecological niche, because the conditions required for the growth of microorganisms in vitro are not known, and some organisms are, therefore, considered to be non-culturable [55]. By using both cultivation and molecular biology techniques (broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry or PCR/ESI-MS), it is possible to gain a more complete understanding of the microbiology of the amniotic cavity [56].

In a study of 46 women with clinical chorioamnionitis at term, amniotic fluid was obtained by transabdominal amniocen-tesis or at the time of Cesarean delivery under sterile conditions [38]. We found that conventional culture identified bacteria in the amniotic fluid from 46% (21/46) of the participants, while PCR/ESI-MS was positive for microorganisms in 59% (27/46) of the patients. The combination of these two approaches re-sulted in the detection of amniotic fluid microorganisms in 61% (28/46) of women with clinical chorioamnionitis at term [38]. Molecular microbiologic techniques identified more mi-croorganisms than cultivation; yet, some bacterial taxa were preferentially identified by each technique [38].

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Ureaplasma spp. and Gardnerella vaginalis are the most commonly identified microorganisms by cultivation and PCR/ESI-MS, respectively [38]. This has clinical implications, because the antibiotics used to treat patients with clinical chorioam-nionitis or puerperal endometritis do not include agents that eradicate infections due to Ureaplasma spp.

The cellular intra-amniotic inflammatory response: neu-trophils and monocytes in the amniotic fluidAn inflammatory response is characterized by cellular and soluble components. The typical changes in the soluble me-ditators are those that occur in the inflammatory cytokine network. However, cellular mediators are prominent, and mainly involve the innate immune response.

Using flow cytometry, we found that neutrophils are the most common leukocyte subset in the amniotic fluid, followed by monocytes; other white blood cells (such as lymphocytes and natural killer cells) are scarce in the amniotic fluid [57] (Figure 2). The absolute counts of neutrophils and monocytes

are significantly higher in patients with microorganisms found in the amniotic fluid than in those without detectable micro-organisms [57]. In addition, the profile of cytokine expression differs between monocytes and neutrophils; while neutrophils predominantly produce tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-1β, monocytes ex-press higher levels of IL-1β and IL-1α [57]. These cytokines are implicated in both the process of parturition and host response against intra-amniotic infection [58-75].

Figure 2

Histological examination of amniotic fluid neutrophils and monocytes in women with clinical chorioamnionitis. A) Hematoxylin & Eosin staining shows the typical morphology of neutrophils (N, green arrow) and mono-cytes (M, red arrow) in the amniotic fluid of women with clinical chorio-amnionitis. Magnification 400X. Scale bars: 50μm. B) Immunofluores-cence of neutrophils and monocytes in the amniotic fluid of women with clinical chorioamnionitis. Neutrophils (CD15+ cells) are green, mono cytes (CD14+ cells) are red, and nuclei are blue (DAPI). Magnification 400X.

31.3%

6.7%

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Intact membranes(n=16)

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No inflammation/infection

Microbial invasion of the amniotic cavity (without inflammation)

Figure 1

Prevalence of microbial-associated intra-amniotic inflammation and intra-amniotic inflammation without detectable microorganisms in patients with clinical signs of chorioamnionitis at term according to the status of the membranes at the time of amniocentesis (intact vs. ruptured). Microbial-associated intra-amniotic inflammation was diag-nosed in 70% (21/30) of the cases with rupture of membranes, and in 25% (4/16) of patients with intact membranes.

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Amniotic fluid white blood cells can be of fetal or maternal origin Although traditionally considered to be of fetal origin [76, 77], neut rophils in amniotic fluid in cases of infection can be of maternal, fetal, or mixed (both) origin, as demonstrated by studies using DNA fingerprinting [78]. In general, we found that patients with intra-amniotic infection and preterm gestations had neutrophils of fetal origin, while those with intra-amniotic infection/inflammation at term had predominantly maternal neutrophils or a cellular infiltrate of mixed origin (maternal and fetal) [78]. Figure 3 shows the DNA fingerprinting of amniotic fluid neutrophils of fetal, maternal, or mixed (maternal and fetal) origin.

The soluble amniotic fluid inflammatory response: changes in the cytokine inflammatory network and lipidomeThe amniotic fluid concentrations of inflammatory cytokines [such as interferon gamma (IFN-γ), TNFα, IL-4, MIP-1β, and IL-8] are higher in patients with clinical chorioamnionitis at term than in women with spontaneous labor at term without intra-amniotic inflammation [75]. Of interest is that once a patient had intra-amniotic inflammation, the changes in the amniotic fluid cytokine network are similar in those with proven infection vs. those with intra-amniotic inflammation without demonstrable microorganisms [75].

