Antonín Pařízek

Steroid metabolome in plasma from the umbilical artery, umbilical vein, maternal cubital vein and in amniotic fluid in normal and preterm labor.

The boost in placental production of CRH in late pregnancy is specific for human. CRH receptors are expressed in the fetal zone of the fetal adrenal (FZFA). Hence, we evaluated the associations between the steroid metabolome and gestational age (GA). The levels of 69 steroids and steroid polar conjugates such as 3beta-hydroxy-5-ene steroids (3betaOH5S), 3-oxo-4-ene steroids (3O4S), progesterone 5alpha/beta-reduced metabolites, 20alpha-hydroxy-metabolites of C21 steroids, C19 5alpha/beta-reduced metabolites, 7alpha/beta-hydroxy-metabolites of 3betaOH5S, estrogens and 16alpha-hydroxy-metabolites of 3betaOH5S and 3O4S, were measured by GC-MS in plasma from the umbilical artery (UA), umbilical vein (UV), and maternal cubital vein (MV) and in amniotic fluid (AF) in 12 women at normal labor and 38 women at preterm labor due to pathologies unrelated to steroid status. Using multivariate regression, prediction models for GA were completed for the individual body fluids. The conjugated 3betaOH5S (the key products of the FZFA), estrogens, some polar conjugates of progesterone 5alpha/beta-reduced metabolites and some steroid 7alpha/beta- and 16alpha-hydroxy-metabolites showed strong positive correlations with the GA. The predictivity decreased in the following sequence UV (R=0.950), UA (R=0.945), MV (R=0.895), and AF (R=0.891). Although the predictivity of steroids in maternal blood was slightly less effective when compared with the UV and UA, it was the best solution for further practice.

Steroid metabolome in fetal and maternal body fluids in human late pregnancy

Despite the extensive research during the last six decades the fundamental questions concerning the role of steroids in the initiation of human parturition and origin and function of some steroids in pregnancy were not definitely answered. Based on steroid metabolomic data found in the literature and our so far unpublished results, we attempted to bring new insights concerning the role of steroids in the sustaining and termination of human pregnancy, and predictive value of these substances for estimation of term. We also aimed to explain enigmas concerning the biosynthesis of progesterone and its bioactive catabolites considering the conjunctions between placental production of CRH, synthesis of bioactive steroids produced by fetal adrenal, localization of placental oxidoreductases and sustaining of human pregnancy. Evaluation of data available in the literature, including our recent findings as well as our new unpublished data indicates increasing progesterone synthesis and its concurrently increasing catabolism with approaching parturition, confirms declining production of pregnancy sustaining 5β-pregnane steroids providing uterine quiescence in late pregnancy, increased sulfation of further neuroinhibiting and pregnancy sustaining steroids. In contrast to the established concept considering LDL cholesterol as the primary substrate for progesterone synthesis in pregnancy, our data demonstrates the functioning of alternative mechanism for progesterone synthesis, which is based on the utilization of fetal pregnenolone sulfate for progesterone production in placenta. Close relationships were found between localization of placental oxidoreductases and consistently higher levels of sex hormones, neuroactive steroids and their metabolites in the oxidized form in the fetus and in the reduced form in the maternal compartment.

Circulating levels of pregnanolone isomers during the third trimester of human pregnancy

This study addresses the question of whether changes in the biosynthesis and metabolism of neuroactive pregnanolone isomers (PIs) might participate in the timing of parturition in humans. The time profiles of unconjugated allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one, P3alpha5alpha), pregnanolone (3alpha-hydroxy-5beta-pregnan-20-one, P3alpha5beta), isopregnanolone (3beta-hydroxy-5alpha-pregnan-20-one, P3beta5alpha) and epipregnanolone (3beta-hydroxy-5beta-pregnan-20-one, P3beta5beta), pregnenolone, their polar conjugates, progesterone, 5alpha-dihydroprogesterone (P5alpha), and 5beta-dihydroprogesterone (P5beta) were monitored in the plasma of 30 healthy women during the third trimester of pregnancy, at 1-week intervals from the 30th week of gestation using GC-MS. Changes in the steroid levels were evaluated by two-way ANOVA with gestational age and subject as independent factors. The mean concentrations of free PIs ranged from 2 to 50 nmol/L, while the mean levels of their polar conjugates were 40-100 x higher. The ratio of 5alpha-PIs to progesterone significantly but inconspicuously culminated in the 35th week. The decelerating biosynthesis of free 5beta-PIs from the 31st week and their escalating sulfation was found from the 30th week. The changes were particularly evident in the second most abundant PI pregnanolone that may, like the allopregnanolone, sustain the pregnancy via attenuation of hypothalamic GABA(A)-receptors and prevent uterine contractility via binding to nuclear pregnane X receptor.

Neuroactive steroids, their precursors, and polar conjugates during parturition and postpartum in maternal and umbilical blood: 1. Identification and simultaneous determination of pregnanolone isomers.

