I. Ahmed

Prevalence and Clinical Significance of Sterile Intra-amniotic Inflammation in Patients with Preterm Labor and Intact Membranes

Inflammation and infection play a major role in preterm birth. The purpose of this study was to (i) determine the prevalence and clinical significance of sterile intra‐amniotic inflammation and (ii) examine the relationship between amniotic fluid (AF) concentrations of high mobility group box‐1 (HMGB1) and the interval from amniocentesis to delivery in patients with sterile intra‐amniotic inflammation.

Sterile and microbial-associated intra-amniotic inflammation in preterm prelabor rupture of membranes

Abstract Objective: The objectives of this study were to: (1) determine the amniotic fluid (AF) microbiology of patients with preterm prelabor rupture of membranes (PROM); and (2) examine the relationship between intra-amniotic inflammation with and without microorganisms (sterile inflammation) and adverse pregnancy outcomes in patients with preterm PROM. Methods: AF samples obtained from 59 women with preterm PROM were analyzed using cultivation techniques (for aerobic and anaerobic bacteria as well as genital mycoplasmas) and with broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). AF concentration of interleukin-6 (IL-6) was determined using ELISA. Results of both tests were correlated with AF IL-6 concentrations and the occurrence of adverse obstetrical/perinatal outcomes. Results: (1) PCR/ESI-MS, AF culture, and the combination of these two tests each identified microorganisms in 36% (21/59), 24% (14/59) and 41% (24/59) of women with preterm PROM, respectively; (2) the most frequent microorganisms found in the amniotic cavity were Sneathia species and Ureaplasma urealyticum; (3) the frequency of microbial-associated and sterile intra-amniotic inflammation was overall similar [ 29% (17/59)]: however, the prevalence of each differed according to the gestational age when PROM occurred; (4) the earlier the gestational age at preterm PROM, the higher the frequency of both microbial-associated and sterile intra-amniotic inflammation; (5) the intensity of the intra-amniotic inflammatory response against microorganisms is stronger when preterm PROM occurs early in pregnancy; and (6) the frequency of acute placental inflammation (histologic chorioamnionitis and/or funisitis) was significantly higher in patients with microbial-associated intra-amniotic inflammation than in those without intra-amniotic inflammation [93.3% (14/15) versus 38% (6/16); p = 0.001]. Conclusions: (1) The frequency of microorganisms in preterm PROM is 40% using both cultivation techniques and PCR/ESI-MS; (2) PCR/ESI-MS identified microorganisms in the AF of 50% more women with preterm PROM than AF culture; and (3) sterile intra-amniotic inflammation was present in 29% of these patients, and it was as or more common than microbial-associated intra-amniotic inflammation among those presenting after, but not before, 24 weeks of gestation.

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

The diagnosis of microbial invasion of the amniotic cavity (MIAC) has been traditionally performed using traditional cultivation techniques, which require growth of microorganisms in the laboratory. Shortcomings of culture methods include the time required (days) for identification of microorganisms, and that many microbes involved in the genesis of human diseases are difficult to culture. A novel technique combines broad‐range real‐time polymerase chain reaction with electrospray ionization time‐of‐flight mass spectrometry (PCR/ESI‐MS) to identify and quantify genomic material from bacteria and viruses.

Clinical chorioamnionitis at term I: Microbiology of the amniotic cavity using cultivation and molecular techniques

Abstract Introduction: The objectives of this study were: 1) to determine the amniotic fluid (AF) microbiology of patients with the diagnosis of clinical chorioamnionitis at term using both cultivation and molecular techniques; and 2) to examine the relationship between intra-amniotic inflammation with and without microorganisms and placental lesions consistent with acute AF infection. Methods: The AF samples obtained by transabdominal amniocentesis from 46 women with clinical signs of chorioamnionitis at term were analyzed using cultivation techniques (for aerobic and anerobic bacteria as well as genital mycoplasmas) and broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). The frequency of microbial invasion of the amniotic cavity (MIAC), intra-amniotic inflammation [defined as an AF interleukin 6 (IL-6) concentration ≥2.6 ng/mL], and placental lesions consistent with acute AF infection (acute histologic chorioamnionitis and/or acute funisitis) were examined according to the results of AF cultivation and PCR/ESI-MS as well as AF IL-6 concentrations. Results: 1) Culture identified bacteria in AF from 46% (21/46) of the participants, whereas PCR/ESI-MS was positive for microorganisms in 59% (27/46) – combining these two tests, microorganisms were detected in 61% (28/46) of patients with clinical chorioamnionitis at term. Eight patients had discordant test results; one had a positive culture and negative PCR/ESI-MS result, whereas seven patients had positive PCR/ESI-MS results and negative cultures. 2) Ureaplasma urealyticum (n=8) and Gardnerella vaginalis (n=10) were the microorganisms most frequently identified by cultivation and PCR/ESI-MS, respectively. 3) When combining the results of AF culture, PCR/ESI-MS and AF IL-6 concentrations, 15% (7/46) of patients did not have intra-amniotic inflammation or infection, 6.5% (3/46) had only MIAC, 54% (25/46) had microbial-associated intra-amniotic inflammation, and 24% (11/46) had intra-amniotic inflammation without detectable microorganisms. 4) Placental lesions consistent with acute AF infection were significantly more frequent in patients with microbial-associated intra-amniotic inflammation than in those without intra-amniotic inflammation [70.8% (17/24) vs. 28.6% (2/7); P=0.04]. Conclusion: Microorganisms in the AF were identified in 61% of patients with clinical chorioamnionitis at term; 54% had microbial-associated intra-amniotic inflammation, whereas 24% had intra-amniotic inflammation without detectable microorganisms.