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Figure 3 DNA fingerprinting of amniotic fluid neutrophils

DNA fingerprinting of purified amniotic fluid neutrophils of fetal (A), maternal (B) or mixed (C) origin, the fetus (umbilical cord), and the mother (buffy coat from peripheral blood) is shown in electropherograms. Each electropherogram contains 16 genetic sites: D3S1358, TH01, D21S11, D18S51, Penta_E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta_D, X- and Y-specific amalogenin genes, vWA, D8S1179, TPOX, and FGA. Each electropherogram was separated into three sections: blue, green, and black. Each color indicates the dye used for the PCR multiplex: blue represents the genes amplified using fluorescein (FL dye), green represents the genes amplified using 6-carboxy-4',5'-dichloro-2',7'-dimethoxyflu-orescein (JOE dye), and black represents the genes amplified using tetramethylrhodamine (TMR dye). Each genetic site has STR alleles, which is represented by peaks. The number next to/above each STR allele (peak) is the number of repeats for each STR allele.

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Given that lipids are important mediators of the inflamma-tory response, we have examined the behavior of bioactive lipids using discovery techniques (i.e. lipidomics) [79]. An elevated leukotriene B4 (LTB4) was associated with the presence of microorganisms in the amniotic cavity [80]. Therefore, lipid mediators can be assessed with mass spectrometry techniques could have value as biomarkers [80].

How well do clinical criteria perform in the identification of proven intra-amniotic infection? Only 54% of patients with the diagnosis of clinical chorio-amnionitis at term have intra-amniotic inflammation and detectable bacteria [38]. These findings indicate that only half of the women with clinical chorioamnionitis at term may benefit from antibiotic administration. Therefore, we evaluated the diagnostic performance and accuracy of clinical signs for the diagnosis of clinical chorioamnionitis using the “gold standard” [a combination of cultivation and molecular biology techniques [56]] for the identification of intra-amniotic infection [81].

We reported that clinical signs of chorioamnionitis do not accurately identify patients with intra-amniotic infection [81]. Maternal and fetal tachycardia, as well as maternal leukocytosis, have low specificity (5%–30%), whereas foul-smelling amniotic fluid and uterine tenderness have poor sensitivity (<15%) for the diagnosis of intra-amniotic infection [81]. These findings call for the development of novel approaches to facilitate the rapid identification of bacteria in the amniotic fluid and reevaluation of the management of clinical chorioamnionitis at term.

The maternal plasma cytokine profileMost of the signs of clinical chorioamnionitis (except for fetal tachycardia and foul-smelling amniotic fluid) are thought to reflect a maternal inflammatory response to microbial invasion of the amniotic cavity [4, 82]. Maternal plasma concentrations of pyrogenic cytokines (IL-1β, IL-2, IL-6, IFN-γ, and TNF-α) are higher in patients with clinical chorioamnionitis at term than in those with spontaneous labor at term without clinical chorioamnionitis [83]. However, the maternal plasma concentrations of cytokines are not significantly different among the 3 major subgroups of patients with clinical chorioamnionitis at term [83]. In addition, women with clinical chorioamnionitis at term without intra-amniotic inflammation have greater maternal plasma concentrations of pyrogenic cytokines than those with spontaneous labor

at term without this clinical condition [83]. These findings indicate that the absolute concentration of cytokines in the maternal plasma cannot be used to identify women who have bacteria in the amniotic fluid and that the assessment of the amniotic fluid is required to identify intra-amniotic inflammation and/or infection.

Umbilical cord plasma cytokine profile in the context of a systemic maternal inflammatory responseNeonates born to mothers with clinical chorioamnionitis are at risk for sepsis [3, 17-19, 32, 84-89], meconium aspiration syndrome [17, 90-94], neonatal encephalopathy [31, 34, 95-97], and long-term neurodevelopmental disabilities including cognitive impairment [98-101] and cerebral palsy [27, 28, 30, 34, 35, 86, 102-104], as well as neonatal death [1, 22, 24, 88, 105]. Such adverse neonatal outcomes may be attributed to the effects of a maternal systemic inflammatory response on the fetus [83] and/or the local inflammatory response in the amniotic cavity [38, 75].

Neonates born to mothers with clinical chorioamnionitis at term have higher umbilical cord plasma concentrations of several inflammatory cytokines (IL-6, IL-12p70, IL-16, IL-13, IL-4, IL-10 and IL-8) compared to those born to mothers with spontaneous labor at term without this clinical condition [106]. Interestingly, neonates born to mothers with clinical chorioamnionitis at term without intra-amniotic inflam-mation also have higher concentrations of inflammatory cytokines (IL-6, IL-12p70, IL-13, IL-4, IL-5, and IL-8) than neonates not exposed to clinical chorioamnionitis, suggesting that maternal fever in the absence of intra-amniotic inflam-mation leads to a change in the fetal cytokine network [106]. In addition, there are significant and positive correlations between maternal and umbilical cord plasma IL-6 and IL-8 concentrations consistent with placental transfer of cytokines [106]. All neonates born to mothers with proven intra-am-niotic infection were diagnosed with fetal inflammatory response syndrome (FIRS) [106].

