Characterizing the steroidal milieu in amniotic fluid of mid-gestation: A LC-MS/MS study.

Growth and development of an embryo or fetus during human pregnancy mainly depend on intact hormone biosynthesis and metabolism in maternal amniotic fluid (AF). We investigated the hormonal milieu in AF and developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of 14 sulfated and 6 unconjugated steroids in AF. 65 A F samples (male: female = 35: 30) of mid-gestation ranging from 16th week of gestation to 25th week of gestation were analyzed. Reference data of 20 steroid levels in AF of healthy women were provided. 13 sulfated and 3 unconjugated steroids were for the first time quantified in AF by LC-MS/MS. Highest concentrations were found for pregnenolone sulfate (PregS: mean ± SD, 8.6 ± 3.7 ng/mL), 17α-hydroxypregnenolone sulfate (17OHPregS: 4.9 ± 2.0 ng/mL), epitestosterone sulfate (eTS: 7.3 ± 3.6 ng/mL), 16α-hydroxydehydroepiandrosterone sulfate (16OH-DHEAS: 21.5 ± 10.7 ng/mL), androsterone sulfate (AnS: 9.2 ± 7.4 ng/mL), estrone sulfate (E1S: 3.0 ± 3.0 ng/mL), estriol 3-sulfate (E3S: 8.1 ± 4.0 ng/mL) and estriol (E3: 1.2 ± 0.4 ng/mL). Only testosterone (T) showed a significant sex difference (p < 0.0001). Correlations between AF steroids mirrored the steroid metabolism of the feto-placental unit, and not only confirmed the classical steroid pathway, but also pointed to a sulfated steroid pathway.