SummaryTo establish normal parameters, we have measured 14 proteins in 120 amniotic fluid samples from normal second trimester pregnancies. Pregnancy-associated macroglobulin (PAM), identified in 15 samples, has served to indicate contamination with maternal blood; this represents the first time that PAM has been used for this purpose. Albumin, transferrin, α-1-antitrypsin, orosomucoid, IgG, and C3 show great variability in individual concentrations but are generally in proportion to each other, particularly transferrin/albumin, α-1-antitrypsin/albumin, and IgG/albumin. The first two ratios are similar to proportions in adult serum, supporting the concept that most amniotic fluid protein is maternal in origin and moves directly across the amnionic membrane. Levels of low density lipoproteine, C4, IgA, IgM, α-2-macroglobulin, and haptoglobin, all low in amniotic fluid, vary independently both of albumin and of each other. α-1-Fetoprotein levels are normal in all samples, suggesting freedom from gross fetal blood contamination. In the presence of maternal blood contamination, 10 of the 15 samples show clear elevation of at least one protein level; C3, IgA, and α-2-macroglobulin are most frequently abnormal.Similar-protein analyses have also been carried out on 11 samples from pregnancies with anencephaly, 5 with spina bifida, and 3 with fetal demise. Results have been compared with normal and blood-contaminated samples. Most protein concentrations are either high normal or abnormally elevated with anencephaly and fetal demise but not with spina bifida. Of all proteins studied, low density lipoprotein is most reliably elevated with neural tube defects and demise and is least affected by blood contamination. IgM and α-2-macroglobulin are also elevated in a significant percentage of uncontaminated samples. Increased IgA and haptoglobin levels correlate with blood contamination but not with neural tube defects. Low density lipoprotein, IgM, and α-2-macroglobulin may be useful adjuncts in the second trimester prenatal diagnosis of neural tube defects.SpeculationWell defined normal ranges for second trimester amniotic fluid proteins are a prerequisite for applying such measurements to diagnosing fetal dysfunction and understanding transamniotic interactions between mother and fetus. In defining such parameters, the present study first re-enforces the necessity to identify blood contamination and then establishes low density lipoprotein, IgM, and α-2-macroglobulin measurements as potentially useful ancillary studies for the prenatal diagnosis of neural tube defects. Similar testing on samples associated with omphalocele, congenital nephrosis, esophageal atresia, and other major fetal malformations must be carried out to determine whether these measurements may be of further help in second trimester differential diagnosis.