Tomas Fait

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.

Oral but not transdermal estrogen replacement therapy changes the composition of plasma lipoproteins

The role of hormone replacement therapy and estrogen replacement therapy (ERT) in cardiovascular disease prevention has not been unambiguously defined yet. The metabolic effects of estrogens may vary depending upon the route of administration. Therefore, we compared the impact of unopposed oral or transdermal ERT on plasma lipids and lipoproteins in 41 hysterectomized women. This was an open-label, randomized, crossover study (with 2 treatments and 2 periods). The 41 hysterectomized women were randomized to receive oral or transdermal 17beta-estradiol in the first or second of two 12-week study periods. Plasma lipid and lipoprotein levels were assayed before and after each treatment using standard automated methods. Lipid content of lipoprotein subclasses was assessed by sequential ultracentrifugation. The atherogenic index of plasma (AIP) was calculated as log(triglyceride [TG]/high-density lipoprotein [HDL] cholesterol). The difference between the 2 forms of administration was tested using a linear mixed model. The change from baseline for each of the forms was tested using paired t test. Oral ERT resulted in a significant increase in HDL cholesterol and apolipoprotein A-I levels, whereas it significantly decreased total and low-density lipoprotein (LDL) cholesterol and increased TG concentrations. Transdermal ERT had no such effect. Oral ERT led to a significant TG enrichment of HDL (0.19 +/- 0.06 vs 0.27 +/- 0.07 mmol/L, P < .001) and LDL particles (0.23 +/- 0.08 vs 0.26 +/- 0.10 mmol/L, P < .001) compared with baseline, whereas transdermal therapy did not have any effect on lipoprotein subclasses composition. The difference between the 2 treatments was statistically significant for HDL-TG and LDL-TG (0.27 +/- 0.07 vs 0.19 +/- 0.05 mmol/L, P < .001 and 0.26 +/- 0.10 vs 0.22 +/- 0.07 mmol/L, P< .001, respectively). The transdermal but not oral ERT significantly reduced the AIP compared with baseline (-0.17 +/- 0.26 vs -0.23 +/- 0.25, P = .023), making the difference between the therapies statistically significant (-0.23 +/- 0.25 vs -0.18 +/- 0.22, P = .017). Oral administration of ERT resulted in TG enrichment of LDL and HDL particles. Transdermal ERT did not change the composition of the lipoproteins and produced a significant improvement of AIP. Compared with transdermal ERT, orally administered ERT changes negatively the composition of plasma lipoproteins.

Relationships of circulating pregnanolone isomers and their polar conjugates to the status of sex, menstrual cycle, and pregnancy

Pregnanolone isomers (PIs) and their polar conjugates (PICs) modulate ionotropic receptors such as gamma-aminobutyric acid or pregnane X receptors. Besides, brain synthesis, PI penetrates the blood-brain barrier. We evaluated the physiological importance of PI respecting the status of sex, menstrual cycle, and pregnancy. Accordingly, circulating levels of allopregnanolone (P3alpha 5alpha ), isopregnanolone (P3beta 5alpha ), pregnanolone (P3alpha 5beta ), epipregnanolone (P3beta 5beta ), their polar conjugates, and related steroids were measured in 15 men (M), 15 women in the follicular phase (F), 16 women in the luteal phase (L), and 30 women in the 36th week of gestation (P) using GC-MS. The steroid levels were similar in M and F, increased about thrice in L and escalated in P (38-410 times compared with F). The PICs were prevalent over the PIs (16-150 times). Higher ratios of 5alpha-PIC to 5alpha-PI found in P indicate the more intensive conjugation of 5alpha-PI during pregnancy. This mechanism probably provides for the elimination of neuroinhibitory P3alpha 5alpha in the maternal compartment. Additionally, our result points to a limited sulfation capacity for neuroinhibitory P3alpha 5beta in P. In contrast to the situation in M, F, and L where the P3alpha 5beta C is the most abundant PIC, and P3alpha 5beta is present in minor quantities compared with the P3alpha 5alpha, P3alpha 5beta may acquire physiological importance during pregnancy, contributing to the sustaining thereof. On the other hand, the declining formation of P3alpha 5beta may participate in the initiation of parturition, given the relative abundance of the steroid, its potency to suppress the activity of oxytocin-producing cells and its effectiveness in uterine relaxation.