Sterile intra-amniotic inflammation in asymptomatic patients with a sonographic short cervix: prevalence and clinical significance

Abstract Objective: To determine the frequency and clinical significance of sterile and microbial-associated intra-amniotic inflammation in asymptomatic patients with a sonographic short cervix. Methods: Amniotic fluid (AF) samples obtained by transabdominal amniocentesis from 231 asymptomatic women with a sonographic short cervix [cervical length (CL) ≤25 mm] were analyzed using cultivation techniques (for aerobic and anaerobic as well as genital mycoplasmas) and broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). The frequency and magnitude of intra-amniotic inflammation [defined as an AF interleukin (IL)-6 concentration ≥2.6 ng/mL], acute histologic placental inflammation, spontaneous preterm delivery (sPTD), and the amniocentesis-to-delivery interval were examined according to the results of AF cultures, PCR/ESI-MS and AF IL-6 concentrations. Results: Ten percent (24/231) of patients with a sonographic short cervix had sterile intra-amniotic inflammation (an elevated AF IL-6 concentration without evidence of microorganisms using cultivation and molecular methods). Sterile intra-amniotic inflammation was significantly more frequent than microbial-associated intra-amniotic inflammation [10.4% (24/231) versus 2.2% (5/231); p < 0.001]. Patients with sterile intra-amniotic inflammation had a significantly higher rate of sPTD <34 weeks of gestation [70.8% (17/24) versus 31.6% (55/174); p < 0.001] and a significantly shorter amniocentesis-to-delivery interval than patients without intra-amniotic inflammation [median 35, (IQR: 10–70) versus median 71, (IQR: 47–98) days, (p < 0.0001)]. Conclusion: Sterile intra-amniotic inflammation is more common than microbial-associated intra-amniotic inflammation in asymptomatic women with a sonographic short cervix, and is associated with increased risk of sPTD (<34 weeks). Further investigation is required to determine the causes of sterile intra-amniotic inflammation and the mechanisms whereby this condition is associated with a short cervix and sPTD.

Clinical chorioamnionitis at term III: how well do clinical criteria perform in the identification of proven intra-amniotic infection?

Abstract Objective: The diagnosis of clinical chorioamnionitis is based on a combination of signs [fever, maternal or fetal tachycardia, foul-smelling amniotic fluid (AF), uterine tenderness and maternal leukocytosis]. Bacterial infections within the amniotic cavity are considered the most frequent cause of clinical chorioamnionitis and an indication for antibiotic administration to reduce maternal and neonatal morbidity. Recent studies show that only 54% of patients with the diagnosis of clinical chorioamnionitis at term have bacteria in the AF and evidence of intra-amniotic inflammation. The objective of this study was to examine the performance of the clinical criteria for the diagnosis of chorioamnionitis to identify patients with microbial-associated intra-amniotic inflammation (also termed intra-amniotic infection). Materials and methods: This retrospective cross-sectional study included 45 patients with the diagnosis of clinical chorioamnionitis at term, whose AF underwent analysis for: 1) the presence of microorganisms using both cultivation and molecular biologic techniques [polymerase chain reaction (PCR) with broad primers], and 2) interleukin (IL)-6 concentrations by enzyme-linked immunosorbent assay. The diagnostic performance (sensitivity, specificity, accuracy, and likelihood ratios) of each clinical sign and their combination to identify clinical chorioamnionitis were determined using microbial-associated intra-amniotic inflammation [presence of microorganisms in the AF using cultivation or molecular techniques and elevated AF IL-6 concentrations (≥2.6 ng/mL)] as the gold standard. Results: The accuracy of each clinical sign for the identification of microbial-associated intra-amniotic inflammation (intra-amniotic infection) ranged between 46.7% and 57.8%. The combination of fever with three or more clinical criteria did not substantially improve diagnostic accuracy. Conclusion: In the presence of a fever during labor at term, signs used to diagnose clinical chorioamnionitis do not accurately identify the patient with proven intra-amniotic infection (i.e., those with microorganisms detected by culture or molecular microbiologic techniques and an associated intra-amniotic inflammatory response).

