The fetal inflammatory response syndrome: the origins of a concept, pathophysiology, diagnosis, and obstetrical implications

  • Eunjung Jung
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA
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  • Roberto Romero
    Correspondence
    Corresponding author. Perinatology Research Branch, NICHD/NIH/DHHS, Hutzel Women's Hospital, 3990 John R Street, 4 Brush, Detroit, MI 48201, USA.
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA

    Department of Epidemiology and Biostatistics, Michigan State University, East Lansing, MI, USA

    Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, USA

    Detroit Medical Center, Detroit, MI, USA

    Department of Obstetrics and Gynecology, Florida International University, Miami, FL, USA
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  • Lami Yeo
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA
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  • Ramiro Diaz-Primera
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA
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  • Julio Marin-Concha
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA
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  • Robert Para
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA
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  • Ashley M. Lopez
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA
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  • Percy Pacora
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA
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  • Nardhy Gomez-Lopez
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA

    Department of Biochemistry, Microbiology and Immunology, Wayne State University School of Medicine, Detroit, MI, USA
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  • Bo Hyun Yoon
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Republic of Korea
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  • Chong Jai Kim
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea
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  • Stanley M. Berry
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA
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  • Chaur-Dong Hsu
    Affiliations
    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 (NICHD/NIH/DHHS), Bethesda, MD, and Detroit, MI, USA

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA

    Department of Physiology, Wayne State University School of Medicine, Detroit, MI, USA
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Open AccessPublished:October 22, 2020DOI:https://doi.org/10.1016/j.siny.2020.101146

      Abstract

      The fetus can deploy a local or systemic inflammatory response when exposed to microorganisms or, alternatively, to non-infection-related stimuli (e.g., danger signals or alarmins). The term “Fetal Inflammatory Response Syndrome” (FIRS) was coined to describe a condition characterized by evidence of a systemic inflammatory response, frequently a result of the activation of the innate limb of the immune response. FIRS can be diagnosed by an increased concentration of umbilical cord plasma or serum acute phase reactants such as C-reactive protein or cytokines (e.g., interleukin-6). Pathologic evidence of a systemic fetal inflammatory response indicates the presence of funisitis or chorionic vasculitis. FIRS was first described in patients at risk for intraamniotic infection who presented preterm labor with intact membranes or preterm prelabor rupture of the membranes. However, FIRS can also be observed in patients with sterile intra-amniotic inflammation, alloimmunization (e.g., Rh disease), and active autoimmune disorders. Neonates born with FIRS have a higher rate of complications, such as early-onset neonatal sepsis, intraventricular hemorrhage, periventricular leukomalacia, and death, than those born without FIRS. Survivors are at risk for long-term sequelae that may include bronchopulmonary dysplasia, neurodevelopmental disorders, such as cerebral palsy, retinopathy of prematurity, and sensorineuronal hearing loss. Experimental FIRS can be induced by intra-amniotic administration of bacteria, microbial products (such as endotoxin), or inflammatory cytokines (such as interleukin-1), and animal models have provided important insights about the mechanisms responsible for multiple organ involvement and dysfunction. A systemic fetal inflammatory response is thought to be adaptive, but, on occasion, may become dysregulated whereby a fetal cytokine storm ensues and can lead to multiple organ dysfunction and even fetal death if delivery does not occur (“rescued by birth”). Thus, the onset of preterm labor in this context can be considered to have survival value. The evidence so far suggests that FIRS may compound the effects of immaturity and neonatal inflammation, thus increasing the risk of neonatal complications and long-term morbidity. Modulation of a dysregulated fetal inflammatory response by the administration of antimicrobial agents, anti-inflammatory agents, or cell-based therapy holds promise to reduce infant morbidity and mortality.

      Keywords

      1. The term "fetal inflammatory response syndrome" (FIRS)

      The human fetus can deploy an inflammatory response when exposed to microbial invasion with bacteria [
      • Wendel Jr., G.D.
      • Sanchez P.J.
      • Peters M.T.
      • Harstad T.W.
      • Potter L.L.
      • Norgard M.V.
      Identification of Treponema pallidum in amniotic fluid and fetal blood from pregnancies complicated by congenital syphilis.
      ], viruses [
      • Morgan-Capner P.
      • Rodeck C.
      • Nicolaides K.
      • Cradock-Watson J.
      Prenatal detection of rubella-specific IgM in fetal sera.
      ,
      • Thilaganathan B.
      • Carroll S.G.
      • Plachouras N.
      • Makrydimas G.
      • Nicolaides K.H.
      Fetal immunological and haematological changes in intrauterine infection.
      ], fungi [
      • Donders G.G.
      • Moerman P.
      • Caudron J.
      • Van Assche F.A.
      Intra-uterine Candida infection: a report of four infected fetusses from two mothers.
      ,
      • Carroll S.G.
      • Nicolaides K.H.
      Fetal haematological response to intra-uterine infection in preterm prelabour amniorrhexis.
      ], and protozoa [
      • Desmonts G.
      • Forestier F.
      • Thulliez P.
      • Daffos F.
      • Capella-Pavlovsky M.
      • Chartier M.
      Prenatal diagnosis of congenital toxoplasmosis.
      ,
      • Pratlong F.
      • Boulot P.
      • Issert E.
      • Msika M.
      • Dupont F.
      • Bachelard B.
      • et al.
      Fetal diagnosis of toxoplasmosis in 190 women infected during pregnancy.
      ,
      • Abrams E.T.
      • Kwiek J.J.
      • Mwapasa V.
      • Kamwendo D.D.
      • Tadesse E.
      • Lema V.M.
      • et al.
      Malaria during pregnancy and foetal haematological status in Blantyre, Malawi.
      ], or non-infection related stimuli. The inflammatory process can be localized to an organ (e.g., the lung following fetal aspiration of amniotic fluid) or become systemic when inflammatory mediators enter the circulation.
      We coined the term “Fetal Inflammatory Response Syndrome” (FIRS) while studying the role of intra-amniotic infection in spontaneous preterm labor to describe the presence of systemic inflammation akin to that observed in adult patients with a systemic inflammatory response syndrome [
      • Gomez R.
      • Romero R.
      • Ghezzi F.
      • Yoon B.H.
      • Mazor M.
      • Berry S.M.
      The fetal inflammatory response syndrome.
      ].
      The term “Systemic Inflammatory Response Syndrome” (SIRS) emerged from the consensus conference of the American College of Chest Physicians and the Society for Critical Care Medicine in 1992, as participants recognized that clinical manifestations of sepsis could also be observed in patients without infection (e.g., burns, trauma, pancreatitis, ischemia, immune-mediated injury), and that they resulted from a systemic inflammatory process [
      American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis.
      ]. The group also proposed the term “Multiple Organ Dysfunction Syndrome” (MODS) to refer to the presence of altered organ function in an acutely ill patient, (e.g., homeostasis cannot be maintained without intervention) [
      American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis.
      ].
      SIRS has been diagnosed in adults using the criteria displayed in Table 1. Yet, the definition could not be applied to the human fetus because vital signs (except for the fetal heart rate) and white blood cell counts cannot be readily determined before birth. For this reason, we elected to define FIRS as an elevated concentration of fetal plasma interleukin-6 (IL-6).
      Table 1Criteria for systemic inflammatory response syndrome
      Modified with permission from American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med. 1992; 20:864–74.
      .
      Two or more of the following criteria should be met:
      • Temperature >38 °C or <36 °C
      • Heart rate >90 beats/min
      • Respiratory rate >20 breaths/min or PaCO2 <32 mm Hg
      • White blood cell count >12,000/mm3 or <4000/mm3 or >10% immature bands
      a Modified with permission from American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med. 1992; 20:864–74.
      In 2001, the American College of Chest Physicians and the Society of Critical Care Medicine reaffirmed the initial criteria for the diagnosis of SIRS. The group of experts noted that an elevation of plasma concentration of certain mediators, such as IL-6, could be associated with SIRS and speculated that this observation could bring about a new definition of the syndrome in adult patients. Concerns at the time were that the clinical and laboratory findings originally proposed to characterize SIRS were non-specific. The terms used to assess patients with suspected sepsis have continued to evolve [
      • Singer M.
      • Deutschman C.S.
      • Seymour C.W.
      • Shankar-Hari M.
      • Annane D.
      • Bauer M.
      • et al.
      The third international consensus definitions for sepsis and septic shock (Sepsis-3).
      ]. Yet, it is clear that the host immune response is key in the recovery of patients as well as in the predisposition to secondary infections and morbidity/mortality.
      The immunological response to infection or tissue injury varies over time and involves both pro- and anti-inflammatory responses. The initial concept was that SIRS was followed by compensatory anti-inflammatory response syndrome and that this process led to MODS and predisposed to death. In the early stages of the study of SIRS, it was believed that most patients died because of an excessive inflammatory response. However, subsequent observations uncovered the importance of the counter-inflammatory response that leads to progressive immune suppression and predisposition to secondary infection. It is now recognized that both pro- and anti-inflammatory responses are activated in early sepsis; however, the pro-inflammatory response is predominant [
      • Hotchkiss R.S.
      • Karl I.E.
      The pathophysiology and treatment of sepsis.
      ,
      • Hotchkiss R.S.
      • Monneret G.
      • Payen D.
      Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy.
      ,
      • Hotchkiss R.S.
      • Moldawer L.L.
      • Opal S.M.
      • Reinhart K.
      • Turnbull I.R.
      • Vincent J.L.
      Sepsis and septic shock.
      ]. As the disorder progresses, the anti-inflammatory limb of the immune response becomes predominant, and patients recovering from sepsis are more susceptible to secondary infections from bacteria, or even reactivation of latent viral infections (e.g., cytomegalvirus, herpes simplex virus) [
      • Luyt C.E.
      • Combes A.
      • Deback C.
      • Aubriot-Lorton M.H.
      • Nieszkowska A.
      • Trouillet J.L.
      • et al.
      Herpes simplex virus lung infection in patients undergoing prolonged mechanical ventilation.
      ,
      • Limaye A.P.
      • Kirby K.A.
      • Rubenfeld G.D.
      • Leisenring W.M.
      • Bulger E.M.
      • Neff M.J.
      • et al.
      Cytomegalovirus reactivation in critically ill immunocompetent patients.
      ]. Indeed, patients who have recovered from sepsis remain at risk of death for approximately one year after the sepsis episode, secondary to the prolonged period of immunosuppression [
      • Hotchkiss R.S.
      • Monneret G.
      • Payen D.
      Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy.
      ,
      • Hotchkiss R.S.
      • Moldawer L.L.
      • Opal S.M.
      • Reinhart K.
      • Turnbull I.R.
      • Vincent J.L.
      Sepsis and septic shock.
      ]. The early pro-inflammatory phase has been attributed predominantly to activity of the innate immune system, while the counter-immune response has been attributed to a dysregulated adaptive immune system: this is probably an oversimplification of the complex nature of the evolution and interaction of different components of the immune system. However, the conceptual framework of an anti-inflammatory response in neonates is important because it may explain why some neonates are born with FIRS [
      • Stampalija T.
      • Romero R.
      • Korzeniewski S.J.
      • Chaemsaithong P.
      • Miranda J.
      • Yeo L.
      • et al.
      Soluble ST2 in the fetal inflammatory response syndrome: in vivo evidence of activation of the anti-inflammatory limb of the immune response.
      ,
      • Gotsch F.
      • Romero R.
      • Kusanovic J.P.
      • Erez O.
      • Espinoza J.
      • Kim C.J.
      • et al.
      The anti-inflammatory limb of the immune response in preterm labor, intra-amniotic infection/inflammation, and spontaneous parturition at term: a role for interleukin-10.
      ], improve clinically, and then become affected by late-onset neonatal sepsis [
      • Hibbert J.E.
      • Currie A.
      • Strunk T.
      Sepsis-induced immunosuppression in neonates.
      ].

      2. Why focus on systemic fetal inflammation or FIRS?

      In 1998 when the initial work in this field was reported, intra-amniotic infection in patients with preterm labor and intact membranes, as well as preterm prelabor rupture of the membranes (PROM), was known to be associated with impending delivery. However, it was not clear whether labor was associated with an intra-amniotic or a fetal systemic inflammatory response. Moreover, the frequency with which intraamniotic infection led to fetal infection and sepsis was unknown.
      We had observed that of preterm neonates born to mothers with intra-amniotic inflammation/infection only a fraction had proven neonatal sepsis; yet, many of these neonates had morbidity which could be attributed at least in part to a systemic inflammatory process, but not necessarily to sepsis. Therefore, an important question emerged: does fetal inflammation predispose to multiple organ dysfunction and result in a higher rate of neonatal morbidity?
      The operative definition of FIRS was an elevation in the fetal plasma concentration of IL-6. Since our group was studying pregnancy outcomes using cytokines to define the presence or absence of intra-amniotic inflammation [
      • Gomez R.
      • Romero R.
      • Ghezzi F.
      • Yoon B.H.
      • Mazor M.
      • Berry S.M.
      The fetal inflammatory response syndrome.
      ], we chose IL-6 because this cytokine could be measured in both amniotic fluid and umbilical cord blood; therefore, we could ascertain the presence and intensity of the intra-amniotic and fetal inflammatory responses based on one analyte. Other cytokines (e.g., TNF-α and IL-1β) were not consistently detected in peripheral blood with the assays available at the time. We also chose IL-6 as a marker of inflammation because this cytokine is a major mediator of the acute phase response to infection or tissue injury (e.g., IL-6 induces production of C-reactive protein).
      We used the term syndrome during the course of initial studies given our observation that both intra-amniotic inflammation and fetal systemic inflammation could be caused by infection and non-infection-related etiologies. We predicted that FIRS would: 1) be associated with the spontaneous onset of labor; 2) be associated with a higher rate of neonatal morbidity, since the fetuses were already affected in utero; and 3) lead to changes/dysfunction in multiple organ systems.

