Amniocentesis Fluid Analysis Protocol

Enrolled subjects underwent transabdominal, ultrasound-guided amniocentesis, which is within the standard of care at Hutzel Women's Hospital for evaluating possible microbial invasion of the amniotic cavity of patients with spontaneous preterm labor. Amniotic fluid was immediately transported in a capped sterile syringe to the clinical laboratory where it was cultured for aerobic and anaerobic bacteria, including genital mycoplasmas. White blood cell (WBC) count and Gram stain of amniotic fluid were also performed shortly after collection. Amniotic fluid not required for clinical assessment was centrifuged for 10 minutes at 4°C shortly after the amniocentesis, and the supernatant was aliquoted and stored at −70°C until analysis. Amniotic fluid IL-6 concentrations were determined after delivery for research purposes, and these results were not used in patient management. A flowchart of our overall approach to amniotic fluid analysis is illustrated in Supporting Figure S1; detailed experimental methods, including microbiologic techniques and IL-6 quantitation, appear in Supporting Materials and Methods S1.