Amnion

Gestational age was determined by the last menstrual period and was confirmed by ultrasound examination; the date derived from ultrasound was used if inconsistent with menstrual dating. Clinical chorioamnionitis was diagnosed by the presence of maternal fever (temperature > 37.8°C) accompanied by two or more of the following criteria: 1) uterine tenderness; 2) malodorous vaginal discharge; 3) fetal tachycardia (heart rate > 160 beats/min); 4) maternal tachycardia (heart rate > 100 beats/min); and 5) maternal leukocytosis (leukocyte count > 15,000 cells/mm³) [6 (link), 16 (link)]. Spontaneous term labor was defined as the presence of regular uterine contractions with a frequency of at least 1 every 10 min and cervical changes after 37 weeks of gestation.
Microbial invasion of the amniotic cavity was defined according to the results of AF culture and polymerase chain reaction with electrospray ionization mass spectrometry (PCR/ESI-MS) (Ibis® Technology – Athogen, Carlsbad, CA) [51 (link), 55 (link), 61 (link), 62 (link)]. Intra-amniotic inflammation was diagnosed when AF interleukin (IL)-6 concentration was ≥ 2.6 ng/mL [63 (link), 64 ]. Based on the results of AF cultures, PCR/ESI-MS and AF concentration of IL-6, patients were classified as having: 1) no intra-amniotic inflammation/infection (either using AF culture or PCR/ESI-MS); 2) MIAC (identification of microorganisms by either AF cultures or PCR/ESI-MS without intra-amniotic inflammation); 3) microbial-associated intra-amniotic inflammation (combination of MIAC and intra-amniotic inflammation); or 4) intra-amniotic inflammation without detectable microorganisms (an elevated AF IL-6 concentration without evidence of microorganisms using cultivation or molecular methods). Acute histologic chorioamnionitis was diagnosed based on the presence of inflammatory cells in the chorionic plate and/or chorioamniotic membranes [65 (link)], and acute funisitis was diagnosed by the presence of neutrophils in the wall of the umbilical vessels and/or Wharton’s jelly, using criteria previously described [65 (link), 66 (link)].

Preterm labor was diagnosed by the presence of regular uterine contractions (at least 3 in 30 minutes) and documented cervical changes in patients with a gestational age between 20 and 36 6/7 weeks. Preterm delivery was defined as birth prior to the 37^th week of gestation. MIAC was defined as either a positive culture for bacteria in AF or the detection of microbial footprints for either viruses or bacteria, using polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (ESI-MS) (Ibis® technology - Athogen, Carlsbad, CA). Intra-amniotic inflammation was diagnosed when AF IL-6 concentration was ≥ 2.6 ng/mL.^{72 (link),155 (link)} Microbial-associated intra-amniotic inflammation was defined as the presence of MIAC with intra-amniotic inflammation. Sterile intra-amniotic inflammation was diagnosed when the AF IL-6 concentration was ≥ 2.6 ng/mL and there was no evidence of microbial footprints for viruses or bacteria (negative AF culture and no detection of microbial footprints using PCR/ESI-MS).
Composite neonatal morbidity was defined as the presence of: respiratory distress syndrome, bronchopulmonary dysplasia, grade III or IV intraventricular hemorrhage, periventricular leukomalacia, proven neonatal sepsis, necrotizing enterocolitis or perinatal mortality. The diagnostic criteria of these complications have been previously reported.^{156 (link)} Acute placental inflammation was diagnosed based on the presence of inflammatory cells in the chorionic plate, chorioamniotic membranes (histologic chorioamnionitis),^{157 (link)-159 (link)} and/or umbilical cord (funisitis).^{157 (link),158 (link)}

Gestational age was determined by the last menstrual period and confirmed by ultrasound examination, or by ultrasound examination alone if the sonographic determination of gestational age was not consistent with menstrual dating. Preterm prelabor rupture of membranes was diagnosed with a sterile speculum examination with documentation of pooling of amniotic fluid in the vagina in association with a positive nitrazine test and/or and positive ferning tests when necessary. Clinical chorioamnionitis was diagnosed when maternal temperature was elevated to 37.8° C and two or more of the following criteria were present: uterine tenderness, malodorous vaginal discharge, maternal leukocytosis (>15,000 cells/mm³), maternal tachycardia (>100 beats/min), and fetal tachycardia (>160 beats/min) [58 (link),59 (link)].
The presence of microorganisms in the amniotic cavity was defined according to the results of AF culture and PCR/ESI-MS (Ibis^® Technology - Athogen, Carlsbad, CA) [60 (link)–63 (link)]. Intra-amniotic inflammation was diagnosed when AF interleukin (IL)-6 concentration was ≥ 2.6 ng/mL [64 (link),65 (link)]. Based on the results of AF culture, PCR/ESI-MS and AF concentration of IL-6, patients were classified as: 1) no intra-amniotic inflammation/infection (either using AF culture or PCR/ESI-MS); 2) microbial invasion of the amniotic cavity (MIAC) (identification of microorganisms by either AF cultures or PCR/ESI-MS without intra-amniotic inflammation); 3) microbial-associated intra-amniotic inflammation (combination of MIAC and intra-amniotic inflammation); or 4) sterile intra-amniotic inflammation (an elevated AF IL-6 concentration without evidence of microorganisms using cultivation or molecular methods). Acute histologic chorioamnionitis was diagnosed based on the presence of inflammatory cells in the chorionic plate and/or chorioamniotic membranes [66 (link),67 (link)], and acute funisitis was diagnosed by the presence of neutrophils in the wall of the umbilical vessels and/or Wharton’s jelly, using criteria previously described [66 (link)–68 (link)]. For all newborns, data records regarding morbidity and mortality were reviewed. Neonatal outcome was assessed by measuring composite neonatal morbidity and mortality, defined as the presence of one or more of the following: bronchopulmonary dysplasia, respiratory distress syndrome, necrotizing enterocolitis, intraventricular hemorrhage ≥ grade III, and respiratory failure requiring mechanical ventilation. Perinatal mortality (stillbirth and neonatal death) were documented separately.