      3. FIRS is followed by the spontaneous onset of preterm labor

      Preterm labor in the setting of infection results from the action of pro-inflammatory cytokines secreted by the mother and/or fetus in response to intra-amniotic infection [
      • Romero R.
      • Gomez R.
      • Ghezzi F.
      • Yoon B.H.
      • Mazor M.
      • Edwin S.S.
      • et al.
      A fetal systemic inflammatory response is followed by the spontaneous onset of preterm parturition.
      ]. Delivery would allow the mother to maximize reproductive fitness and the fetus to exit a hostile intrauterine environment. The mechanisms of parturition require cooperation between the mother and the conceptus as the effector organs of parturition are maternal (myometrium, decidua, and cervix), but there is a substantial contribution of the conceptus (chorioamniotic membranes).
      In a study of patients with preterm PROM who were not in labor upon admission, we found that FIRS was associated with a higher rate of spontaneous preterm delivery within 48 and 72 h of amniocentesis, compared to those without FIRS (48 h: 88% vs. 29.7%; and 72 h: 88% vs. 35%; p-value <0.05 for both) [
      • Romero R.
      • Gomez R.
      • Ghezzi F.
      • Yoon B.H.
      • Mazor M.
      • Edwin S.S.
      • et al.
      A fetal systemic inflammatory response is followed by the spontaneous onset of preterm parturition.
      ]. Moreover, patients with initiation of labor and delivery within 48 h of amniocentesis had a higher proportion of fetuses with plasma IL-6 values > 11 pg/mL than patients delivered >48 h [58% (7/12) vs. 8% (1/13), respectively, p-value <0.05] (Fig. 1). Multivariate analysis showed that plasma IL-6 was the only factor associated with pregnancy duration after adjusting for gestational age, amniotic fluid IL-6, and the microbiologic state of the amniotic cavity. The relationship between intra-amniotic inflammation and fetal systemic inflammation and the onset of preterm labor is displayed in Fig. 2. These findings led to the conclusion that FIRS was followed by the onset of preterm parturition in patients with preterm PROM.
      Fig. 1
      Fig. 1Fetuses with fetal inflammatory response syndrome (FIRS) have a shorter intrauterine stay than those without FIRS. Mothers were admitted with preterm premature rupture of membranes and patients were not in labor at admission. The interval between the procedure and delivery reflects duration of pregnancy and spontaneous onset of labor. Fetuses with fetal plasma IL-6 concentrations greater than 11 pg/mL have a shorter procedure-to-delivery interval than those with plasma IL-6 concentrations of 11 pg/mL or less (median 0.8 days [range 0.1–5 days] vs. median 6 days [range 0.2–33.6 days]; respectively; P < 0.05). (Modified with permission from Romero R, Gomez R, Ghezzi F et al.: A fetal systemic inflammatory response is followed by the spontaneous onset of preterm parturition. Am J Obstet Gynecol 1998; 179:186–193.).
      Fig. 2
      Fig. 2Duration of pregnancy according to whether or not there is intraamniotic inflammation and fetal systemic inflammation (FIRS). The key determinant of pregnancy duration is fetal systemic inflammation regardless of the inflammatory status of the amniotic cavity, as reflected by the procedure-to-delivery interval. The inflammatory status of the amniotic cavity and the fetus was assessed with interleukin-6 (IL-6) concentrations. A white color fetus represents no inflammation [defined as fetal plasma (FP) IL-6 less than 11 pg/mL]. A red color fetus represents fetal systemic inflammation (FP IL-6 greater than 11 pg/mL). The amniotic fluid compartment is either white (no intraamniotic inflammation) or yellow in color (intraamniotic inflammation present). The cut-off value of 7.9 ng/ml was used to define intra-amniotic inflammation. The number of patients in each group is depicted (n). (Reproduced with permission from Romero R, Gomez R, Ghezzi F et al: A fetal systemic inflammatory response is followed by the spontaneous onset of preterm parturition. Am J Obstet Gynecol 179:186–193, 1998.).

      4. FIRS is associated with a higher rate of neonatal morbidity and mortality

      We then tested the hypothesis of whether fetuses with systemic inflammation would have a higher rate of neonatal morbidity. Just as adult patients with SIRS are critically ill, we reasoned that fetuses with FIRS were more likely to present morbidity after birth. To address this question, we studied patients with preterm labor and intact membranes as well as those with preterm PROM. Severe neonatal morbidity was defined as the presence of respiratory distress syndrome, suspected or proven neonatal sepsis, pneumonia, bronchopulmonary dysplasia, intraventricular hemorrhage, or necrotizing enterocolitis. The presence of FIRS (fetal plasma IL-6 >11 pg/mL) conferred a higher rate of neonatal morbidity than in those without FIRS (77.8% vs. 29%; p < 0.001) (Fig. 3) [
      • Gomez R.
      • Romero R.
      • Ghezzi F.
      • Yoon B.H.
      • Mazor M.
      • Berry S.M.
      The fetal inflammatory response syndrome.
      ]. Multivariate analysis showed that FIRS was an independent predictor of severe neonatal morbidity after adjusting for gestational age, the obstetrical cause of preterm delivery (preterm labor or preterm PROM), clinical chorioamnionitis, presence of microorganisms in the amniotic cavity, and amniotic fluid IL-6 results. Importantly, the determination of the presence/absence of fetal systemic inflammation was done before delivery, thus the findings cannot be attributed to an intrapartum phenomenon.
      Fig. 3
      Fig. 3Fetal inflammatory response syndrome (FIRS) was associated with a higher severe neonatal morbidity than the absence of FIRS. FIRS was defined as a fetal plasma IL-6 >11 pg/mL. This was calculated using logistic regression to adjust for gestational age and other covariates. (Reproduced and modified with permission from Gomez R, Romero R, Ghezzi F et al.: The fetal inflammatory response syndrome. Am J Obstet Gynecol 179:194–202, 1998.).

      5. The clinical significance of FIRS

      A systematic review and meta-analysis of observational studies including 1116 neonates has shown that FIRS was associated with a higher frequency of adverse outcomes – specifically, early-onset sepsis (RR = 3.1), bronchopulmonary dysplasia (RR = 5.9), intraventricular hemorrhage (RR = 4.9), periventricular leukomalacia (RR = 3.3), respiratory distress syndrome (RR = 2.4), and neonatal death (RR = 7.0), when compared to neonates without FIRS [
      • Tang Q.
      • Zhang L.
      • Li H.
      • Shao Y.
      The fetal inflammation response syndrome and adverse neonatal outcomes: a meta-analysis.
      ]. In preterm neonates, FIRS was significantly and independently associated with an increased risk of retinopathy of prematurity and its progression [
      • Park Y.J.
      • Woo S.J.
      • Kim Y.M.
      • Hong S.
      • Lee Y.E.
      • Park K.H.
      Immune and inflammatory proteins in cord blood as predictive biomarkers of retinopathy of prematurity in preterm infants.
      ]. Moreover, FIRS is also associated with a neonatal systemic inflammatory response, which manifests as clinically suspected neonatal sepsis with negative blood and cerebrospinal fluid cultures [
      • Andrews W.W.
      • Goldenberg R.L.
      • Faye-Petersen O.
      • Cliver S.
      • Goepfert A.R.
      • Hauth J.C.
      The Alabama Preterm Birth study: polymorphonuclear and mononuclear cell placental infiltrations, other markers of inflammation, and outcomes in 23- to 32-week preterm newborn infants.
      ,
      • Goldenberg R.L.
      • Andrews W.W.
      • Faye-Petersen O.M.
      • Cliver S.P.
      • Goepfert A.R.
      • Hauth J.C.
      The Alabama preterm birth study: corticosteroids and neonatal outcomes in 23- to 32-week newborns with various markers of intrauterine infection.
      ,
      • Goldenberg R.L.
      • Andrews W.W.
      • Goepfert A.R.
      • Faye-Petersen O.
      • Cliver S.P.
      • Carlo W.A.
      • et al.
      The Alabama Preterm Birth Study: umbilical cord blood Ureaplasma urealyticum and Mycoplasma hominis cultures in very preterm newborn infants.
      ]. Future studies using a uniform definition of FIRS would strengthen the accuracy of these observations.

      6. Evidence of multi-systemic involvement in FIRS

      We had predicted that the fetus with FIRS will have evidence of multisystemic involvement in utero or in the immediate neonatal period (Fig. 4). However, there are important limitations to the study of this hypothesis in humans. Improved understanding of FIRS comes from studies in animal models—in particular, the pioneering contributions of the laboratories of Kallapur, Jobe, Adams, and Waldorf in the United States, Newnham in Australia, Kramer in the Netherlands, and Hallman in Finland.
      Fig. 4
      Fig. 4The fetal inflammatory response syndrome (FIRS) is associated with multi-systemic involvement. Clinical and/or experimental evidence suggests that there is involvement of the organs displayed in the figure. The data to support this conclusion is reviewed in the article. (Modified with permission from Gotsch F, Romero R, Kusanovic JP, Mazaki-Tovi S, Pineles B, Erez O, Espinoza J, Hassan SS. Fetal Inflammatory Response Syndrome. Clinical Obstetrics and Gynecology 50:652–683; 2007).

      6.1 Hematopoietic system

      Neutrophils, a major component of the innate immune system, play a critical role in the host response against infection and other insults. During human fetal development, neutrophils first appear in the clavicular bone marrow at 12–13 weeks of gestation [
      • Slayton W.B.
      • Li Y.
      • Calhoun D.A.
      • Juul S.E.
      • Iturraspe J.
      • Braylan R.C.
      • et al.
      The first-appearance of neutrophils in the human fetal bone marrow cavity.
      ]. However, lymphocytes are the predominant circulating white blood cells in the preterm fetus [
      • De Waele M.
      • Foulon W.
      • Renmans W.
      • Segers E.
      • Smet L.
      • Jochmans K.
      • et al.
      Hematologic values and lymphocyte subsets in fetal blood.
      ]. After 32 weeks of gestation, the proportion of neutrophils increases in fetal blood to become the predominant leukocyte at term [
      • De Waele M.
      • Foulon W.
      • Renmans W.
      • Segers E.
      • Smet L.
      • Jochmans K.
      • et al.
      Hematologic values and lymphocyte subsets in fetal blood.
      ,
      • Davies N.P.
      • Buggins A.G.
      • Snijders R.J.
      • Jenkins E.
      • Layton D.M.
      • Nicolaides K.H.
      Blood leucocyte count in the human fetus.
      ].
      The initial study of the hematologic profile in FIRS reported that affected fetuses had a higher median corrected white blood cell count and corrected neutrophil count than unaffected fetuses. Neutrophilia (neutrophil count >95th percentile for gestational age) was found in 71% (30/42) of cases, while neutropenia (neutrophil count <5th percentile for gestational age) was present in 4.8% (2/42) [
      • Romero R.
      • Savasan Z.A.
      • Chaiworapongsa T.
      • Berry S.M.
      • Kusanovic J.P.
      • Hassan S.S.
      • et al.
      Hematologic profile of the fetus with systemic inflammatory response syndrome.
      ]. No detectable changes in lymphocyte, monocyte, basophil or eosinophil counts were observed in the initial report.
      In a subsequent study of preterm neonates born after spontaneous preterm labor and preterm PROM, Kim et al. reported the hematologic profiles in umbilical cord blood. FIRS was defined as the presence of funisitis, and the authors reported that preterm neonates with funisitis had significantly higher rates of neutrophilia (>95th percentile for gestational age) [93.2% (41/44) vs. 77.8% (119/153); p = 0.027] and monocytosis (>95th percentile for gestational age) [81.8% (36/44) vs. 64.1% (98/153); p = 0.026] than those without funisitis [
      • Kim E.N.
      • Kim C.J.
      • Park J.W.
      • Yoon B.H.
      Acute funisitis is associated with distinct changes in fetal hematologic profile.
      ].
      In addition to the change in number, FIRS (defined by the presence of funisitis) has also been associated with phenotypic evidence of monocyte and granulocyte activation [
      • Berry S.M.
      • Romero R.
      • Gomez R.
      • Puder K.S.
      • Ghezzi F.
      • Cotton D.B.
      • et al.
      Premature parturition is characterized by in utero activation of the fetal immune system.
      ]. Indeed, the umbilical cord blood of neonates with acute funisitis had a higher mean channel brightness for CD14 and CD64 on granulocytes and of CD64 on monocytes and a higher basal level of intracellular reactive oxygen species and oxidative burst in monocytes [
      • Kim S.K.
      • Romero R.
      • Chaiworapongsa T.
      • Kusanovic J.P.
      • Mazaki-Tovi S.
      • Mittal P.
      • et al.
      Evidence of changes in the immunophenotype and metabolic characteristics (intracellular reactive oxygen radicals) of fetal, but not maternal, monocytes and granulocytes in the fetal inflammatory response syndrome.
      ].
      The mechanisms responsible for the quantitative and qualitative changes in neutrophils in the presence of FIRS have not been elucidated. However, the concentration of granulocyte colony stimulating factor (G-CSF), a cytokine and major physiologic regulator of neutrophil production released during stressful conditions, as well as the activation of neutrophils [
      • Nicola N.A.
      Granulocyte colony stimulating factor.
      ,
      • Uzumaki H.
      • Okabe T.
      • Sasaki N.
      • Hagiwara K.
      • Takaku F.
      • Tobita M.
      • et al.
      Identification and characterization of receptors for granulocyte colony-stimulating factor on human placenta and trophoblastic cells.
      ,
      • Demetri G.D.
      • Griffin J.D.
      Granulocyte colony-stimulating factor and its receptor.
      ], is higher in fetuses with FIRS compared to those without FIRS [
      • Chaiworapongsa T.
      • Romero R.
      • Berry S.M.
      • Hassan S.S.
      • Yoon B.H.
      • Edwin S.
      • et al.
      The role of granulocyte colony-stimulating factor in the neutrophilia observed in the fetal inflammatory response syndrome.
      ].
      Changes in the red blood cell lineage in FIRS (defined by an elevated fetal plasma IL-6 concentration) have also been observed. Affected fetuses have a slightly higher median nucleated red blood cell count (adjusted for gestational age) compared to those without FIRS [
      • Romero R.
      • Savasan Z.A.
      • Chaiworapongsa T.
      • Berry S.M.
      • Kusanovic J.P.
      • Hassan S.S.
      • et al.
      Hematologic profile of the fetus with systemic inflammatory response syndrome.
      ]. An elevated nucleated red blood cell count has also been observed in neonates delivered from patients with prolonged (>24 h) rupture of the fetal membranes [
      • Mandel D.
      • Oron T.
      • Mimouni G.S.
      • Littner Y.
      • Dollberg S.
      • Mimouni F.B.
      The effect of prolonged rupture of membranes on circulating neonatal nucleated red blood cells.
      ], histologic chorioamnionitis [
      • Leikin E.
      • Garry D.
      • Visintainer P.
      • Verma U.
      • Tejani N.
      Correlation of neonatal nucleated red blood cell counts in preterm infants with histologic chorioamnionitis.
      ], a high acute placental inflammation score [
      • Salafia C.M.
      • Ghidini A.
      • Pezzullo J.C.
      • Rosenkrantz T.S.
      Early neonatal nucleated erythrocyte counts in preterm deliveries: clinical and pathologic correlations.
      ], and early-onset neonatal sepsis [
      • Dulay A.T.
      • Buhimschi I.A.
      • Zhao G.
      • Luo G.
      • bdel-Razeq S.
      • Cackovic M.
      • et al.
      Nucleated red blood cells are a direct response to mediators of inflammation in newborns with early-onset neonatal sepsis.
      ]. Since changes in nucleated red blood cells in FIRS are not associated with a lower pH or PO2 [
      • Romero R.
      • Soto E.
      • Berry S.M.
      • Hassan S.S.
      • Kusanovic J.P.
      • Yoon B.H.
      • et al.
      Blood pH and gases in fetuses in preterm labor with and without systemic inflammatory response syndrome.
      ], metabolic acidemia/hypoxemia is unlikely to be the cause of the observed changes [
      • Baschat A.A.
      • Gembruch U.
      • Reiss I.
      • Gortner L.
      • Harman C.R.
      • Weiner C.P.
      Neonatal nucleated red blood cell counts in growth-restricted fetuses: relationship to arterial and venous Doppler studies.
      ]. Given that the IL-6 concentration in umbilical cord blood correlates with the nucleated red blood cell count [
      • Ferber A.
      • Minior V.K.
      • Bornstein E.
      • Divon M.Y.
      Fetal "nonreassuring status" is associated with elevation of nucleated red blood cell counts and interleukin-6.
      ], IL-6 may mediate these changes [
      • Ulich T.R.
      • del Castillo J.
      • Guo K.Z.
      In vivo hematologic effects of recombinant interleukin-6 on hematopoiesis and circulating numbers of RBCs and WBCs.
      ,
      • Spiropoulos A.
      • Goussetis E.
      • Margeli A.
      • Premetis E.
      • Skenderi K.
      • Graphakos S.
      • et al.
      Effect of inflammation induced by prolonged exercise on circulating erythroid progenitors and markers of erythropoiesis.
      ], either directly or indirectly, through erythropoietin. The latter possibility must be considered since the serum erythropoietin concentration is higher in newborns with funisitis than in those without funisitis [
      • Maier R.F.
      • Gunther A.
      • Vogel M.
      • Dudenhausen J.W.
      • Obladen M.
      Umbilical venous erythropoietin and umbilical arterial pH in relation to morphologic placental abnormalities.
      ].
      Experimental fetal systemic inflammation induced by the administration of IL-1α, an alarmin, into the amniotic cavity of pregnant sheep has been associated with a decreased number of circulating neutrophils within 24 h (early response), followed by an increase in neutrophil numbers at 3 and 7 days (late response) [
      • Kallapur S.G.
      • Kramer B.W.
      • Nitsos I.
      • Pillow J.J.
      • Collins J.J.
      • Polglase G.R.
      • et al.
      Pulmonary and systemic inflammatory responses to intra-amniotic IL-1alpha in fetal sheep.
      ]. Fetal systemic inflammation induced by exposure to bacterial endotoxin in ewes is associated with an increased number of circulating neutrophils 7 days after exposure [
      • Kramer B.W.
      • Moss T.J.
      • Willet K.E.
      • Newnham J.P.
      • Sly P.D.
      • Kallapur S.G.
      • et al.
      Dose and time response after intraamniotic endotoxin in preterm lambs.
      ]. Consistent with these findings, preterm pigs exposed to intra-amniotic bacterial endotoxin for 3 days before delivery also demonstrated increased blood neutrophil counts at birth [
      • Nguyen D.N.
      • Thymann T.
      • Goericke-Pesch S.K.
      • Ren S.
      • Wei W.
      • Skovgaard K.
      • et al.
      Prenatal intra-amniotic endotoxin induces fetal gut and lung immune responses and postnatal systemic inflammation in preterm pigs.
      ]. It is possible that the frequency of neutrophilia in human umbilical cord blood reflects at least, in part, the duration of exposure to an inflammatory stimulus.

