1H HR‐MAS spectroscopy for quantitative measurement of choline concentration in amniotic fluid as a marker of fetal lung maturity: Inter‐ and intraobserver reproducibility study

Respiratory distress syndrome (RDS) is a major cause of neonatal morbidity and mortality due to insufficient surfactant production in the lungs (1–3). The disease primarily affects premature infants and is the seventh leading cause of death in infants under 1 year of age (1,2). Excreted surfactant aggregates to form a monolayer complex that reduces alveolar surface tension and facilitates alveolar inflation (4). Insufficient surfactant at birth results in collapse of the fetal alveoli and RDS. Surfactant is composed predominantly of phosphatidylcholine (lecithin) (70%) with lesser amounts of other phospholipids, including phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylinositol (5). Measurement of the lecithin-to-sphingomyelin (LS) ratio in amniotic fluid samples is traditionally considered to be the gold standard for fetal lung maturity testing, but the assay is time-consuming and technically challenging to perform. Consequently, many hospitals now measure the surfactant-to-albumin (SA) ratio, which is a faster and simpler test, although there is not good concordance between the two measurements (6). These traditional methods of evaluating fetal lung maturity have a number of disadvantages. Analysis of amniotic fluid requires amniocentesis, with the associated risks of this invasive procedure including infection and miscarriage (7,8). The tests are also associated with a high rate of false-positive results in fetuses with intermediate lung maturity, even when several different measurements are incorporated into a more comprehensive assessment (9–11). In these cases the laboratory tests can indicate fetal lung maturity yet the fetus may still develop RDS (12). Choline, a major component of surfactant, can be easily detected and quantified by 1H MR spectroscopy ex vivo. Case reports of 1H MRS of amniotic fluid pockets in utero have generated interest in pursuing this technique for the potential noninvasive evaluation of fetal lung maturity in vivo (13–15). However, in vivo studies are lower in spectroscopic resolution and sensitivity, and are expensive and technically challenging to perform (16). Accordingly, a systematic study of amniotic fluid samples ex vivo would be useful to establish metabolic markers of fetal lung maturity (such as choline) and provide proof of concept to justify pursuing in vivo protocol development. High-resolution magic angle spinning (HR-MAS) spectroscopy is an ex vivo MRS technique that can be applied to intact tissues, cells, and biofluids. Although HR-MAS spectroscopy is normally used to improve spectral resolution, in the case of amniotic fluid, which contains mostly water, the small sample volume relative to the coil size allows for much better water suppression than a conventional liquids probe. Because reproducibility of measurements is an important fundamental parameter that needs to be established in such studies, the reproducibility of the MRS quantification technique first needs to be evaluated. Thus, the purpose of this study was to determine the intra- and interobserver reproducibility of metabolite concentration measurements (including choline concentration) detectable by 1H HR-MAS spectroscopy using a semiautomated software program for peak fitting.