Differences in testosterone and its precursors by sex of the offspring in meconium

Abstract

Prenatal metabolism exerts profound effects on development. The first stool of the newborn, meconium, provides a window into the prenatal metabolic environment. The objective of this study was to examine the feasibility of meconium as a novel matrix to quantify prenatal steroid levels. We quantified parameters of analytical interest regarding the use of meconium, including sample stability. We hypothesized that meconium steroid content would differ by sex, prompting analysis of meconium to test effects of prenatal steroid metabolism. Meconium from 193 newborns enrolled in the Early Autism Risk Longitudinal Investigation (EARLI) study, including 107 males, and 86 females, were analyzed by isotope dilution-liquid chromatography-high resolution mass spectrometry (ID-LC-HRMS) while blinded to identity for testosterone (T), androstenedione (AD), and dehydroepiandrosterone (DHEA). Steroid levels were compared by sex, and investigations of potential trends resulting from sample storage or processing was conducted. The unconjugated steroid content of meconium in ng/g (mean, standard deviation) was for males: T (2.67, 8.99), AD (20.01, 28.12), DHEA (13.96, 23.57) and for females: T (0.82, 1.63), AD (22.32, 24.38), DHEA (21.06, 43.49). T was higher in meconium from males (p=0.0333), and DHEA was higher in meconium from females (p=0.0202). 6 female and 3 male T values were below the limit of detection. No extreme variability in hydration or trend in steroid levels by storage time was detected. Sexually dimorphic levels of hormones may reflect gestational differentiation, and future studies should consider meconium analysis.

Keywords: Androgens; Mass spectrometry; Meconium; Prenatal development; Sexual differentiation; Steroids.

Figure 1

The hydration of meconium. Vacuum desiccation of meconium gave consistent dehydration. No significant differences found by collection time by one-way ANOVA, or by multiple comparisons.

Figure 2

Representative LC-HRMS and LC-MS/HRMS chromatograms. Chromatograms of steroid analysis from meconium extract for (A) DHEA, (B) T, (C) AD with HRMS (top chromatogram), stable isotope internal standard co-elution (bottom inverted chromatogram), MS/HRMS (dashed lines).

Figure 3

Standard curves from a comparison of surrogate matrices. Calibration curves prepared with standards spiked into water (triangle, dashed line), charcoal stripped serum (square, dotted line), dichloromethane stripped meconium (circle, solid line) were linear across physiologic ranges for both detected derivatives of DHEA (top), T (middle), and AD (bottom).

Figure 4

Recovery of steroids from meconium. (A) Analyte recovery from pooled meconium was tested via extraction from pooled meconium (white), dichloromethane stripped meconium (solid black), re-extraction of pooled meconium (vertical stripe) and as a negative control double charcoal stripped serum (cross-hatch). (B) Quantitative recovery from control meconium spiked with 5000 pg DHEA (white), 1000 pg AD (cross-hatch), and 1000 pg T (solid black). N = 3 for all experimental groups. SEM, standard error of measurement.

Figure 5

Steroids are stable in meconium under common storage conditions. Aliquots of homogenized pooled meconium were quantified for AD, DHEA and T content after bench room temperature (RT) storage for 24 hours, and either one (horizontal striped bar) or three (vertical striped bar) freeze/thaw (F/T) cycles as indicated.