A rapid method for the identification and measurement of four pregnanolone isomers and their polar conjugates in human plasma was developed using a simple quadrupole GC/MS system with electron impact ionization. Steroid levels were measured in the plasma of 13 and three women at delivery with subarachnoidal and epidural analgesia, respectively, and in corresponding samples of umbilical plasma. A good correlation (r=0.94, P<0.001, n=8) was found between the allopregnanolone in maternal plasma determined by GC/MS and that measured by RIA. Epipregnanolone (3beta-hydroxy-5beta-pregnan-20-one) was identified and measured for the first time in human plasma; its concentration in both maternal and umbilical plasma was much lower than that of other pregnanolone isomers. The levels of 3beta-hydroxy-pregnanolone isomers were significantly higher in the umbilical plasma than in the maternal plasma, while the differences in 3alpha-hydroxy-isomers were insignificant. The differences in conjugates were insignificant except in the case of allopregnanolone, the levels of which were lower in umbilical plasma. In all of the pregnanolone isomers, a significantly lower conjugated/unconjugated steroid ratio was found in the umbilical plasma than in the maternal plasma. The possible role of the sulfatation of pregnanolone isomers around parturition is discussed.

Neuroactive steroids, their precursors and polar conjugates during parturition and postpartum in maternal blood: 2. Time profiles of pregnanolone isomers

Time profiles of the pregnanolone isomers epipregnanolone (3 beta-hydroxy-5 beta-pregnan-20-one), allopregnanolone (3 alpha-hydroxy-5 alpha-pregnan-20-one), pregnanolone (3 alpha-hydroxy-5 beta-pregnan-20-one), and isopregnanolone (3 beta-hydroxy-5 alpha-pregnan-20-one) were measured around parturition and in the postpartum period in the serum of 13 and three women with subarachnoidal and epidural analgesia, respectively. In addition, the levels of polar conjugates of all pregnanolone isomers were followed during parturition. GC/MS analysis was used for the measurement of steroid levels. Changes in concentrations of free steroids exhibited a similar pattern, with a fall primarily within the first hour after delivery. The decrease in conjugated steroids was shifted to the interval within the first hour and first day after delivery, and the changes were more pronounced. The time profile of the conjugated/free steroid ratio exhibited a significant decrease within the first hour and the first day after delivery in all of the isomers investigated. A decrease was also observed in the ratio of 3 alpha/3 beta-isomers and 5 alpha/5 beta-isomers around parturition. The possible physiological consequences of the findings are indicated.

Soluble receptor for advanced glycation end products in physiological and pathological pregnancy

OBJECTIVE The receptor for advanced glycation end products, RAGE, has been implicated in pathogenesis of many diseases. Soluble RAGE, sRAGE, extracellular domain of RAGE, is new biomarker. The aim of the study was to determine sRAGE levels in physiological pregnancy and their changes in pregnancies complicated by preterm labor or preeclampsia. DESIGN AND METHODS Serum levels of sRAGE were determined in 79 healthy pregnant women, 42 pregnant women in preterm labor or with preeclampsia and 24 non-pregnant controls. RESULTS sRAGE serum levels are decreased in physiological pregnancy compared to healthy non-pregnant controls (p<0.001). Serum sRAGE concentrations are higher in the 2nd trimester of physiological pregnancy, compared to the 1st and 3rd trimesters of pregnancy (p<0.001). sRAGE levels in women with preterm labor are decreased (p<0.05) and correlate negatively with the leukocyte count (r=-0.47, p<0.05). In women with preeclampsia, sRAGE is elevated (p<0.05) and correlates with serum creatinine concentration (r=0.54, p<0.05) and with uric acid concentration (r=0.51, p<0.05). CONCLUSION Our results clearly demonstrate significant differences in serum sRAGE levels in physiological pregnancy and in pathological states in pregnancy, however, further studies are required demonstrate the usefulness and significance of sRAGE.

Determination of 17α-hydroxypregnenolone sulfate and its application in diagnostics

New combined radioimmunoassay for determination of 17-hydroxypregnenolone sulfate (17-PregS) involving the hydrolysis of analyte by methanolysis was developed. 17-PregS, in addition to being secreted by the adrenals, is also formed by peripheral sulfoconjugation of 17-hydroxypregnenolone (17-Preg) or directly by hydroxylation of pregnenolone sulfate with 17alpha-hydroxylase/C17-20lyase. The measurement of 17-PregS can be used as a tool for detection of enzymatic deficiency particularly in pregnancy and for detection of congenital adrenal hyperplasia or gonadal dysfunction. The serum levels of 17-PregS, 17-Preg, dehydroepiandrosterone, dehydroepiandrosterone sulfate, pregnenolone and pregnenolone sulfate were measured in different age groups of human and in pregnant women respecting the age of gestation. The levels of 17-PregS are approximately three times higher than the levels of free 17-Preg in all subject groups. The levels of 17-PregS during pregnancy reached the local minimum in the 3rd month of gestation. The ratio of 17-PregS to free 17-Preg showed increasing profile during pregnancy with a maximum in the 8th month of gestation. These findings indicate that, the conversion of pregnenolone sulfate to 17-PregS is the major metabolic pathway for biosynthesis of 17-PregS.