      6.2 Thymus

      Thymic involution occurs after infection in the fetus and neonate [
      • De Felice C.
      • Toti P.
      • Santopietro R.
      • Stumpo M.
      • Pecciarini L.
      • Bagnoli F.
      Small thymus in very low birth weight infants born to mothers with subclinical chorioamnionitis.
      ,
      • Toti P.
      • De Felice C.
      • Stumpo M.
      • Schürfeld K.
      • Di Leo L.
      • Vatti R.
      • et al.
      Acute thymic involution in fetuses and neonates with chorioamnionitis.
      ,
      • Glavina-Durdov M.
      • Springer O.
      • Capkun V.
      • Saratlija-Novaković Z.
      • Rozić D.
      • Barle M.
      The grade of acute thymus involution in neonates correlates with the duration of acute illness and with the percentage of lymphocytes in peripheral blood smear. Pathological study.
      ,
      • Yinon Y.
      • Zalel Y.
      • Weisz B.
      • Mazaki-Tovi S.
      • Sivan E.
      • Schiff E.
      • et al.
      Fetal thymus size as a predictor of chorioamnionitis in women with preterm premature rupture of membranes.
      ,
      • El-Haieg D.O.
      • Zidan A.A.
      • El-Nemr M.M.
      The relationship between sonographic fetal thymus size and the components of the systemic fetal inflammatory response syndrome in women with preterm prelabour rupture of membranes.
      ,
      • Musilova I.
      • Hornychova H.
      • Kostal M.
      • Jacobsson B.
      • Kacerovsky M.
      Ultrasound measurement of the transverse diameter of the fetal thymus in pregnancies complicated by the preterm prelabor rupture of membranes.
      ]. Subclinical chorioamnionitis has been associated with a small thymus in very-low-birth-weight infants [
      • De Felice C.
      • Toti P.
      • Santopietro R.
      • Stumpo M.
      • Pecciarini L.
      • Bagnoli F.
      Small thymus in very low birth weight infants born to mothers with subclinical chorioamnionitis.
      ], possibly from acute fetal [
      • Toti P.
      • De Felice C.
      • Stumpo M.
      • Schürfeld K.
      • Di Leo L.
      • Vatti R.
      • et al.
      Acute thymic involution in fetuses and neonates with chorioamnionitis.
      ] and neonatal [
      • Toti P.
      • De Felice C.
      • Stumpo M.
      • Schürfeld K.
      • Di Leo L.
      • Vatti R.
      • et al.
      Acute thymic involution in fetuses and neonates with chorioamnionitis.
      ,
      • Glavina-Durdov M.
      • Springer O.
      • Capkun V.
      • Saratlija-Novaković Z.
      • Rozić D.
      • Barle M.
      The grade of acute thymus involution in neonates correlates with the duration of acute illness and with the percentage of lymphocytes in peripheral blood smear. Pathological study.
      ] involution. In neonates, thymic involution correlates with the duration of acute illness and with the percentage of lymphocytes in peripheral blood [
      • Glavina-Durdov M.
      • Springer O.
      • Capkun V.
      • Saratlija-Novaković Z.
      • Rozić D.
      • Barle M.
      The grade of acute thymus involution in neonates correlates with the duration of acute illness and with the percentage of lymphocytes in peripheral blood smear. Pathological study.
      ]. The proposed mechanism for thymic involution is thought to be stress (via glucocorticoid production) and the effects of proinflammatory cytokines [
      • Morrissey P.J.
      • Charrier K.
      • Alpert A.
      • Bressler L.
      In vivo administration of IL-1 induces thymic hypoplasia and increased levels of serum corticosterone.
      ,
      • Gruver A.L.
      • Sempowski G.D.
      Cytokines, leptin, and stress-induced thymic atrophy.
      ,
      • Billard M.J.
      • Gruver A.L.
      • Sempowski G.D.
      Acute endotoxin-induced thymic atrophy is characterized by intrathymic inflammatory and wound healing responses.
      ].
      A sonographically small thymus for gestational age has been reported by Di Naro et al. [
      • Di Naro E.
      • Cromi A.
      • Ghezzi F.
      • Raio L.
      • Uccella S.
      • D'Addario V.
      • et al.
      Fetal thymic involution: a sonographic marker of the fetal inflammatory response syndrome.
      ] and other investigators [
      • Yinon Y.
      • Zalel Y.
      • Weisz B.
      • Mazaki-Tovi S.
      • Sivan E.
      • Schiff E.
      • et al.
      Fetal thymus size as a predictor of chorioamnionitis in women with preterm premature rupture of membranes.
      ,
      • El-Haieg D.O.
      • Zidan A.A.
      • El-Nemr M.M.
      The relationship between sonographic fetal thymus size and the components of the systemic fetal inflammatory response syndrome in women with preterm prelabour rupture of membranes.
      ,
      • Musilova I.
      • Hornychova H.
      • Kostal M.
      • Jacobsson B.
      • Kacerovsky M.
      Ultrasound measurement of the transverse diameter of the fetal thymus in pregnancies complicated by the preterm prelabor rupture of membranes.
      ,
      • Sciaky-Tamir Y.
      • Hershkovitz R.
      • Mazor M.
      • Shelef I.
      • Erez O.
      The use of imaging technology in the assessment of the fetal inflammatory response syndrome-imaging of the fetal thymus.
      ] in the presence of intra-amniotic infection and inflammation (preterm labor and intact membranes or preterm PROM). Another study reported that infants born preterm (<28 weeks of gestation) with sonographic signs of cerebral white matter damage have thymic involution more frequently than a control group matched for gestational age [
      • Kuban J.D.
      • Allred E.N.
      • Leviton A.
      Thymus involution and cerebral white matter damage in extremely low gestational age neonates.
      ]. This has been interpreted as indicating that neurologic injury and thymic involution may have a common origin, namely fetal systemic inflammation.
      Intra-amniotic administration of bacterial endotoxin in sheep results in both decreased thymic weight and corticomedullary ratio. The effects can be observed as early as 5 h after exposure to endotoxin [
      • Kunzmann S.
      • Glogger K.
      • Been J.V.
      • Kallapur S.G.
      • Nitsos I.
      • Moss T.J.
      • et al.
      Thymic changes after chorioamnionitis induced by intraamniotic lipopolysaccharide in fetal sheep.
      ,
      • Kuypers E.
      • Wolfs T.G.
      • Collins J.J.
      • Jellema R.K.
      • Newnham J.P.
      • Kemp M.W.
      • et al.
      Intraamniotic lipopolysaccharide exposure changes cell populations and structure of the ovine fetal thymus.
      ]. Increased expression of IL-6 and type I interferons, as well as glucocorticoids, has been implicated in thymic involution [
      • Kuypers E.
      • Wolfs T.G.
      • Collins J.J.
      • Jellema R.K.
      • Newnham J.P.
      • Kemp M.W.
      • et al.
      Intraamniotic lipopolysaccharide exposure changes cell populations and structure of the ovine fetal thymus.
      ,
      • Tarcic N.
      • Ovadia H.
      • Weiss D.W.
      • Weidenfeld J.
      Restraint stress-induced thymic involution and cell apoptosis are dependent on endogenous glucocorticoids.
      ,
      • Sempowski G.D.
      • Rhein M.E.
      • Scearce R.M.
      • Haynes B.F.
      Leukemia inhibitory factor is a mediator of Escherichia coli lipopolysaccharide-induced acute thymic atrophy.
      ,
      • Démoulins T.
      • Abdallah A.
      • Kettaf N.
      • Baron M.L.
      • Gerarduzzi C.
      • Gauchat D.
      • et al.
      Reversible blockade of thymic output: an inherent part of TLR ligand-mediated immune response.
      ,
      • Anz D.
      • Thaler R.
      • Stephan N.
      • Waibler Z.
      • Trauscheid M.J.
      • Scholz C.
      • et al.
      Activation of melanoma differentiation-associated gene 5 causes rapid involution of the thymus.
      ]. In addition, an increase in the percentage of CD3+ cells, but a decrease in the percentage of CD8+ (cytotoxic lymphocytes) and FOXP3+ cells, occurs after exposure to endotoxin [
      • Kunzmann S.
      • Glogger K.
      • Been J.V.
      • Kallapur S.G.
      • Nitsos I.
      • Moss T.J.
      • et al.
      Thymic changes after chorioamnionitis induced by intraamniotic lipopolysaccharide in fetal sheep.
      ,
      • Kuypers E.
      • Wolfs T.G.
      • Collins J.J.
      • Jellema R.K.
      • Newnham J.P.
      • Kemp M.W.
      • et al.
      Intraamniotic lipopolysaccharide exposure changes cell populations and structure of the ovine fetal thymus.
      ,
      • Melville J.M.
      • Bischof R.J.
      • Meeusen E.N.
      • Westover A.J.
      • Moss T.J.
      Changes in fetal thymic immune cell populations in a sheep model of intrauterine inflammation.
      ]. Such findings suggest that endotoxin exposure has an effect not only on the innate but also on the adaptive immune response. Taken together, FIRS can induce structural, functional, and immunologic changes in the thymus.
      Activation of T cells (belonging to the adaptive limb of the immune response) has been observed in term and preterm infants born to mothers with clinical chorioamnionitis. Such infants demonstrate overexpression of CD25, HLA-DR, or CD69 markers in T cells [
      • Luciano A.A.
      • Yu H.
      • Jackson L.W.
      • Wolfe L.A.
      • Bernstein H.B.
      Preterm labor and chorioamnionitis are associated with neonatal T cell activation.
      ], a higher number of memory T cells (CD45RO+), and a decreased number of naive T cells [
      • Luciano A.A.
      • Yu H.
      • Jackson L.W.
      • Wolfe L.A.
      • Bernstein H.B.
      Preterm labor and chorioamnionitis are associated with neonatal T cell activation.
      ,
      • Brüning T.
      • Daiminger A.
      • Enders G.
      Diagnostic value of CD45RO expression on circulating T lymphocytes of fetuses and newborn infants with pre-, peri- or early post-natal infections.
      ,
      • Peoples J.D.
      • Cheung S.
      • Nesin M.
      • Lin H.
      • Tatad A.M.
      • Hoang D.
      • et al.
      Neonatal cord blood subsets and cytokine response to bacterial antigens.
      ,
      • Matsuda Y.
      • Kato H.
      • Imanishi K.
      • Mitani M.
      • Ohta H.
      • Uchiyama T.
      T cell activation in abnormal perinatal events.
      ]. Further studies are required to comprehensively describe changes in cells involved in adaptive immunity in the presence of fetal systemic inflammation from different etiologies (e.g., bacterial and viral infections, non-infection related causes) and at varying gestational ages (i.e., preterm and term).
      It is noteworthy that studies of fetal rhesus macaques report that the number of T regulatory cells (FOXP3+) is decreased, while there is an increase in the number of IL-17 (pro-inflammatory cytokine) producing cells in lymphoid tissues after intra-amniotic administration of IL-1β [
      • Kallapur S.G.
      • Presicce P.
      • Senthamaraikannan P.
      • Alvarez M.
      • Tarantal A.F.
      • Miller L.M.
      • et al.
      Intra-amniotic IL-1β induces fetal inflammation in rhesus monkeys and alters the regulatory T cell/IL-17 balance.
      ]. However, these changes were not observed in fetal blood [
      • Kallapur S.G.
      • Presicce P.
      • Senthamaraikannan P.
      • Alvarez M.
      • Tarantal A.F.
      • Miller L.M.
      • et al.
      Intra-amniotic IL-1β induces fetal inflammation in rhesus monkeys and alters the regulatory T cell/IL-17 balance.
      ].