Can placental pathology identify the patient with intra-amniotic infection?Examination of the placenta, the largest human biopsy, can provide information about fetal exposure to intra-amniotic inflammation or infection [10, 11, 13, 24, 26, 33, 107-124]. Indeed, inflammation of the umbilical cord or chorionic vessels represents a fetal response and is indicative that

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the fetus was exposed to inflammatory stimuli before birth [113]. Therefore, we examined the diagnostic performance of placental histologic features of acute inflammation in the identification of intra-amniotic infection and FIRS in patients with clinical chorioamnionitis at term [125]. Acute histologic chorioamnionitis and funisitis are indeed associated with the presence of intra-amniotic infection and FIRS [125]. However, current placental histopathologic methods have limitations in identifying whether the fetus was exposed to microorganisms in the amniotic cavity [125]. The available data suggest that placental examination is neither sensitive nor specific for the identification of intra-amniotic infection, because acute histologic chorioamnionitis and funisitis can be observed when there is intra-amniotic inflammation in the absence of demonstrable microorganisms.

A rapid MMP-8 test for the identification of intra-amniotic inflammationA rapid test which utilizes neutrophil collagenase or MMP-8 has been made available for the diagnosis of intra-amniotic inflammation [126-133]. This test in patients with intra-amniotic inflammation and clinical chorioamnionitis has the following diagnostic indices, predictive values, and likelihood ratios: sensitivity of 82.4% (28/34), specificity of 90% (9/10), positive predictive value of 96.6% (28/29), negative predictive value of 60% (9/15), positive likelihood ratio 8.2 (95% CI 1.3–53.2), and negative likelihood ratio 0.2 (95% CI 0.1–0.4) [134].

In patients with intra-amniotic infection and clinical cho-rioamnionitis, the rapid MMP-8 test has a sensitivity of 91.7% (22/24), specificity of 65% (13/20), positive predictive value of 75.9% (22/29), negative predictive value of 86.7% (13/15), positive likelihood ratio of 2.6 (95% CI 1.4–4.8), and negative likelihood ratio of 0.1 (95% CI 0.03–0.5) [134].

In addition, the rapid MMP-8 test has a significantly higher specificity than the ELISA IL-6 test in the identification of intra-amniotic inflammation [66.7% (14/21) vs. 42.9% (9/21); p=0.03] [134]. Importantly, the rapid MMP-8 test has 100% sensitivity and 100% negative predictive value in the identifi-cation of neonates affected with FIRS [134].

This study showed that a rapid MMP-8 point-of-care test can be used for the diagnosis of intra-amniotic inflammation and to identify the mother at risk of delivering a neonate with systemic inflammation among patients with clinical chorio-amnionitis at term [134].

ConclusionsA series of systematic studies in patients with clinical chorio-amnionitis at term have revealed the following:

• Clinical chorioamnionitis at term is a heterogeneous condition, and only a fraction of these patients have proven intra-amniotic infection

• The frequency of infection is higher when the chorioamni-otic membranes are ruptured, rather than intact (70% vs 25%)

• The most common organisms are Ureaplasma spp. and Gardnerella vaginalis – this is important because most treat-ment approaches in clinical obstetrics and neonatology do not provide appropriate coverage for genital mycoplasmas

• The cellular intra-amniotic inflammatory response is characterized by an influx of neutrophils and monocytes

• The soluble intra-amniotic inflammatory response is char-acterized by elevated concentrations of the major inflammatory cytokines

• The maternal plasma cytokines are elevated in patients with clinical chorioamnionitis at term; however, the con-centrations of these proteins cannot distinguish between patients who have proven intra-amniotic infection and those with intra-amniotic inflammation without demonstrable microorganisms

• The fetal concentrations of inflammatory cytokines are elevated in neonates born to mothers with clinical chorio-amnionitis compared to those without clinical chorioamni-onitis at term. There is indirect evidence that elevations in neonatal inflammatory concentrations may reflect a systemic inflammatory response in the mother, even in the absence of intra-amniotic inflammation (this may occur in cases of epidural-induced fever)

• Placental pathologic examination to detect acute histologic chorioamnionitis, funisitis, or chorionic vasculitis is neither specific nor sensitive for the identification of patients with proven intra-amniotic infection

• Analysis of amniotic fluid is the only definitive way to make a diagnosis of intra-amniotic infection in patients with clinical chorioamnionitis at term

• Potential biomarkers for intra-amniotic inflammation, which can be implemented at the bedside, is an MMP-8 rapid test which detects neutrophil collagenase

• Future studies are required to determine if noninvasive collection of amniotic fluid with a special device in patients who have ruptured membranes can be of clinical value to diagnose intra-amniotic infection/inflammation

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