The frequency of acute atherosis in normal pregnancy and preterm labor, preeclampsia, small-for-gestational age, fetal death and midtrimester spontaneous abortion

Abstract Objective: Acute atherosis is characterized by subendothelial lipid-filled foam cells, fibrinoid necrosis and perivascular lymphocytic infiltration. This lesion is generally confined to non-transformed spiral arteries and is frequently observed in patients with preeclampsia. However, the frequency of acute atherosis in the great obstetrical syndromes is unknown. The purpose of this study was to determine the frequency and topographic distribution of acute atherosis in placentas and placental bed biopsy samples obtained from women with normal pregnancy and those affected by the “great obstetrical syndromes”. We also examined the relationship between acute atherosis and pregnancy outcome in patients with preeclampsia. Material and methods: A retrospective cohort study of pregnant women who delivered between July 1998 and July 2014 at Hutzel Women’s Hospital/Detroit Medical Center was conducted to examine 16 345 placentas. Patients were classified into the following groups: (1) uncomplicated pregnancy; (2) spontaneous preterm labor (sPTL) and preterm prelabor rupture of membranes (PPROM); (3) preeclampsia; (4) gestational hypertension; (5) small-for-gestational age (SGA); (6) chronic hypertension; (5) fetal death; (6) spontaneous abortion and (7) others. A subset of patients had placental bed biopsy. The incidence of acute atherosis was compared among the different groups. Results: (1) The prevalence of acute atherosis in uncomplicated pregnancies was 0.4% (29/6961) based upon examination of nearly 7000 placentas; (2) the frequency of acute atherosis was 10.2% (181/1779) in preeclampsia, 9% (26/292) in fetal death, 2.5% (3/120) in midtrimester spontaneous abortion, 1.7% (22/1,298) in SGA neonates and 1.2% (23/1,841) in sPTL and PPROM; (3) among patients with preeclampsia, those with acute atherosis than in those without the lesion had significantly more severe disease, earlier onset, and a greater frequency of SGA neonates (p < 0.05 all) and (4) the lesion was more frequently observed in the decidua (parietalis or basalis) than in the decidual segment of the spiral arteries in patients with placental bed biopsies. Conclusions: Acute atherosis is rare in normal pregnancy, and occurs more frequently in patients with pregnancy complications, including preeclampsia, sPTL, preterm PROM, midtrimester spontaneous abortion, fetal death and SGA.

Clinical chorioamnionitis at term VI: acute chorioamnionitis and funisitis according to the presence or absence of microorganisms and inflammation in the amniotic cavity

Abstract Objective: Neonates born to mothers with clinical chorioamnionitis at term are at an increased risk of infection. Acute subchorionitis, chorioamnionitis, and funisitis are considered placental histologic features consistent with acute inflammation according to the Society for Pediatric Pathology. The objectives of this study were to examine the performance of placental histologic features in the identification of: 1) microbial-associated intra-amniotic inflammation (intra-amniotic infection); and 2) fetal inflammatory response syndrome (FIRS). Methods: This retrospective cohort study included women with the diagnosis of clinical chorioamnionitis at term (n=45), who underwent an amniocentesis to determine: 1) the presence of microorganisms using both cultivation and molecular biologic techniques [polymerase chain reaction (PCR) with broad range primers]; and 2) interleukin (IL)-6 concentrations by enzyme-linked immunosorbent assay (ELISA). The diagnostic performance (sensitivity, specificity, accuracy, and likelihood ratios) of placental histologic features consistent with acute inflammation was determined for the identification of microbial-associated intra-amniotic inflammation and FIRS. Results: 1) The presence of acute histologic chorioamnionitis and funisitis was associated with the presence of proven intra-amniotic infection assessed by amniotic fluid analysis; 2) funisitis was also associated with the presence of FIRS; 3) the negative predictive value of acute funisitis ≥stage 2 for the identification of neonates born to mothers with intra-amniotic infection was <50%, and therefore, suboptimal to exclude fetal exposure to bacteria in the amniotic cavity; and 4) acute funisitis ≥stage 2 had a negative predictive value of 86.8% for the identification of FIRS in a population with a prevalence of 20%. Conclusion: Acute histologic chorioamnionitis and funisitis are associated with intra-amniotic infection and the presence of FIRS. However, current pathologic methods have limitations in the identification of the fetus exposed to microorganisms present in the amniotic cavity. Further studies are thus required to determine whether molecular markers can enhance the performance of placental pathology in the identification of neonates at risk for neonatal sepsis.