      6.3 Spleen

      The spleen is the largest secondary lymphoid organ, and plays an important role in immunological processes, hematopoiesis, and red blood cell clearance [
      • Lewis S.M.
      • Williams A.
      • Eisenbarth S.C.
      Structure and function of the immune system in the spleen.
      ]. The unique physical organization of the organ is thought to facilitate filtering of microorganisms and abnormal cells [
      • Lewis S.M.
      • Williams A.
      • Eisenbarth S.C.
      Structure and function of the immune system in the spleen.
      ,
      • Bronte V.
      • Pittet M.J.
      The spleen in local and systemic regulation of immunity.
      ]. In adults affected by sepsis, there is a loss of B and CD4 T cells in the spleen, and similar findings have been reported in fetuses and neonates. In a study of 20 fetuses exposed to intra-amniotic infection/inflammation (diagnosed by acute histologic chorioamnionitis or funisitis), 10 neonates died of proven sepsis and showed splenic depletion [
      • Toti P.
      • De Felice C.
      • Occhini R.
      • Schuerfeld K.
      • Stumpo M.
      • Epistolato M.C.
      • et al.
      Spleen depletion in neonatal sepsis and chorioamnionitis.
      ]. Indeed, the percentage of red and white pulp areas occupied by CD45RA+, CD45RO+, and CD20+ cells was reduced [
      • Toti P.
      • De Felice C.
      • Occhini R.
      • Schuerfeld K.
      • Stumpo M.
      • Epistolato M.C.
      • et al.
      Spleen depletion in neonatal sepsis and chorioamnionitis.
      ].
      Changes in the fetal splenic circulation have also been observed in the context of intra-amniotic infection/inflammation [
      • Musilova I.
      • Kacerovsky M.
      • Andrys C.
      • Kostal M.
      • Slaba K.
      • Jacobsson B.
      The fetal splenic vein flow pattern and fetal inflammatory response in the preterm prelabor rupture of membranes.
      ,
      • Musilova I.
      • Spacek R.
      • Stranik J.
      • Jacobsson B.
      • Kacerovsky M.
      Fetal portal system flowmetry and intra-amniotic inflammation in preterm prelabor rupture of membranes.
      ]. Under normal circumstances, the fetal splenic vein has a non-pulsatile Doppler waveform. However, Musilova et al. reported a pulsatile flow pattern in the fetal splenic vein in 84% (16/19) of cases with funisitis but in only 15% (9/60) of cases without fetal inflammation [
      • Musilova I.
      • Kacerovsky M.
      • Hornychova H.
      • Kostal M.
      • Jacobsson B.
      Pulsation of the fetal splenic vein--a potential ultrasound marker of histological chorioamnionitis and funisitis in women with preterm prelabor rupture of membranes.
      ]. In addition, fetuses with a pulsatile splenic vein flow pattern exhibited a higher concentration of umbilical cord blood IL-6 than those with continuous flow [median 56.7 pg/mL versus 5.6 pg/mL; p < 0.0001] [
      • Musilova I.
      • Kacerovsky M.
      • Andrys C.
      • Kostal M.
      • Slaba K.
      • Jacobsson B.
      The fetal splenic vein flow pattern and fetal inflammatory response in the preterm prelabor rupture of membranes.
      ]. Collectively, this suggests that non-invasive interrogation of the fetal circulation may help to identify the fetus with systemic inflammation. The mechanism whereby pulsatile flow in the splenic vein is generated is thought to result from increased intrasplenic pressure secondary to microbial product-induced impairment of splenic drainage into the lymphatic circulation.
      Several experimental studies have examined the effect of cytokines and bacterial endotoxin on the fetal spleen [
      • Kramer B.W.
      • Moss T.J.
      • Willet K.E.
      • Newnham J.P.
      • Sly P.D.
      • Kallapur S.G.
      • et al.
      Dose and time response after intraamniotic endotoxin in preterm lambs.
      ,
      • Kuypers E.
      • Willems M.G.
      • Jellema R.K.
      • Kemp M.W.
      • Newnham J.P.
      • Delhaas T.
      • et al.
      Responses of the spleen to intraamniotic lipopolysaccharide exposure in fetal sheep.
      ]. Kramer et al. demonstrated a two-fold increase in TNF-α expression in the fetal spleen 24 h after intra-amniotic injection of LPS [
      • Kramer B.W.
      • Moss T.J.
      • Willet K.E.
      • Newnham J.P.
      • Sly P.D.
      • Kallapur S.G.
      • et al.
      Dose and time response after intraamniotic endotoxin in preterm lambs.
      ]. A subsequent study showed that intra-amniotic LPS-induced changes in the splenic cytokine profile and increased CD3 expression, a marker for T cells [
      • Kuypers E.
      • Willems M.G.
      • Jellema R.K.
      • Kemp M.W.
      • Newnham J.P.
      • Delhaas T.
      • et al.
      Responses of the spleen to intraamniotic lipopolysaccharide exposure in fetal sheep.
      ]. Cytokine changes in the fetal spleen have been detected as early as 5 h after the injection of LPS and for up to 15 days after the onset of inflammation, indicating that intrauterine exposure to an inflammatory stimulus caused a rapid and sustained response in the fetal spleen [
      • Kuypers E.
      • Willems M.G.
      • Jellema R.K.
      • Kemp M.W.
      • Newnham J.P.
      • Delhaas T.
      • et al.
      Responses of the spleen to intraamniotic lipopolysaccharide exposure in fetal sheep.
      ]. Taken together, the available evidence suggests that intra-amniotic infection or intra-amniotic inflammation can induce changes in the developing fetal spleen. The long-term consequences of these changes in the spleen require investigation.

      6.4 Adrenal glands

      The adrenal glands are involved in the response to sepsis and systemic inflammation. Adult patients admitted to the intensive care unit with sterile inflammatory processes, such as burns or pancreatitis, present endocrine evidence of stress, as indicated by an elevation in the cortisol and dehydroepiandrosterone sulfate ratio [
      • Yoon B.H.
      • Romero R.
      • Jun J.K.
      • 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.
      ]. The same has been reported in the human fetus [
      • Yoon B.H.
      • Romero R.
      • Jun J.K.
      • 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.
      ] and has been implicated in the mechanisms for preterm labor. Specifically, Yoon et al. reported a significant association between the fetal plasma cortisol and dehydroepiandrosterone sulfate ratio and pregnancy duration in patients with preterm PROM (hazard ratio 2.9; 95% CI: 1 to 8.4) [
      • Yoon B.H.
      • Romero R.
      • Jun J.K.
      • 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.
      ]. Patients with preterm PROM who went into spontaneous labor and delivered within 7 days of sampling had a significantly higher median fetal plasma concentration of cortisol than those who delivered after 7 days (p < 0.0001). Indeed, a fetal plasma cortisol, but not a maternal cortisol, concentration was an independent predictor of pregnancy duration after adjusting for both gestational age and the results of amniotic fluid cultures (hazard ratio 2.9; 95% CI 1.3 to 6.7). Interestingly, there is a significant correlation between fetal plasma cortisol and IL-6 concentrations (r = 0.3; p < 0.05) [
      • Yoon B.H.
      • Romero R.
      • Jun J.K.
      • 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.
      ]. Such endocrine changes may have short- and long-term implications given observations about the effects of glucocorticoids in fetal programming of several metabolic functions [
      • Seckl J.R.
      • Cleasby M.
      • Nyirenda M.J.
      Glucocorticoids, 11beta-hydroxysteroid dehydrogenase, and fetal programming.
      ,
      • Nathanielsz P.W.
      • Berghorn K.A.
      • Derks J.B.
      • Giussani D.A.
      • Docherty C.
      • Unno N.
      • et al.
      Life before birth: effects of cortisol on future cardiovascular and metabolic function.
      ,
      • Seckl J.R.
      • Meaney M.J.
      Glucocorticoid programming.
      ,
      • Moritz K.M.
      • Boon W.M.
      • Wintour E.M.
      Glucocorticoid programming of adult disease.
      ,
      • Seckl J.R.
      • Holmes M.C.
      Mechanisms of disease: glucocorticoids, their placental metabolism and fetal 'programming' of adult pathophysiology.
      ].

      6.5 Thyroid

      Transient hypothyroxinemia of prematurity occurs in 30–80% of preterm infants (<30 weeks of gestation) and is characterized by a transient reduction in the serum concentrations of T4 and T3 as well as a low or normal TSH [
      • Williams F.L.
      • Visser T.J.
      • Hume R.
      Transient hypothyroxinaemia in preterm infants.
      ]. De Felice et al. reported that preterm neonates with acute histologic chorioamnionitis had a significantly lower T4 concentration on post-delivery day 4 in comparison to those without this placental lesion [
      • De Felice C.
      • Bagnoli F.
      • Toti P.
      • Musaro M.A.
      • Peruzzi L.
      • Paffetti P.
      • et al.
      Transient hypothyroxinemia of prematurity and histological chorioamnionitis.
      ]. Low T4 concentrations in preterm infants have been linked to persistent neurodevelopmental deficits in cognitive and motor function at 2 years of age [
      • Reuss M.L.
      • Paneth N.
      • Pinto-Martin J.A.
      • Lorenz J.M.
      • Susser M.
      The relation of transient hypothyroxinemia in preterm infants to neurologic development at two years of age.
      ]. The mechanism responsible for these findings has not been established. Sepsis in adults has been associated with lower concentrations of T3 (“low T3 syndrome”), and this is thought to result from apoptosis of thyroid cells [
      • Luo B.
      • Yu Z.
      • Li Y.
      Thyroid hormone disorders and sepsis.
      ,
      • Kim J.G.
      • Shin H.
      • Kim W.
      • Lim T.H.
      • Jang B.
      • Cho Y.
      • et al.
      The value of decreased thyroid hormone for predicting mortality in adult septic patients: a systematic review and meta-analysis.
      ].

