ESPE Abstracts (2018) 89 P-P1-011

Characterizing the Steroidome in Ammniotic Fluid of Mid-gestation by LC-MS/MS

Rong Wanga, Dov Tiosanob, Michaela F Hartmanna & Stefan A Wudya


aSteroid Research & Mass Spectrometry Unit, Pediatric Endocrinology, Center of Child and Adolescent Medicine, Justus-Liebig-University, Giessen, Germany; bDivision of Pediatric Endocrinology, Meyer Children’s Hospital, Rambam Medical Center, Haifa, Israel


The amniotic fluid (AF) milieu is complex and essential to fetal well-being. Here we present a new LC-MS/MS method for the targeted metabolomics analysis of 20 unconjugated and conjugated steroids in 65 AF samples during mid-gestation. Sample preparation included protein precipitation, centrifugation, solid phase extraction and derivatization. We measured progesterone (Prog), 17α-hydroxyprogesterone (17OHProg), testosterone (T), estrone (E1), estradiol (E2), estriol (E3), estrone sulfate(E1S), 17β-estradiol 3-sulfate (E2S), estriol 3-sulfate(E3S), 17β-estradiol 17-sulfate (E2-17S), 16α-hydroxydehydroepiandrosterone sulfate (16OHDHEAS), dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate (PregS), 17α-hydroxypregnenolone sulfate (17OHPregS), testosterone sulfate (TS), epitestosterone sulfate (eTS), dihydrotestosterone sulfate (DHTS), androsterone sulfate (AnS), epiandrosterone sulfate (epiAnS), and 3,17β-androstenediol 3-sulfate (AnDiolS). Except DHEAS, all other sulfated steroids were quantified by LC-MS/MS in AF for the first time. The compounds with highest concentrations were Prog (16.39–78.55 ng/ml), 17OHProg (0.38–2.04 ng/ml), PregS (2.57–20.25 ng/ml), 17OHPregS (2.08–13.18 ng/ml), epiTS (2.92–17.81 ng/ml), DHEAS (1.54–12.27 ng/ml), 16OHDHEAS (6.87–62.91 ng/ml), AnS (0.90–39.39 ng/ml), E1S (0.38–25.26 ng/ml), E3 (0.59–2.60 ng/ml), E3S (2.16–20.99 ng/ml). We have produced MS based reference values for 20 steroids in AF of mid-gestation. DHEAS and E3S were both strongly correlated with 16OHDHEAS, thus confirming the classic concept of the feto-placental unit. Our LC-MS/MS method can be used for prenatal diagnosis of congenital adrenal hyperplasia and low-E3 diseases such as aromatase and ORD deficiencies, X-linked steroid sulfatase deficiency or Smith-Lemli-Opitz Syndrom.

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