Clinical chorioamnionitis at term IV: the maternal plasma cytokine profile.

Abstract Introduction: Fever is a major criterion for clinical chorioamnionitis; yet, many patients with intrapartum fever do not have demonstrable intra-amniotic infection. Some cytokines, such as interleukin (IL)-1, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α), can induce a fever. The objective of this study was to determine whether maternal plasma concentrations of cytokines could be of value in the identification of patients with the diagnosis of clinical chorioamnionitis at term who have microbial-associated intra-amniotic inflammation. Methods: A retrospective cross-sectional study was conducted, including patients with clinical chorioamnionitis at term (n=41; cases) and women in spontaneous labor at term without clinical chorioamnionitis (n=77; controls). Women with clinical chorioamnionitis were classified into three groups according to the results of amniotic fluid culture, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), and amniotic fluid IL-6 concentration: 1) no intra-amniotic inflammation; 2) intra-amniotic inflammation without detectable microorganisms; or 3) microbial-associated intra-amniotic inflammation. The maternal plasma concentrations of 29 cytokines were determined with sensitive and specific V-PLEX immunoassays. Nonparametric statistical methods were used for analysis, adjusting for a false discovery rate of 5%. Results: 1) The maternal plasma concentrations of pyrogenic cytokines (IL-1β, IL-2, IL-6, IFN-γ, and TNF-α) were significantly higher in patients with clinical chorioamnionitis at term than in those with spontaneous term labor without clinical chorioamnionitis; 2) the maternal plasma concentrations of cytokines were not significantly different among the three subgroups of patients with clinical chorioamnionitis (intra-amniotic inflammation with and without detectable bacteria and those without intra-amniotic inflammation); and 3) among women with the diagnosis of clinical chorioamnionitis, but without evidence of intra-amniotic inflammation, the maternal plasma concentrations of pyrogenic cytokines were significantly higher than in patients with spontaneous labor at term. These observations suggest that a fever can be mediated by increased circulating concentrations of these cytokines, despite the absence of a local intra-amniotic inflammatory response. Conclusions: 1) The maternal plasma concentrations of pyrogenic cytokines (e.g. IL-1β, IL-2, IL-6, IFN-γ, and TNF-α) are higher in patients with intra-partum fever and the diagnosis of clinical chorioamnionitis at term than in those in spontaneous labor at term without a fever; and 2) maternal plasma cytokine concentrations have limited value in the identification of patients with bacteria in the amniotic cavity. Accurate assessment of the presence of intra-amniotic infection requires amniotic fluid analysis.

Placental lesions associated with acute atherosis

Abstract Objective: Acute atherosis is a lesion of the spiral arteries characterized by fibrinoid necrosis of the vessel wall, an accumulation of fat-containing macrophages, and a mononuclear perivascular infiltrate, which can be found in patients with preeclampsia, fetal death, small-for-gestational age, spontaneous preterm labor/premature prelabor rupture of membrane, and spontaneous mid-trimester abortion. This lesion is thought to decrease blood flow to the intervillous space which may lead to other vascular lesions of the placenta. The objective of this study was to test whether there is an association between acute atherosis and placental lesions that are consistent with maternal vascular underperfusion (MVU), amniotic fluid infection (AFI), fetal vascular thrombo-occlusive disease (FVTOD) or chronic inflammation. Material and methods: A retrospective cohort study of pregnant women who delivered between July 1998 and July 2014 at Hutzel Women’s Hospital/Detroit Medical Center was conducted examine 16 457 placentas. The frequency of placenta lesions (diagnosed using the criteria of the Perinatal Section of the Society for Pediatric Pathology) was compared between pregnancies with and without acute atherosis. Results: Among 16 457 women who were enrolled, 10.2% (1671/16 457) were excluded, leaving 14 786 women who contributed data for analysis. Among them, the prevalence of acute atherosis was 2.2% (326/14 786). Women with acute atherosis were more than six times as likely as those without to have placental lesions consistent with maternal underperfusion (adjusted odds ratio – aOR: 6.7; 95% CI 5.2–8.6). To a lesser degree, acute atherosis was also associated with greater risks of having either lesions consistent with FVTOD (aOR 1.7; 95% CI 1.2–2.3) or chronic chorioamnionitis (aOR 1.9; 95% CI 1.3–3), but not with other chronic inflammatory lesions, after adjusting for gestational age at delivery. In contrast, women with acute atherosis were 60% less likely to have lesions consistent with AFI, adjusting for gestational age at delivery (aOR 0.4; 95% CI 0.3–0.5). Conclusions: Acute atherosis is associated with increased risks of having placental lesions consistent with MVU, and to a lesser extent, chronic chorioamnionitis and those consistent with FVTOD.