In total, 113 women with PPROM were recruited. Results regarding histopathological assessment of the Tipifarnib placenta and PCR analysis for genital mycoplasmas were not available for 8 (7%) and 2 (2%) women, respectively. Therefore, the remaining 103 women were included in the replication cohort. In all pregnancies, the gestational age was established based on first trimester ultrasound evaluation. Women who met the following criteria were eligible for enrollment in the replication cohort: a singleton pregnancy with PPROM, maternal age higher than 18 years, no signs of small for gestational age (estimated fetal weight by ultrasound below 10th percentiles for gestational age), no fetal structural malformations or chromosomal abnormalities, no maternal complications (hypertension, preeclampsia, diabetes mellitus, and thyroid disease), and no other medical complications during pregnancies.

Exclusion criteria were vaginal bleeding or signs of fetal hypoxia. Altogether, 40 women confirmed to have both MIAC and HCA were compared against 63 women in whom at least one of these conditions was ruled out. Proteomics Exploratory Phase Generation of pooled samples Protein concentration was determined using the bicinchoninic acid assay kit (Pierce, Rockford, IL) in each of 38 exploratory cohort samples. An equal amount of protein was taken from each sample to create a pooled MIAC- and HCA-positive and a pooled negative sample, both in duplicate (Fig. S1). Each pooled sample contained 2 mg of protein.

The volume was adjusted to 4 ml using the Multiple Affinity Removal System (MARS) buffer A (Agilent, Palo Alto, CA), and samples were concentrated using Amicon Ultra filters (Millipore, Bedford, MA) with a 3 kDa molecular weight cut-off. The retenates were collected and adjusted to 200 ��l with MARS buffer A. Immunoaffinity depletion of high-abundance amniotic fluid proteins The 14 abundance proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha-2-macroglobulin, alpha-1-acid glycoprotein, IgM, apolipoprotein Al, apolipoprotein All, complement C3, and transthyretin) were removed using the MARS Hu-14 column (Agilent) on an Alliance 2695 HPLC system (Waters, Milford, MA) according to the manufacturer��s instruction. The flow-through Cilengitide fraction was collected between 5th and 22nd minute of separation. MARS buffer was exchanged three times for water using the 3 kDa cut-off Amicon Ultra filters. The retenates were collected, and total protein concentration was determined by the bicinchoninic acid assay. Trypsin digestion From each replicate, 200 ��g of protein was brought to 40 ��l of 250 mM triethylammonium bicarbonate buffer, pH 8.5 (Sigma, St. Louis, MO) containing 0.1% RapiGest (Waters).