      6.6 Lung

      Amniotic fluid is inhaled by the fetus and reaches the airways, as demonstrated by color Doppler ultrasound imaging [
      • Stephens J.D.
      • Birnholz J.C.
      Noninvasive verification of fetal respiratory movements in normal pregnancy.
      ,
      • Stephens J.D.
      • Birnholz J.C.
      Verification of human fetal breathing with phased array ultrasound imaging.
      ,
      • Isaacson G.
      • Birnholz J.C.
      Human fetal upper respiratory tract function as revealed by ultrasonography.
      ,
      • Kalache K.D.
      • Chaoui R.
      • Bollmann R.
      Doppler assessment of tracheal and nasal fluid flow during fetal breathing movements: preliminary observations.
      ]. Congenital pneumonia, which is frequently diagnosed in cases of fetal death, is thought to be secondary to fetal aspiration of microorganisms. Meconium may be detected in the alveoli in stillbirths, indicating that amniotic fluid can reach the alveoli [
      • Burgess A.M.
      • Hutchins G.M.
      Inflammation of the lungs, umbilical cord and placenta associated with meconium passage in utero. Review of 123 autopsied cases.
      ,
      • Mortensen E.
      • Kearney M.S.
      Meconium aspiration in the midtrimester fetus: an autopsy study.
      ]. Tracheobronchial fluid obtained from neonates via an endotracheal tube placed shortly after birth often shows the presence of white blood cells and microorganisms in patients affected by intra-amniotic infection [
      • Cassell G.H.
      • Waites K.B.
      • Crouse D.T.
      • Rudd P.T.
      • Canupp K.C.
      • Stagno S.
      • et al.
      Association of Ureaplasma urealyticum infection of the lower respiratory tract with chronic lung disease and death in very-low-birth-weight infants.
      ]. Interestingly, fetuses with intra-amniotic infection often have a dramatic decrease in fetal “breathing movements,” which may decrease the likelihood of bacterial entry into the lung [
      • Vintzileos A.M.
      • Campbell W.A.
      • Nochimson D.J.
      • Connolly M.E.
      • Fuenfer M.M.
      • Hoehn G.J.
      The fetal biophysical profile in patients with premature rupture of the membranes--an early predictor of fetal infection.
      ,
      • Vintzileos A.M.
      • Campbell W.A.
      • Nochimson D.J.
      • Weinbaum P.J.
      Fetal breathing as a predictor of infection in premature rupture of the membranes.
      ,
      • Goldstein I.
      • Romero R.
      • Merrill S.
      • Wan M.
      • O'Connor T.Z.
      • Mazor M.
      • et al.
      Fetal body and breathing movements as predictors of intraamniotic infection in preterm premature rupture of membranes.
      ,
      • Sherer D.M.
      • Spong C.Y.
      • Salafia C.M.
      Fetal breathing movements within 24 hours of delivery in prematurity are related to histologic and clinical evidence of amnionitis.
      ].
      The relationship between intra-amniotic inflammation/infection and the development of respiratory complications, such as respiratory distress syndrome (RDS) and chronic lung disease/bronchopulmonary dysplasia (BPD) has been the subject of intensive investigation [
      • Speer C.P.
      Inflammatory mechanisms in neonatal chronic lung disease.
      ,
      • Speer C.P.
      New insights into the pathogenesis of pulmonary inflammation in preterm infants.
      ,
      • Thebaud B.
      • Goss K.N.
      • Laughon M.
      • Whitsett J.A.
      • Abman S.H.
      • Steinhorn R.H.
      • et al.
      Bronchopulmonary dysplasia.
      ]. The Watterberg hypothesis proposes that intra-amniotic inflammation/infection is associated with a decreased rate of RDS (early protective effect) but an increased rate of BPD [
      • Watterberg K.L.
      • Demers L.M.
      • Scott S.M.
      • Murphy S.
      Chorioamnionitis and early lung inflammation in infants in whom bronchopulmonary dysplasia develops.
      ]. A systematic review and meta-analysis, which included 158 studies and 244,096 preterm infants, showed that there is a significant association between clinical and histologic chorioamnionitis and subsequent development of BPD [
      • Villamor-Martinez E.
      • Alvarez-Fuente M.
      • Ghazi A.M.T.
      • Degraeuwe P.
      • Zimmermann L.J.I.
      • Kramer B.W.
      • et al.
      Association of chorioamnionitis with bronchopulmonary dysplasia among preterm infants: a systematic review, meta-analysis, and metaregression.
      ]. However, chorioamnionitis was not a risk factor for the development of RDS. A multivariate meta-regression revealed that a model combining the difference in gestational age and the odds of RDS explained 64% of the variance in the association between chorioamnionitis and BPD (36 weeks’ post-menstrual age) across studies [
      • Villamor-Martinez E.
      • Alvarez-Fuente M.
      • Ghazi A.M.T.
      • Degraeuwe P.
      • Zimmermann L.J.I.
      • Kramer B.W.
      • et al.
      Association of chorioamnionitis with bronchopulmonary dysplasia among preterm infants: a systematic review, meta-analysis, and metaregression.
      ].
      Watterberg et al. reported that low-birth-weight infants exposed to chorioamnionitis had higher concentrations of IL-1β in tracheal lavage samples and a lower incidence of RDS, but a higher rate of BPD in comparison to the control group [
      • Watterberg K.L.
      • Demers L.M.
      • Scott S.M.
      • Murphy S.
      Chorioamnionitis and early lung inflammation in infants in whom bronchopulmonary dysplasia develops.
      ]. Ghezzi et al. reported that IL-8 concentrations in amniotic fluid were higher in women who presented with spontaneous preterm labor (intact or ruptured membranes) and delivered infants at 24–28 weeks of gestation who later developed BPD [
      • Ghezzi F.
      • Gomez R.
      • Romero R.
      • Yoon B.H.
      • Edwin S.S.
      • David C.
      • et al.
      Elevated interleukin-8 concentrations in amniotic fluid of mothers whose neonates subsequently develop bronchopulmonary dysplasia.
      ]. Indeed, preterm neonates born at 33 weeks of gestation or earlier who developed BPD had median amniotic fluid concentrations of IL-1β and IL-8 that were significantly higher than those in whom BPD did not develop [
      • Yoon B.H.
      • Romero R.
      • Jun J.K.
      • Park K.H.
      • Park J.D.
      • Ghezzi F.
      • et al.
      Amniotic fluid cytokines (interleukin-6, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-8) and the risk for the development of bronchopulmonary dysplasia.
      ]. Collectively, the evidence suggests that exposure to intra-amniotic inflammation is a risk factor for BPD.
      Subsequently, Yoon et al. reported that neonates born between 25 and 34 weeks of gestation and in whom BPD developed had a significantly higher median umbilical cord plasma IL-6 concentration in comparison to matched preterm infants without BPD. This indicates that the risk for BPD is higher in the offspring of pregnancies in which there is intra-amniotic inflammation and fetal systemic inflammation [
      • Yoon B.H.
      • Romero R.
      • Kim K.S.
      • Park J.S.
      • Ki S.H.
      • Kim B.I.
      • et al.
      A systemic fetal inflammatory response and the development of bronchopulmonary dysplasia.
      ].
      This set of clinical observations led to systematic investigation of the effect of microbial products and pro-inflammatory cytokines on fetal lung development. The models include intra-amniotic administration of endotoxin, administration of IL-1β, or the inoculation of bacteria in the amniotic cavity. The major findings have been that inflammation stimulates surfactant protein production but alters lung development [
      • Jobe A.H.
      • Newnham J.P.
      • Willet K.E.
      • Sly P.
      • Ervin M.G.
      • Bachurski C.
      • et al.
      Effects of antenatal endotoxin and glucocorticoids on the lungs of preterm lambs.
      ,
      • Kallapur S.G.
      • Willet K.E.
      • Jobe A.H.
      • Ikegami M.
      • Bachurski C.J.
      Intra-amniotic endotoxin: chorioamnionitis precedes lung maturation in preterm lambs.
      ,
      • Moss T.J.
      • Nitsos I.
      • Ikegami M.
      • Jobe A.H.
      • Newnham J.P.
      Experimental intrauterine Ureaplasma infection in sheep.
      ].
      Bry et al. first reported that intra-amniotic administration of IL-1α to pregnant rabbits increased mRNA and protein expression of surfactant proteins A and B, as well as surfactant lipids [
      • Bry K.
      • Lappalainen U.
      • Hallman M.
      Intraamniotic interleukin-1 accelerates surfactant protein synthesis in fetal rabbits and improves lung stability after premature birth.
      ]. This was accompanied by improved neonatal lung function. Injection of IL-1α also induced preterm labor and delivery; therefore, the effect of inflammation on surfactant production could be interpreted as promoting lung maturation in anticipation of preterm delivery.
      The laboratories of Jobe and Newnham have systematically studied endotoxin-induced fetal lung injury in sheep and rhesus macaques [
      • Kallapur S.G.
      • Willet K.E.
      • Jobe A.H.
      • Ikegami M.
      • Bachurski C.J.
      Intra-amniotic endotoxin: chorioamnionitis precedes lung maturation in preterm lambs.
      ,
      • Willet K.E.
      • Jobe A.H.
      • Ikegami M.
      • Newnham J.
      • Brennan S.
      • Sly P.D.
      Antenatal endotoxin and glucocorticoid effects on lung morphometry in preterm lambs.
      ,
      • Bachurski C.J.
      • Ross G.F.
      • Ikegami M.
      • Kramer B.W.
      • Jobe A.H.
      Intra-amniotic endotoxin increases pulmonary surfactant proteins and induces SP-B processing in fetal sheep.
      ,
      • Newnham J.P.
      • Moss T.J.
      • Padbury J.F.
      • Willet K.E.
      • Ikegami M.
      • Ervin M.G.
      • et al.
      The interactive effects of endotoxin with prenatal glucocorticoids on short-term lung function in sheep.
      ,
      • Moss T.J.
      • Nitsos I.
      • Kramer B.W.
      • Ikegami M.
      • Newnham J.P.
      • Jobe A.H.
      Intra-amniotic endotoxin induces lung maturation by direct effects on the developing respiratory tract in preterm sheep.
      ,
      • Kallapur S.G.
      • Jobe A.H.
      • Ikegami M.
      • Bachurski C.J.
      Increased IP-10 and MIG expression after intra-amniotic endotoxin in preterm lamb lung.
      ,
      • Kallapur S.G.
      • Bachurski C.J.
      • Le Cras T.D.
      • Joshi S.N.
      • Ikegami M.
      • Jobe A.H.
      Vascular changes after intra-amniotic endotoxin in preterm lamb lungs.
      ,
      • Kallapur S.G.
      • Moss T.J.
      • Ikegami M.
      • Jasman R.L.
      • Newnham J.P.
      • Jobe A.H.
      Recruited inflammatory cells mediate endotoxin-induced lung maturation in preterm fetal lambs.
      ,
      • Kramer B.W.
      • Joshi S.N.
      • Moss T.J.
      • Newnham J.P.
      • Sindelar R.
      • Jobe A.H.
      • et al.
      Endotoxin-induced maturation of monocytes in preterm fetal sheep lung.
      ,
      • Kallapur S.G.
      • Nitsos I.
      • Moss T.J.
      • Polglase G.R.
      • Pillow J.J.
      • Cheah F.C.
      • et al.
      IL-1 mediates pulmonary and systemic inflammatory responses to chorioamnionitis induced by lipopolysaccharide.
      ,
      • Shah T.A.
      • Hillman N.H.
      • Nitsos I.
      • Polglase G.R.
      • Pillow J.J.
      • Newnham J.P.
      • et al.
      Pulmonary and systemic expression of monocyte chemotactic proteins in preterm sheep fetuses exposed to lipopolysaccharide-induced chorioamnionitis.
      ,
      • Polglase G.R.
      • Hooper S.B.
      • Gill A.W.
      • Allison B.J.
      • Crossley K.J.
      • Moss T.J.
      • et al.
      Intrauterine inflammation causes pulmonary hypertension and cardiovascular sequelae in preterm lambs.
      ,
      • Kramer B.W.
      • Kallapur S.G.
      • Moss T.J.
      • Nitsos I.
      • Polglase G.P.
      • Newnham J.P.
      • et al.
      Modulation of fetal inflammatory response on exposure to lipopolysaccharide by chorioamnion, lung, or gut in sheep.
      ,
      • Collins J.J.
      • Kuypers E.
      • Nitsos I.
      • Jane Pillow J.
      • Polglase G.R.
      • Kemp M.W.
      • et al.
      LPS-induced chorioamnionitis and antenatal corticosteroids modulate Shh signaling in the ovine fetal lung.
      ,
      • Kemp M.W.
      • Kannan P.S.
      • Saito M.
      • Newnham J.P.
      • Cox T.
      • Jobe A.H.
      • et al.
      Selective exposure of the fetal lung and skin/amnion (but not gastro-intestinal tract) to LPS elicits acute systemic inflammation in fetal sheep.
      ]. Intra-amniotic administration of LPS induces fetal lung inflammation, and this is accompanied by a dramatic increase in the number of mononuclear cells and granuocytes in bronchoalveolar lavage fluid and mRNA expression for IL-1, IL-8, and IL-6 in the lung tissue [
      • Kallapur S.G.
      • Willet K.E.
      • Jobe A.H.
      • Ikegami M.
      • Bachurski C.J.
      Intra-amniotic endotoxin: chorioamnionitis precedes lung maturation in preterm lambs.
      ]. An increase in surfactant production, and structural changes in the developing fetal lungs, such as decreased alveolar number, thinning of alveolar septae, and increased alveolar size has been consistently reported [
      • Willet K.E.
      • Jobe A.H.
      • Ikegami M.
      • Newnham J.
      • Brennan S.
      • Sly P.D.
      Antenatal endotoxin and glucocorticoid effects on lung morphometry in preterm lambs.
      ]. In addition, there is down-regulation of the expression of elastin [
      • Collins J.J.
      • Kuypers E.
      • Nitsos I.
      • Jane Pillow J.
      • Polglase G.R.
      • Kemp M.W.
      • et al.
      LPS-induced chorioamnionitis and antenatal corticosteroids modulate Shh signaling in the ovine fetal lung.
      ] and several genes involved in vascular development (vascular endothelial growth factor A, vascular endothelial growth factor receptor 2, and endothelial nitric oxide synthase) [
      • Kallapur S.G.
      • Jobe A.H.
      • Ikegami M.
      • Bachurski C.J.
      Increased IP-10 and MIG expression after intra-amniotic endotoxin in preterm lamb lung.
      ,
      • Kallapur S.G.
      • Bachurski C.J.
      • Le Cras T.D.
      • Joshi S.N.
      • Ikegami M.
      • Jobe A.H.
      Vascular changes after intra-amniotic endotoxin in preterm lamb lungs.
      ]. The latter is thought to predispose to pulmonary hypertension [
      • Polglase G.R.
      • Hooper S.B.
      • Gill A.W.
      • Allison B.J.
      • Crossley K.J.
      • Moss T.J.
      • et al.
      Intrauterine inflammation causes pulmonary hypertension and cardiovascular sequelae in preterm lambs.
      ]. The structural changes reported after endotoxin administration have also been observed after intra-amniotic inoculation with U. urealyticum in sheep and rhesus macques [
      • Moss T.J.
      • Nitsos I.
      • Ikegami M.
      • Jobe A.H.
      • Newnham J.P.
      Experimental intrauterine Ureaplasma infection in sheep.
      ,
      • Moss T.J.
      • Knox C.L.
      • Kallapur S.G.
      • Nitsos I.
      • Theodoropoulos C.
      • Newnham J.P.
      • et al.
      Experimental amniotic fluid infection in sheep: effects of Ureaplasma parvum serovars 3 and 6 on preterm or term fetal sheep.
      ,
      • Moss T.J.
      • Nitsos I.
      • Knox C.L.
      • Polglase G.R.
      • Kallapur S.G.
      • Ikegami M.
      • et al.
      Ureaplasma colonization of amniotic fluid and efficacy of antenatal corticosteroids for preterm lung maturation in sheep.
      ,
      • Novy M.J.
      • Duffy L.
      • Axthelm M.K.
      • Sadowsky D.W.
      • Witkin S.S.
      • Gravett M.G.
      • et al.
      Ureaplasma parvum or Mycoplasma hominis as sole pathogens cause chorioamnionitis, preterm delivery, and fetal pneumonia in rhesus macaques.
      ,
      • Collins J.J.
      • Kallapur S.G.
      • Knox C.L.
      • Nitsos I.
      • Polglase G.R.
      • Pillow J.J.
      • et al.
      Inflammation in fetal sheep from intra-amniotic injection of Ureaplasma parvum.
      ].
      Fetal lung inflammation is characterized by robust expression of proinflammatory mediators such as IL-1β, IL-8, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein 1, and serum amyloid A3 in both sheep and monkeys [
      • Kallapur S.G.
      • Presicce P.
      • Senthamaraikannan P.
      • Alvarez M.
      • Tarantal A.F.
      • Miller L.M.
      • et al.
      Intra-amniotic IL-1β induces fetal inflammation in rhesus monkeys and alters the regulatory T cell/IL-17 balance.
      ,
      • Kallapur S.G.
      • Willet K.E.
      • Jobe A.H.
      • Ikegami M.
      • Bachurski C.J.
      Intra-amniotic endotoxin: chorioamnionitis precedes lung maturation in preterm lambs.
      ,
      • Kramer B.W.
      • Joshi S.N.
      • Moss T.J.
      • Newnham J.P.
      • Sindelar R.
      • Jobe A.H.
      • et al.
      Endotoxin-induced maturation of monocytes in preterm fetal sheep lung.
      ,
      • Shah T.A.
      • Hillman N.H.
      • Nitsos I.
      • Polglase G.R.
      • Pillow J.J.
      • Newnham J.P.
      • et al.
      Pulmonary and systemic expression of monocyte chemotactic proteins in preterm sheep fetuses exposed to lipopolysaccharide-induced chorioamnionitis.
      ]. It is noteworthy that IL-1β is the major cytokine involved in fetal lung injury, as TNF-α and interferon-γ do not elicit the same degree of inflammation [
      • Kallapur S.G.
      • Presicce P.
      • Senthamaraikannan P.
      • Alvarez M.
      • Tarantal A.F.
      • Miller L.M.
      • et al.
      Intra-amniotic IL-1β induces fetal inflammation in rhesus monkeys and alters the regulatory T cell/IL-17 balance.
      ,
      • Ikegami M.
      • Moss T.J.
      • Kallapur S.G.
      • Mulrooney N.
      • Kramer B.W.
      • Nitsos I.
      • et al.
      Minimal lung and systemic responses to TNF-alpha in preterm sheep.
      ]. Moreover, pre-treatment with intra-amniotic injection of an IL-1α antagonist before the administration of bacterial endotoxin prevents lung inflammation and maturation [
      • Kallapur S.G.
      • Moss T.J.
      • Ikegami M.
      • Jasman R.L.
      • Newnham J.P.
      • Jobe A.H.
      Recruited inflammatory cells mediate endotoxin-induced lung maturation in preterm fetal lambs.
      ].
      In summary, exposure to microbial products and intra-amniotic inflammation induces fetal lung maturity, which favors survival in the context of preterm delivery [
      • Kallapur S.G.
      • Presicce P.
      • Rueda C.M.
      • Jobe A.H.
      • Chougnet C.A.
      Fetal immune response to chorioamnionitis.
      ]. However, acceleration of lung maturity is accompanied by dramatic changes in the anatomy of the lung (e.g., reduction in the number of alveoli, impaired microvascular development, and thickening of the arteriolar walls, which collectively resemble changes observed in infants with bronchopulmonary dysplasia) [
      • Jj C
      ]. Therefore, the short-term gain in lung maturity appears to predispose to the development of chronic lung disease.