The steroid metabolome in lamotrigine-treated women with epilepsy.

BACKGROUND Epilepsy in women may be associated with reproductive disorders and alterations in serum steroid levels. Some steroids can be induced by epilepsy and/or treatment with antiepileptic drugs; however, there are still limited data available concerning this effect on the levels of other neuroactive steroid metabolites such as 3a-hydroxy-5a/b-reduced androstanes. AIM To evaluate steroid alterations in women with epilepsy (WWE) on lamotrigine monotherapy. SUBJECTS AND METHODS Eleven WWE and 11 age-matched healthy women underwent blood sampling in both phases of their menstrual cycles (MCs). The steroid metabolome, which included 30 unconjugated steroids, 17 steroid polar conjugates, gonadotropins, and sex hormone-binding globulin (SHBG), was measured using gas chromatography-mass spectrometry (GC-MS) and radioimmunoassay (RIA). RESULTS WWE had lower cortisol levels (status p<0.001), but elevated levels of unconjugated 17-hydroxypregnenolone (status p<0.001). Progesterone was higher in the follicular menstrual phase (FP) in WWE than in the controls (status×menstrual phase p<0.05, Bonferroni multiple comparisons p<0.05), whereas 17-hydroxyprogesterone was higher in WWE in both menstrual phases (status p<0.001). The steroid conjugates were mostly elevated in WWE. The levels of 5α/β-reduced androstanes in WWE that were significantly higher than the controls were etiocholanolone (status p<0.001), 5α-androstane-3α,17β-diol (status p<0.001), and the 5α/β-reduced androstane polar conjugates (status p<0.001). CONCLUSIONS WWE showed a trend toward higher circulating 3α-hydroxy-5α/β-reduced androstanes, increased activity of 17α-hydroxylase/17,20 lyase in the Δ(5)-steroid metabolic pathway, and increased levels of the steroid polar conjugates.

Neuroactive steroids, their precursors and polar conjugates during parturition and postpartum in maternal and umbilical blood: 3.3β-hydroxy-5-ene steroids

Five 3beta-hydroxy-5-ene steroids involved in the metabolic route from pregnenolone sulfate to dehydroepiandrosterone and its sulfate, of which three are known allosteric modulators of neurotransmitter receptors, were monitored in the serum of 20 women around parturition. In addition, their levels in maternal and umbilical serum were compared at delivery. On the basis of these data, a scheme of steroid biosynthesis in maternal organism during the critical stages around parturition is proposed. In maternal serum, all the steroids except dehydroepiandrosterone sulfate decreased during labor and even first day after delivery, although their changes were less distinct the more distant from pregnenolone sulfate (PregS) in the metabolic pathway. Calculation of product/immediate precursor ratios in maternal serum over all stages around parturition enabled identification of the respective changes in the activities of the relevant enzymes. The ratio of 17-hydroxypregnenolone/pregnenolone did not change significantly, while that of dehydroepiandrosterone/17-hydroxypregnenolone grew, indicating increased C17,20 side chain cleavage on the account of C17-hydroxylation both catalyzed by C17-hydroxylase-C17,20-lyase. As was shown by factor analysis, the changes in the maternal steroids were associated with a single common factor, which strongly correlated with all the steroids except dehydroepiandrosterone sulfate. The lack of change in the pregnenolone sulfate/pregnenolone ratio and a marked increase of the ratio dehydroepiandrosterone sulfate to unconjugated dehydroepiandrosterone indicate a different means of formation of both steroid sulfates. On the basis of these data, a scheme of steroid biosynthesis in maternal organism during the critical stages around parturition is proposed.

Fetal complications due to intrahepatic cholestasis of pregnancy

Abstract Intrahepatic cholestasis of pregnancy (ICP) is the most common liver disorder of pregnancy. Diagnosis is based on the clinical picture, particularly the presence of pruritus with a deterioration of liver function tests, and typically elevated serum levels of total bile acids. ICP manifests in the second half of pregnancy, predominantly during the third trimester. Symptoms of the disease resolve spontaneously after delivery. Etiology is still not fully understood. Genetic defects in specific transport proteins, elevated levels of sex hormones, and various environmental factors are thought to play a role in the development of this disorder. Although practically benign for the pregnant woman, ICP represents a serious threat to the fetus. It increases the risk of preterm delivery, meconium excretion into the amniotic fluid, respiratory distress syndrome, and sudden intrauterine fetal death. Identifying fetuses at risk of ICP complications remains challenging. The ideal obstetrical management of ICP needs to be definitively determined. The aim of this review is to summarize the current knowledge on fetal complications of ICP and describe management options for their prevention.