Pregnanolone

Neuroactive steroids are metabolites of progesterone, synthesised during pregnancy by the placenta. Here, we describe development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantitation of allopregnanolone, pregnanolone, isopregnanolone, epipregnanolone and allopregnan-20α-ol-3-one in maternal serum. Following addition of deuterated internal standards, 200 μL of serum was subjected to solid phase extraction. Chromatography was performed using a pentafluorophenyl column, and LC-MS/MS on a Sciex 6500+. Sample injection volume was 20 μL, and injection-to-injection time 10.0 min. The assay was validated according to published guidelines; assay linearity and lower limit of quantification were suitable for analysis of each steroid in maternal serum, for all analytes mean recoveries were 100 % ± 15 %, intra- and inter-assay imprecision <15 %, and matrix effects negligible, and specificity experiments confirmed nil interference from a wide range of endogenous metabolites of progesterone. The method was applied to human serum samples obtained from a large cohort of third trimester pregnancies which were subsequently characterised by normal fetal and maternal outcomes, and relationships between maternal neuroactive steroid concentrations and fetal gestational age assessed. Positive correlations between maternal serum concentration and fetal gestational age were observed for isopregnanolone, allopregnanolone and allopregnan-20α-ol-3-one. The LC-MS/MS method offers significant advantages over previously published approaches for quantitation of neuroactive steroids in human maternal serum, notably obviating the need for derivatisation, whilst achieving exceptional specificity. Characterisation of normal maternal neuroactive steroid concentrations will aid future research as dysregulated placental progesterone metabolism is observed in pregnancies with poor outcomes.

Pregnane derivatives such as pregnenolone or progesterone and many other metabolites are important in mammals where many of them act as hormones including sexual hormones. Much less is known about the presence and functions of pregnane derivatives in plants. The main objectives of this work were (1) to determine the presence of pregnane derivatives in winter wheat (2) verify if there are changes of concentration of pregnane derivatives during wheat growth/development with special attention to vernalisation process (3) to answer the question of whether selected pregnane derivatives are stimulators of wheat development and whether the potential stimulation of this development is accompanied by the expression of the Vrn1 (Vernalisation1) gene. To the best of our knowledge, this is the first report that demonstrates the presence of pregnenolone and 5α-dihydroprogesterone in the leaves and intact crowns of winter wheat. The levels of some of the pregnane derivatives changed during plant growth/development, it was demonstrated that pregnenolone, pregnanolone and 17α-hydroxypregnenolone stimulated wheat development. The changes in the Vrn1 expression are discussed in light of the stimulation of generative development by the pregnane derivatives.