      6.7 Heart

      Cardiac dysfunction can occur during the course of a systemic inflammatory response and sepsis in adults [
      • Parker M.M.
      • Shelhamer J.H.
      • Bacharach S.L.
      • Green M.V.
      • Natanson C.
      • Frederick T.M.
      • et al.
      Profound but reversible myocardial depression in patients with septic shock.
      ,
      • Parker M.M.
      • McCarthy K.E.
      • Ognibene F.P.
      • Parrillo J.E.
      Right ventricular dysfunction and dilatation, similar to left ventricular changes, characterize the cardiac depression of septic shock in humans.
      ,
      • Vincent J.L.
      • Gris P.
      • Coffernils M.
      • Leon M.
      • Pinsky M.
      • Reuse C.
      • et al.
      Myocardial depression characterizes the fatal course of septic shock.
      ,
      • Kumar A.
      • Haery C.
      • Parrillo J.E.
      Myocardial dysfunction in septic shock.
      ,
      • Rudiger A.
      • Singer M.
      Mechanisms of sepsis-induced cardiac dysfunction.
      ]. Impaired cardiac performance results from a combination of systolic and diastolic dysfunction, which was originally attributed to the presence of circulating myocardial depressant factors and, more recently, to direct effects of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 [
      • Court O.
      • Kumar A.
      • Parrillo J.E.
      • Kumar A.
      Clinical review: myocardial depression in sepsis and septic shock.
      ]. The pattern of myocardial depression is characterized by left ventricular dilation, decreased left ventricular ejection fraction, and a normal or increased cardiac index [
      • Parker M.M.
      • Shelhamer J.H.
      • Bacharach S.L.
      • Green M.V.
      • Natanson C.
      • Frederick T.M.
      • et al.
      Profound but reversible myocardial depression in patients with septic shock.
      ]. Acute ventricular dilation within the first days of septic shock is more frequently observed among survivors, and this is believed to represent compensatory cardiac dilation to maintain stroke volume despite the loss of myocardial contractility [
      • Parker M.M.
      • Shelhamer J.H.
      • Bacharach S.L.
      • Green M.V.
      • Natanson C.
      • Frederick T.M.
      • et al.
      Profound but reversible myocardial depression in patients with septic shock.
      ].
      In preterm PROM and intra-amniotic infection, human fetal echocardiographic studies have also reported evidence of cardiac diastolic dysfunction consistent with increased left ventricular compliance, when compared to those with uncomplicated pregnancies [
      • Romero R.
      • Espinoza J.
      • Gonçalves L.F.
      • Gomez R.
      • Medina L.
      • Silva M.
      • et al.
      Fetal cardiac dysfunction in preterm premature rupture of membranes.
      ]. It is thought that these changes reflect a compensatory mechanism similar to that observed in adults with sepsis.
      Another investigation has shown impairment in both fetal diastolic and systolic cardiac performance of the right ventricle through tissue Doppler and strain rate imaging in fetuses with preterm PROM and proven intra-amniotic infection [
      • Di Naro E.
      • Cromi A.
      • Ghezzi F.
      • Giocolano A.
      • Caringella A.
      • Loverro G.
      Myocardial dysfunction in fetuses exposed to intraamniotic infection: new insights from tissue Doppler and strain imaging.
      ]. These observations are consistent with the study of Yanowitz et al., who found that umbilical cord IL-6 concentrations inversely correlated with systolic, mean, and diastolic blood pressure, and that the presence of funisitis was also associated with changes in right ventricular cardiac output, consistent with lower systemic vascular resistance [
      • Yanowitz T.D.
      • Jordan J.A.
      • Gilmour C.H.
      • Towbin R.
      • Bowen A.
      • Roberts J.M.
      • et al.
      Hemodynamic disturbances in premature infants born after chorioamnionitis: association with cord blood cytokine concentrations.
      ]. These hemodynamic abnormalities are thought to predispose infants to brain injury, thereby buttressing the case for the importance of multisystem organ involvement in the pathophysiology of neonatal complications in the context of FIRS.
      Cardiotocography of fetuses exposed to intra-amniotic inflammation (defined as histologic chorioamnionitis) has shown an increased fetal heart baseline, a higher number of low variation episodes, and a higher number of short-term variations [
      • Vandenbroucke L.
      • Doyen M.
      • Le Lous M.
      • Beuchee A.
      • Loget P.
      • Carrault G.
      • et al.
      Chorioamnionitis following preterm premature rupture of membranes and fetal heart rate variability.
      ].
      Several experimental studies conducted in mice, pregnant sheep, and rhesus macaques have shown that intra-amniotic administration of endotoxin or Candida albicans leads to functional abnormalities in the fetal heart and a change in the gene regulatory networks that program cardiac development [
      • Rounioja S.
      • Räsänen J.
      • Glumoff V.
      • Ojaniemi M.
      • Mäkikallio K.
      • Hallman M.
      Intra-amniotic lipopolysaccharide leads to fetal cardiac dysfunction. A mouse model for fetal inflammatory response.
      ,
      • Rounioja S.
      • Räsänen J.
      • Ojaniemi M.
      • Glumoff V.
      • Autio-Harmainen H.
      • Hallman M.
      Mechanism of acute fetal cardiovascular depression after maternal inflammatory challenge in mouse.
      ,
      • Tare M.
      • Bensley J.G.
      • Moss T.J.
      • Lingwood B.E.
      • Kim M.Y.
      • Barton S.K.
      • et al.
      Exposure to intrauterine inflammation leads to impaired function and altered structure in the preterm heart of fetal sheep.
      ,
      • Durosier L.D.
      • Herry C.L.
      • Cortes M.
      • Cao M.
      • Burns P.
      • Desrochers A.
      • et al.
      Does heart rate variability reflect the systemic inflammatory response in a fetal sheep model of lipopolysaccharide-induced sepsis?.
      ,
      • Stock S.J.
      • Patey O.
      • Thilaganathan B.
      • White S.
      • Furfaro L.L.
      • Payne M.S.
      • et al.
      Intrauterine Candida albicans infection causes systemic fetal candidiasis with progressive cardiac dysfunction in a sheep model of early pregnancy.
      ,
      • Mitchell T.
      • MacDonald J.W.
      • Srinouanpranchanh S.
      • Bammler T.K.
      • Merillat S.
      • Boldenow E.
      • et al.
      Evidence of cardiac involvement in the fetal inflammatory response syndrome: disruption of gene networks programming cardiac development in nonhuman primates.
      ]. Specifically, in non-human primates, intra-amniotic administration of group B streptococcus or E. coli led to increased concentrations of IL-6 and IL-8 in the fetal myocardium without significant evidence of histopathologic inflammation of the heart [
      • Mitchell T.
      • MacDonald J.W.
      • Srinouanpranchanh S.
      • Bammler T.K.
      • Merillat S.
      • Boldenow E.
      • et al.
      Evidence of cardiac involvement in the fetal inflammatory response syndrome: disruption of gene networks programming cardiac development in nonhuman primates.
      ]. This was associated with changes in the gene set involving cardiac morphogenesis and vasculogenesis. This observation is important given that epidemiologic studies have shown a 17-fold increased risk of heart failure after preterm birth. Moreover, adults who were born prematurely have exhibited substantial changes in myocardial structure and function [
      • Carr H.
      • Cnattingius S.
      • Granath F.
      • Ludvigsson J.F.
      • Edstedt Bonamy A.K.
      Preterm birth and risk of heart failure up to early adulthood.
      ]. Thus, exposure to intra-amniotic inflammation can explain, at least in part, the predisposition of preterm neonates to develop long-term cardiovascular disorders and, specifically, heart failure [
      • Bonamy A.K.
      • Martin H.
      • Jörneskog G.
      • Norman M.
      Lower skin capillary density, normal endothelial function and higher blood pressure in children born preterm.
      ,
      • Bensley J.G.
      • Stacy V.K.
      • De Matteo R.
      • Harding R.
      • Black M.J.
      Cardiac remodelling as a result of pre-term birth: implications for future cardiovascular disease.
      ,
      • Lewandowski A.J.
      • Bradlow W.M.
      • Augustine D.
      • Davis E.F.
      • Francis J.
      • Singhal A.
      • et al.
      Right ventricular systolic dysfunction in young adults born preterm.
      ,
      • Lewandowski A.J.
      • Augustine D.
      • Lamata P.
      • Davis E.F.
      • Lazdam M.
      • Francis J.
      • et al.
      Preterm heart in adult life: cardiovascular magnetic resonance reveals distinct differences in left ventricular mass, geometry, and function.
      ].

