Chorion

In each case, hematoxylin and eosin (H&E) stained sections of the chorioamniotic membranes roll (n=1), umbilical cord (n=1), and placental disc (n=3) were examined. Pathologists were masked to the clinical diagnosis except for the gestational age at delivery. The diagnosis of CCA was made when lymphocytic infiltration into the chorionic trophoblast layer or chorioamniotic connective tissue was observed. The severity of CCA was scored based upon on two parameters. The extent of inflammation was graded 0 when there was no inflammation, 1 when there were more than two foci of or patchy inflammation, and 2 when diffuse inflammation was present. The stage of inflammation was scored as stage 1 if amniotropic lymphocytic infiltration was limited to the chorionic trophoblast layer sparing the chorioamniotic connective tissue, and stage 2 if lymphocytic infiltration into the chorioamniotic connective tissue was noted. Histopathological screening for other lesions of the placenta was performed according to the diagnostic criteria proposed by the Perinatal Section of the Society for Pediatric Pathology. Such classification encompasses lesions consistent with amniotic fluid infection, maternal vascular underperfusion, and fetal vascular obstruction.^{10 (link),25 (link),26 (link)} The diagnosis of chronic deciduitis with plasma cells was given when lymphoplasmacytic infiltrate was present in the decidua of the basal plate.^{27 (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.