      6.8 Gut

      The fetal intestines can be exposed to microorganisms and inflammatory mediators after amniotic fluid is swallowed. FIRS is a risk factor for necrotizing enterocolitis [
      • Andrews W.W.
      • Goldenberg R.L.
      • Faye-Petersen O.
      • Cliver S.
      • Goepfert A.R.
      • Hauth J.C.
      The Alabama Preterm Birth study: polymorphonuclear and mononuclear cell placental infiltrations, other markers of inflammation, and outcomes in 23- to 32-week preterm newborn infants.
      ,
      • Been J.V.
      • Lievense S.
      • Zimmermann L.J.
      • Kramer B.W.
      • Wolfs T.G.
      Chorioamnionitis as a risk factor for necrotizing enterocolitis: a systematic review and meta-analysis.
      ]. A systematic review and meta-analysis, inclusive of 22,601 patients from 12 studies, demonstrated that clinical chorioamnionitis (OR 1.24; 95% CI: 1.01–1.52) and histological chorioamnionitis with acute funisitis (OR 3.29; 95% CI: 1.87–5.78) were significantly associated with necrotizing enterocolitis [
      • Been J.V.
      • Lievense S.
      • Zimmermann L.J.
      • Kramer B.W.
      • Wolfs T.G.
      Chorioamnionitis as a risk factor for necrotizing enterocolitis: a systematic review and meta-analysis.
      ]. The mechanism for these associations is thought to involve impaired gut barrier function and immune dysfunction [
      • Neu J.
      • Mshvildadze M.
      • Mai V.
      A roadmap for understanding and preventing necrotizing enterocolitis.
      ].
      Experimental evidence suggests that immaturity and prenatal inflammation alters maturation of the fetal intestine [
      • Nguyen D.N.
      • Thymann T.
      • Goericke-Pesch S.K.
      • Ren S.
      • Wei W.
      • Skovgaard K.
      • et al.
      Prenatal intra-amniotic endotoxin induces fetal gut and lung immune responses and postnatal systemic inflammation in preterm pigs.
      ,
      • Wolfs T.G.
      • Buurman W.A.
      • Zoer B.
      • Moonen R.M.
      • Derikx J.P.
      • Thuijls G.
      • et al.
      Endotoxin induced chorioamnionitis prevents intestinal development during gestation in fetal sheep.
      ,
      • Wolfs T.G.
      • Kallapur S.G.
      • Polglase G.R.
      • Pillow J.J.
      • Nitsos I.
      • Newnham J.P.
      • et al.
      IL-1alpha mediated chorioamnionitis induces depletion of FoxP3+ cells and ileal inflammation in the ovine fetal gut.
      ,
      • Fricke E.M.
      • Elgin T.G.
      • Gong H.
      • Reese J.
      • Gibson-Corley K.N.
      • Weiss R.M.
      • et al.
      Lipopolysaccharide-induced maternal inflammation induces direct placental injury without alteration in placental blood flow and induces a secondary fetal intestinal injury that persists into adulthood.
      ,
      • Elgin T.G.
      • Fricke E.M.
      • Gong H.
      • Reese J.
      • Mills D.A.
      • Kalantera K.M.
      • et al.
      Fetal exposure to maternal inflammation interrupts murine intestinal development and increases susceptibility to neonatal intestinal injury.
      ]. In a sheep model, Wolfs et al. reported that administration of intra-amniotic endotoxin prevented maturation of tight junctions in intestinal epithelial cells [
      • Wolfs T.G.
      • Buurman W.A.
      • Zoer B.
      • Moonen R.M.
      • Derikx J.P.
      • Thuijls G.
      • et al.
      Endotoxin induced chorioamnionitis prevents intestinal development during gestation in fetal sheep.
      ]. Abnormal tight junctional distribution predisposes to easy access of microorganisms and toxins to the mucosa and inner layers of the gut pre- and post-natally.
      In a recent study, intraperitoneal administration of bacterial endotoxin to pregnant rats increased the frequency of necrotizing enterocolitis and neonatal mortality [
      • Yan X.
      • Managlia E.
      • Tan X.D.
      • De Plaen I.G.
      Prenatal inflammation impairs intestinal microvascular development through a TNF-dependent mechanism and predisposes newborn mice to necrotizing enterocolitis.
      ]. The proposed mechanism for this observation was fetal TNF-α-mediated impairment of intestinal microvasculature, which was associated with a decrease in vascular endothelial growth factor (VEGF) and VEGF receptor 2 protein expression [
      • Yan X.
      • Managlia E.
      • Tan X.D.
      • De Plaen I.G.
      Prenatal inflammation impairs intestinal microvascular development through a TNF-dependent mechanism and predisposes newborn mice to necrotizing enterocolitis.
      ]. This was ameliorated by a hypoxia-inducible factor-1α (HIF-1α) stabilizing agent (HIF-1α is a master regulator of VEGF function), and was abrogated by neutralizing TNF-α activity. This body of work indicates that prenatal inflammation increases the risk for necrotizing enterocolitis and that therapeutic approaches to maintain VEGF function may help prevent this complication.
      Meconium-stained amniotic fluid, a common phenomenon (1 in 7 pregnancies), has traditionally been attributed to hypoxia [
      • Lee J.
      • Romero R.
      • Lee K.A.
      • Kim E.N.
      • Korzeniewski S.J.
      • Chaemsaithong P.
      • et al.
      Meconium aspiration syndrome: a role for fetal systemic inflammation.
      ]. Interestingly, such fluid frequently contains bacteria [
      • Romero R.
      • Hanaoka S.
      • Mazor M.
      • Athanassiadis A.P.
      • Callahan R.
      • Hsu Y.C.
      • et al.
      Meconium-stained amniotic fluid: a risk factor for microbial invasion of the amniotic cavity.
      ,
      • Mazor M.
      • Furman B.
      • Wiznitzer A.
      • Shoham-Vardi I.
      • Cohen J.
      • Ghezzi F.
      Maternal and perinatal outcome of patients with preterm labor and meconium-stained amniotic fluid.
      ,
      • Romero R.
      • Yoon B.H.
      • Chaemsaithong P.
      • Cortez J.
      • Park C.W.
      • Gonzalez R.
      • et al.
      Bacteria and endotoxin in meconium-stained amniotic fluid at term: could intra-amniotic infection cause meconium passage?.
      ], endotoxin [
      • Romero R.
      • Yoon B.H.
      • Chaemsaithong P.
      • Cortez J.
      • Park C.W.
      • Gonzalez R.
      • et al.
      Bacteria and endotoxin in meconium-stained amniotic fluid at term: could intra-amniotic infection cause meconium passage?.
      ,
      • Hsieh T.T.
      • Hsieh C.C.
      • Hung T.H.
      • Chiang C.H.
      • Yang F.P.
      • Pao C.C.
      Differential expression of interleukin-1 beta and interleukin-6 in human fetal serum and meconium-stained amniotic fluid.
      ], and higher concentrations of inflammatory mediators, such as IL-1β, TNF-α [
      • Yamada T.
      • Minakami H.
      • Matsubara S.
      • Yatsuda T.
      • Kohmura Y.
      • Sato I.
      Meconium-stained amniotic fluid exhibits chemotactic activity for polymorphonuclear leukocytes in vitro.
      ], IL-8 [
      • Yamada T.
      • Minakami H.
      • Matsubara S.
      • Yatsuda T.
      • Kohmura Y.
      • Sato I.
      Meconium-stained amniotic fluid exhibits chemotactic activity for polymorphonuclear leukocytes in vitro.
      ,
      • Okazaki K.
      • Kondo M.
      • Kato M.
      • Kakinuma R.
      • Nishida A.
      • Noda M.
      • et al.
      Serum cytokine and chemokine profiles in neonates with meconium aspiration syndrome.
      ], and phospholipase-A2. We have proposed that meconium passage in utero, in some cases, may represent accelerated bowel transit (i.e., fetal diarrhea). Most of the time, meconium-stained amniotic fluid is a benign finding and is not associated with adverse neonatal outcome. However, meconium aspiration syndrome is a serious neonatal complication that develops in 5% of cases having meconium-stained amniotic fluid.
      To explore why some infants with meconium-stained amniotic fluid develop this syndrome and others do not, we examined the role of fetal systemic inflammation. We reported that the combination of meconium-stained amniotic fluid and FIRS had a higher frequency of meconium aspiration syndrome than those without FIRS (Fig. 5) [
      • Lee J.
      • Romero R.
      • Lee K.A.
      • Kim E.N.
      • Korzeniewski S.J.
      • Chaemsaithong P.
      • et al.
      Meconium aspiration syndrome: a role for fetal systemic inflammation.
      ]. Thus, we proposed a chain of events in that meconium (with its proinflammatory properties), when aspirated before birth and combined with a fetal systemic inflammatory response involving the fetal lungs, can predispose to meconium aspiration syndrome [
      • Lee J.
      • Romero R.
      • Lee K.A.
      • Kim E.N.
      • Korzeniewski S.J.
      • Chaemsaithong P.
      • et al.
      Meconium aspiration syndrome: a role for fetal systemic inflammation.
      ,
      • Hofer N.
      • Jank K.
      • Strenger V.
      • Pansy J.
      • Resch B.
      Inflammatory indices in meconium aspiration syndrome.
      ,
      • Yokoi K.
      • Iwata O.
      • Kobayashi S.
      • Muramatsu K.
      • Goto H.
      Influence of foetal inflammation on the development of meconium aspiration syndrome in term neonates with meconium-stained amniotic fluid.
      ]. In addition, elevated CRP concentrations, and low white blood cell and neutrophil counts in neonatal blood are associated with the severity of meconium aspiration syndrome during the early phases of the disease [
      • Hofer N.
      • Jank K.
      • Strenger V.
      • Pansy J.
      • Resch B.
      Inflammatory indices in meconium aspiration syndrome.
      ].
      Fig. 5
      Fig. 5Meconium aspiration syndrome (MAS) is more likely to occur in patients with meconium-stained amniotic fluid if intraamniotic inflammation and fetal inflammatory response syndrome ascertained by funisitis are present. Frequency of MAS in the context of intraamniotic inflammation and funisitis. Neonates exposed to both intraamniotic inflammation and funisitis were at significantly greater risk of MAS than newborns exposed to neither of these 2 conditions [28.6% (4/14) vs 0% (0/28), P = 0.009]. In contrast, newborns exposed to only intraamniotic inflammation without funisitis were not at greater risk of MAS than newborns exposed to neither of these 2 conditions [10.9% (5/46) vs 0% (0/28); P = 0.15]. MAS did not occur in the absence of intraamniotic inflammation. (Reproduced with permission from Lee J, Romero R, Lee KA et al. Meconium aspiration syndrome: a role for fetal systemic inflammation. Am J Obstet Gynecol 214:366.e1-9, 2016.).

      6.9 Liver

      The liver is exposed to exogenous agents that reach the fetus either directly from the placenta (via the umbilical vein) or from the amniotic fluid, which is swallowed and resorbed in the intestine (via the portal vessels). Kupffer cells are macrophages lining the hepatic sinusoids, which are capable of recognition and phagocytosis of a wide range of bacteria as well as immune complexes [
      • Cope E.
      • Dilly S.
      Kupffer cell numbers during human development.
      ]. Kupffer cells are able to produce cytokines (IL-6, IL-1β, TNF-α) [
      • Kutteh W.H.
      • Rainey W.E.
      • Carr B.R.
      Regulation of interleukin-6 production in human fetal Kupffer cells.
      ,
      • Kutteh W.H.
      • Rainey W.E.
      • Beutler B.
      • Carr B.R.
      Tumor necrosis factor-alpha and interleukin-1 beta production by human fetal Kupffer cells.
      ], which can modify fetal hematopoiesis. During fetal development, hematopoiesis occurs mainly in the fetal liver, followed by localization to the bone marrow. Fetuses with funisitis exhibit a significant increase in fetal hepatic hematopoiesis and myelopoiesis during the second trimester [
      • Stallmach T.
      • Karolyi L.
      Augmentation of fetal granulopoiesis with chorioamnionitis during the second trimester of gestation.
      ,
      • Pfisterer C.
      • Faber R.
      • Horn L.C.
      Chorioamnionitis-induced changes of fetal extramedullar hematopoiesis in the second trimester of gestation. Is diagnosis from fetal autopsy possible?.
      ,
      • Miranda R.N.
      • Omurtag K.
      • Castellani W.J.
      • De las Casas L.E.
      • Quintanilla N.M.
      • Kaabipour E.
      Myelopoiesis in the liver of stillborns with evidence of intrauterine infection.
      ], indicating that the fetal response to intra-amniotic infection involves the liver.
      Experimental intra-amniotic inflammation induced by bacterial endotoxin in sheep fetuses led to hepatic inflammation and liver dysfunction in the neonatal period. Specifically, there was an increase in the number of CD3+ T lymphocytes as well as in IL-1β and TNFα mRNA in the fetal liver [
      • Bieghs V.
      • Vlassaks E.
      • Custers A.
      • van Gorp P.J.
      • Gijbels M.J.
      • Bast A.
      • et al.
      Chorioamnionitis induced hepatic inflammation and disturbed lipid metabolism in fetal sheep.
      ]. In addition, fetal hepatic inflammation was associated with metabolic disturbances, including increased umbilical cord plasma concentrations of total cholesterol, high-density lipoprotein, low-density lipoprotein, and triglycerides [
      • Bieghs V.
      • Vlassaks E.
      • Custers A.
      • van Gorp P.J.
      • Gijbels M.J.
      • Bast A.
      • et al.
      Chorioamnionitis induced hepatic inflammation and disturbed lipid metabolism in fetal sheep.
      ]. Two weeks after endotoxin injection, inflammation was still detected in the fetal liver, and there were higher glucose concentrations in the blood [
      • Bieghs V.
      • Vlassaks E.
      • Custers A.
      • van Gorp P.J.
      • Gijbels M.J.
      • Bast A.
      • et al.
      Chorioamnionitis induced hepatic inflammation and disturbed lipid metabolism in fetal sheep.
      ]. In a subsequent study focusing on the effects of bacterial endotoxin exposure during fetal life, Vlassaks et al. reported that liver triglycerides and plasma cholesterol concentrations were increased in LPS-exposed lambs compared to controls even though hepatic inflammation had resolved [
      • Vlassaks E.
      • Gavilanes A.W.
      • Bieghs V.
      • Reinartz A.
      • Gassler N.
      • Van Gorp P.J.
      • et al.
      Antenatal exposure to chorioamnionitis affects lipid metabolism in 7-week-old sheep.
      ]. There was also evidence of increased lipid peroxidation, leptin receptor mRNA expression, and expression of cytochrome c oxidase subunit 4. This represents the first evidence that fetal inflammation has an effect on the liver and lipid metabolism. Whether this has long-term consequences by increasing the susceptibility to hepatic inflammation and insulin resistance is yet to be determined.

      6.10 Kidneys

      The fetal kidney is a potential target organ during FIRS [
      • Carroll S.G.
      • Papaioannou S.
      • Nicolaides K.H.
      Assessment of fetal activity and amniotic fluid volume in the prediction of intrauterine infection in preterm prelabor amniorrhexis.
      ,
      • Yoon B.H.
      • Kim Y.A.
      • Romero R.
      • Kim J.C.
      • Park K.H.
      • Kim M.H.
      • et al.
      Association of oligohydramnios in women with preterm premature rupture of membranes with an inflammatory response in fetal, amniotic, and maternal compartments.
      ,
      • Lee S.E.
      • Romero R.
      • Lee S.M.
      • Yoon B.H.
      Amniotic fluid volume in intra-amniotic inflammation with and without culture-proven amniotic fluid infection in preterm premature rupture of membranes.
      ]. Children and adult patients with sepsis can experience oliguria secondary to pre-renal failure (i.e., hypoperfusion) [
      • Rangel-Frausto M.S.
      • Pittet D.
      • Costigan M.
      • Hwang T.
      • Davis C.S.
      • Wenzel R.P.
      The natural history of the systemic inflammatory response syndrome (SIRS). A prospective study.
      ]. Since fetal urine is the main source of amniotic fluid, several investigators have examined the relationship between amniotic fluid volume and intra-amniotic and fetal inflammation. Yoon et al. reported that oligohydramnios in women with preterm PROM was associated with higher IL-6 concentrations in umbilical cord plasma at birth, higher concentrations of amniotic fluid proinflammatory cytokines (IL-6, IL-1β, and TNF-α), and a higher frequency of clinical and acute histologic chorioamnionitis compared to those without oligohydramnios [
      • Yoon B.H.
      • Kim Y.A.
      • Romero R.
      • Kim J.C.
      • Park K.H.
      • Kim M.H.
      • et al.
      Association of oligohydramnios in women with preterm premature rupture of membranes with an inflammatory response in fetal, amniotic, and maternal compartments.
      ]. Similarly, Lee et al. reported that patients with intra-amniotic infection and inflammation had a higher frequency of oligohydramnios than those with a negative amniotic fluid culture [
      • Lee S.E.
      • Romero R.
      • Lee S.M.
      • Yoon B.H.
      Amniotic fluid volume in intra-amniotic inflammation with and without culture-proven amniotic fluid infection in preterm premature rupture of membranes.
      ]. Carroll et al. reported that fetuses with bacteremia diagnosed by cordocentesis had a higher frequency of oligohydramnios than in those with a negative fetal blood culture [
      • Carroll S.G.
      • Papaioannou S.
      • Nicolaides K.H.
      Assessment of fetal activity and amniotic fluid volume in the prediction of intrauterine infection in preterm prelabor amniorrhexis.
      ]. Taken together, the data suggest that oligohydramnios is associated with intra-amniotic inflammation and FIRS.
      In the context of preterm PROM, reduced amniotic fluid volume may be partly attributed to the loss of amniotic fluid through the site of membrane rupture. However, it is possible that fetal urine production is altered in FIRS, and this can result from redistribution of blood flow away from the kidneys to ensure adequate blood flow to critical organs and increase efficiency of oxygen utilization [
      • Schlichtig R.
      • Kramer D.J.
      • Pinsky M.R.
      Flow redistribution during progressive hemorrhage is a determinant of critical O2 delivery.
      ,
      • Kato R.
      • Pinsky M.R.
      Personalizing blood pressure management in septic shock.
      ]. It is interesting that neonates delivered to mothers with intra-amniotic inflammation have higher serum blood urea nitrogen concentrations and that such concentrations correlate with amniotic fluid IL-6 [
      • Azpurua H.
      • Dulay A.T.
      • Buhimschi I.A.
      • Bahtiyar M.O.
      • Funai E.
      • Abdel-Razeq S.S.
      • et al.
      Fetal renal artery impedance as assessed by Doppler ultrasound in pregnancies complicated by intraamniotic inflammation and preterm birth.
      ].
      In a murine model, maternal intraperitoneal endotoxin injection induced fetal leukocyte activation, recruitment, and infiltration of the fetal renal parenchyma [
      • Hudalla H.
      • Karenberg K.
      • Kuon R.J.
      • Poschl J.
      • Tschada R.
      • Frommhold D.
      LPS-induced maternal inflammation promotes fetal leukocyte recruitment and prenatal organ infiltration in mice.
      ]. Moreover, fetal sheep exposed to intra-amniotic endotoxin have a reduction in nephron numbers by 23% as well as low glomerular density [
      • Galinsky R.
      • Moss T.J.
      • Gubhaju L.
      • Hooper S.B.
      • Black M.J.
      • Polglase G.R.
      Effect of intra-amniotic lipopolysaccharide on nephron number in preterm fetal sheep.
      ]. Evidence of renal injury has also been reported in preterm pigs after intra-amniotic injection of endotoxin, which induced an altered postnatal renal biochemical profile (i.e., increased concentration of urinary microalbumin, microalbumin:creatinine ratio, and sodium) [
      • Nguyen D.N.
      • Thymann T.
      • Goericke-Pesch S.K.
      • Ren S.
      • Wei W.
      • Skovgaard K.
      • et al.
      Prenatal intra-amniotic endotoxin induces fetal gut and lung immune responses and postnatal systemic inflammation in preterm pigs.
      ]. In summary, microbial products and inflammatory mediators in the amniotic fluid may predispose preterm fetuses to impaired renal function during the postnatal period and increased risk of hypertension and renal dysfunction in later life [
      • Brenner B.M.
      • Garcia D.L.
      • Anderson S.
      Glomeruli and blood pressure. Less of one, more the other?.
      ,
      • Mendez N.
      • Torres-Farfan C.
      • Salazar E.
      • Bascur P.
      • Bastidas C.
      • Vergara K.
      • et al.
      Fetal programming of renal dysfunction and high blood pressure by chronodisruption.
      ].

      6.11 Brain

      A considerable body of evidence supports the concept that fetal systemic inflammation and often infection is causally related to fetal/neonatal neuroinflammation, brain injury, and neurodevelopmental disorders, including mental illness [
      • Murphy D.J.
      • Sellers S.
      • MacKenzie I.Z.
      • Yudkin P.L.
      • Johnson A.M.
      Case-control study of antenatal and intrapartum risk factors for cerebral palsy in very preterm singleton babies.
      ,
      • Yoon B.H.
      • Kim C.J.
      • Romero R.
      • Jun J.K.
      • Park K.H.
      • Choi S.T.
      • et al.
      Experimentally induced intrauterine infection causes fetal brain white matter lesions in rabbits.
      ,
      • Yoon B.H.
      • Jun J.K.
      • Romero R.
      • Park K.H.
      • Gomez R.
      • Choi J.H.
      • et al.
      Amniotic fluid inflammatory cytokines (interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha), neonatal brain white matter lesions, and cerebral palsy.
      ,
      • Grether J.K.
      • Nelson K.B.
      Maternal infection and cerebral palsy in infants of normal birth weight.
      ,
      • Nelson K.B.
      • Dambrosia J.M.
      • Grether J.K.
      • Phillips T.M.
      Neonatal cytokines and coagulation factors in children with cerebral palsy.
      ,
      • Al-Haddad B.J.S.
      • Oler E.
      • Armistead B.
      • Elsayed N.A.
      • Weinberger D.R.
      • Bernier R.
      • et al.
      The fetal origins of mental illness.
      ].
      In 1955, Eastman and DeLeon reported that intrapartum maternal fever (often from intraamniotic infection with bacteria) conferred a seven-fold increased risk for cerebral palsy [
      • Eastman N.J.
      • Deleon M.
      The etiology of cerebral palsy.
      ]. Subsequently, a solid body of evidence has accumulated over the last six decades showing that inflammation and infection play a role in the genesis of brain injury. In 1971, Leviton and Gilles [
      • Leviton A.
      • Gilles F.H.
      Custering of the morphological components of perinatal telencephalic leucoencephalopathy.
      ] reported on the characteristics of perinatal telencephalic leucoencephalopathy (white matter abnormalities in the fetal brain after midgestation) and that there was a potential link to neonatal bacteremia and endotoxin exposure [
      • Gilles F.H.
      • Leviton A.
      • Kerr C.S.
      Endotoxin leucoencephalopathy in the telencephalon of the newborn kitten.
      ]. This original concept has gained considerable support and its evolution is described in another article in this issue [
      • Gilles F.H.
      • Leviton A.
      Neonatal white matter damage and the fetal inflammatory response.
      ]. Other articles in this issue describe the etiology, mechanisms of brain injury, short- and long-term neurologic outcome after fetal exposure to inflammation, and potential interventions to downregulate the inflammatory response [
      • Nelson K.B.
      The epidemiology of FIRS in term and late preterm births.
      ,
      • Wiswell T.E.
      Evaluation for the etiology of neonatal encephalopathy and the diagnosis of FIRS. Seminars in Fetal and Neonatal Medicine.
      ,
      • Yap V.
      • Perlman J.M.
      Mechanisms of brain injury in newborn infants associated with the fetal inflammatory response syndrome.
      ,
      • Scher M.S.
      Neurologic outcome after fetal inflammatory response syndrome: trimester-specific considerations.
      ]. The work of Nelson has supported the concept that maternal infection plays a role in the genesis of cerebral palsy in infants born at or close to term [
      • Nelson K.B.
      • Grether J.K.
      Causes of cerebral palsy.
      ,
      • Nelson K.B.
      • Willoughby R.E.
      Infection, inflammation and the risk of cerebral palsy.
      ,
      • Nelson K.B.
      • Blair E.
      Prenatal factors in cerebral palsy.
      ]. Other articles in this issue of Seminars in Fetal and Neonatal Medicine describe FIRS and its manifestations in term or late preterm gestation. Therefore, the role of intra-amniotic neuroinflammation does not seem to be limited to preterm neonates, a concept that is important given that most cases of cerebral palsy derive from infants born at term.
      Briefly, the evidence in support of this concept indicates that 1) proven intra-amniotic infection is present in at least 25% of all preterm deliveries [
      • Garite T.J.
      • Freeman R.K.
      • Linzey E.M.
      • Braly P.
      The use of amniocentesis in patients with premature rupture of membranes.
      ,
      • Bobitt J.R.
      • Hayslip C.C.
      • Damato J.D.
      Amniotic fluid infection as determined by transabdominal amniocentesis in patients with intact membranes in premature labor.
      ,
      • Zlatnik F.J.
      • Cruikshank D.P.
      • Petzold C.R.
      • Galask R.P.
      Amniocentesis in the identification of inapparent infection in preterm patients with premature rupture of the membranes.
      ,
      • Broekhuizen F.F.
      • Gilman M.
      • Hamilton P.R.
      Amniocentesis for gram stain and culture in preterm premature rupture of the membranes.
      ,
      • Romero R.
      • Sirtori M.
      • Oyarzun E.
      • Avila C.
      • Mazor M.
      • Callahan R.
      • et al.
      Infection and labor. V. Prevalence, microbiology, and clinical significance of intraamniotic infection in women with preterm labor and intact membranes.
      ,
      • DiGiulio D.B.
      • Romero R.
      • Kusanovic J.P.
      • Gomez R.
      • Kim C.J.
      • Seok K.S.
      • et al.
      Prevalence and diversity of microbes in the amniotic fluid, the fetal inflammatory response, and pregnancy outcome in women with preterm pre-labor rupture of membranes.
      ,
      • Romero R.
      • Miranda J.
      • Chaiworapongsa T.
      • Chaemsaithong P.
      • Gotsch F.
      • Dong Z.
      • et al.
      A novel molecular microbiologic technique for the rapid diagnosis of microbial invasion of the amniotic cavity and intra-amniotic infection in preterm labor with intact membranes.
      ] and in 61% of patients with clinical chorioamnionitis at term [
      • Romero R.
      • Miranda J.
      • Kusanovic J.P.
      • Chaiworapongsa T.
      • Chaemsaithong P.
      • Martinez A.
      • et al.
      Clinical chorioamnionitis at term I: microbiology of the amniotic cavity using cultivation and molecular techniques.
      ]; 2) a fetal inflammatory response (diagnosed by the presence of funisitis, elevated concentrations of IL-6 or C-reactive protein in umbilical cord blood) is present in a large fraction of patients with intra-amniotic infection [
      • Gomez R.
      • Romero R.
      • Ghezzi F.
      • Yoon B.H.
      • Mazor M.
      • Berry S.M.
      The fetal inflammatory response syndrome.
      ,
      • Yoon B.H.
      • Romero R.
      • Shim J.Y.
      • Shim S.S.
      • Kim C.J.
      • Jun J.K.
      C-reactive protein in umbilical cord blood: a simple and widely available clinical method to assess the risk of amniotic fluid infection and funisitis.
      ,
      • Kim C.J.
      • Romero R.
      • Chaemsaithong P.
      • Chaiyasit N.
      • Yoon B.H.
      • Kim Y.M.
      Acute chorioamnionitis and funisitis: definition, pathologic features, and clinical significance.
      ]; 3) there is a strong association between intra-amniotic infection/inflammation and the subsequent development of white matter lesions of the neonatal brain [
      • Gilles F.H.
      • Murphy S.F.
      Perinatal telencephalic leucoencephalopathy.
      ,
      • Leviton A.
      • Gilles F.H.
      An epidemiologic study of perinatal telencephalic leucoencephalopathy in an autopsy population.
      ,
      • Dammann O.
      • Leviton A.
      Infection remote from the brain, neonatal white matter damage, and cerebral palsy in the preterm infant.
      ,
      • Debillon T.
      • Gras-Leguen C.
      • Vérielle V.
      • Winer N.
      • Caillon J.
      • Rozé J.C.
      • et al.
      Intrauterine infection induces programmed cell death in rabbit periventricular white matter.
      ,
      • Duncan J.R.
      • Cock M.L.
      • Scheerlinck J.P.
      • Westcott K.T.
      • McLean C.
      • Harding R.
      • et al.
      White matter injury after repeated endotoxin exposure in the preterm ovine fetus.
      ,
      • Hagberg H.
      • Peebles D.
      • Mallard C.
      Models of white matter injury: comparison of infectious, hypoxic-ischemic, and excitotoxic insults.
      ,
      • Bell M.J.
      • Hallenbeck J.M.
      Effects of intrauterine inflammation on developing rat brain.
      ,
      • Dammann O.
      • Kuban K.C.
      • Leviton A.
      Perinatal infection, fetal inflammatory response, white matter damage, and cognitive limitations in children born preterm.
      ,
      • Debillon T.
      • Gras-Leguen C.
      • Leroy S.
      • Caillon J.
      • Rozé J.C.
      • Gressens P.
      Patterns of cerebral inflammatory response in a rabbit model of intrauterine infection-mediated brain lesion.
      ,
      • Mallard C.
      • Welin A.K.
      • Peebles D.
      • Hagberg H.
      • Kjellmer I.
      White matter injury following systemic endotoxemia or asphyxia in the fetal sheep.
      ,
      • Dammann O.
      • Leviton A.
      Inflammation, brain damage and visual dysfunction in preterm infants.
      ,
      • Gilles F.H.
      • Leviton A.
      • Dooling E.C.
      The developing human brain: growth and epidemiologic neuropathology.
      ,
      • Korzeniewski S.J.
      • Romero R.
      • Cortez J.
      • Pappas A.
      • Schwartz A.G.
      • Kim C.J.
      • et al.
      A "multi-hit" model of neonatal white matter injury: cumulative contributions of chronic placental inflammation, acute fetal inflammation and postnatal inflammatory events.
      ,
      • Lu H.Y.
      • Zhang Q.
      • Wang Q.X.
      • Lu J.Y.
      Contribution of histologic chorioamnionitis and fetal inflammatory response syndrome to increased risk of brain injury in infants with preterm premature rupture of membranes.
      ,
      • Gussenhoven R.
      • Westerlaken R.J.J.
      • Ophelders D.
      • Jobe A.H.
      • Kemp M.W.
      • Kallapur S.G.
      • et al.
      Chorioamnionitis, neuroinflammation, and injury: timing is key in the preterm ovine fetus.
      ,
      • Stojanovska V.
      • Atik A.
      • Nitsos I.
      • Skiold B.
      • Barton S.K.
      • Zahra V.A.
      • et al.
      Effects of intrauterine inflammation on cortical gray matter of near-term lambs.
      ,
      • Disdier C.
      • Awa F.
      • Chen X.
      • Dhillon S.K.
      • Galinsky R.
      • Davidson J.O.
      • et al.
      Lipopolysaccharide-induced changes in the neurovascular unit in the preterm fetal sheep brain.
      ,
      • Zhang Z.
      • Narayan S.
      • Su L.
      • Al-Alawyat H.
      • Liu J.
      • Kannan S.
      Cerebellar injury and impaired function in a rabbit model of maternal inflammation induced neonatal brain injury.
      ,
      • Galinsky R.
      • Dhillon S.K.
      • Dean J.M.
      • Davidson J.O.
      • Lear C.A.
      • Wassink G.
      • et al.
      Tumor necrosis factor inhibition attenuates white matter gliosis after systemic inflammation in preterm fetal sheep.
      ]; 4) a fetal inflammatory response (diagnosed by funisitis) increases the risk of both periventricular leukomalacia and cerebral palsy [
      • Gomez R.
      • Romero R.
      • Ghezzi F.
      • Yoon B.H.
      • Mazor M.
      • Berry S.M.
      The fetal inflammatory response syndrome.