Factors influencing women's selection of combined hormonal contraceptive methods after counselling in 11 countries: results from a subanalysis of the CHOICE study.

Abstract Objectives To investigate which characteristics of women and healthcare professionals (HCPs) were associated with changing to another combined hormonal contraceptive (CHC) method after contraceptive counselling. Methods CHOICE was a cross-sectional survey in which 18,787 women were counselled about combined hormonal contraceptives, during which their contraceptive methods preferred both prior to and after counselling were recorded. In this subanalysis, characteristics associated with changing the method after counselling were determined using logistic regression models. Results The probability of intending to change from the pill to another method was associated with being older; university-educated; being in a steady relationship; a prior unintended pregnancy; a younger HCP or one who recommended methods other than the pill. Changing to the patch was associated with a female HCP or a HCP who recommended the patch or an injectable. Changing to the ring was associated with being over 21 years; university-educated; being in a relationship; previous hormonal method use; and counselling by a female HCP, a HCP < 60 years old, or a HCP who recommended the ring or an implant. The country of residence influenced these changes in a complex pattern. Conclusions Womens choice of CHC methods after contraceptive counselling are influenced by their age, educational background, relationship status, prior unplanned pregnancies and country of residence, as well as age, gender and preferences of their HCP.

Prenatally Diagnosable Differences in the Cellular Immunity of Fetuses with Down’s and Edwards’ Syndrome

Introduction: Lymphocyte subpopulations are identified by the uniform CD classification (Cluster of Differentiation) and can be accurately differentiated with monoclonal antibodies using the method of flow cytometry. With the aid of cordocentesis it is possible to perform studies on the status and development of cellular immunity as early as in the second trimester of pregnancy. Objective: To compare lymphocyte subpopulations present in fetuses with chromosomal abnormalities (Down’s syndrome (DS), Edwards’ syndrome (ES)) and fetuses with normal karyotype. Study Design: Prospective observational study. Methods: We examined a total of 61 pregnant women with an average age of 31.5 years (20– 46 years). Results: In fetuses with DS we found a significant decrease in B lymphocytes (CD19),a decrease in the subpopulations of multi-reactive B-cells (CD5+CD19+, B-CLL),and a decrease in the index of CD4/CD8 and class II HLA-DR. In contrast, the representation of NK cells expressing /CD3-CD (16 + 56)+/ was greatly increased. In ES we found a decrease in T lymphocytes (CD3), a decrease in T-helper lymphocytes (monocytes CD4), a decreased index of CD4/CD8 and a greater representation of NK cells /CD3-CD (16 + 56)+/. Conclusion: We determined the normal values of lymphocyte subpopulations in physiological fetuses. We demonstrated that the immunological defect of the affected fetuses is already present antenatally, and can be reliably diagnosed in the second trimester of pregnancy.

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.

Changes in Hemostatic Variables Induced by Estrogen Replacement Therapy: Comparison of Transdermal and Oral Administration in a Crossover-Designed Study

Aim: The purpose of this study was to determine the changes of biochemical risk factors for thromboembolisms using different administration routes of early estrogen replacement therapy. Methods: In a 12-week prospective, randomized crossover trial, estradiol was administered orally (2 mg daily) or transdermally (0.05 mg daily). Forty-five healthy early postmenopausal women were included into the study within 12 weeks after hysterectomy and oophorectomy. Forty-one women (age 49 ± 6 years) completed the study, and their data were analyzed. The hemocoagulation parameters were determined prior to beginning of the study and at the end of each treatment period, separated by a 1-week washout period. Results: After oral therapy, the average tissue factor pathway inhibitor levels decreased statistically significantly (p < 0.0001) from 87.5 ± 39.1 to 68 ± 37.49 ng/ml. The plaminogen activator inhibitor-1 levels also decreased statistically significantly (p = 0.001) after the oral estrogen therapy from 11.39 ± 12.02 to 5.0 ± 5.27 IU/l. These changes were also significant when compared with the nonsignificant changes after the transdermal therapy. No significant changes occurred in the levels of D-dimers. After both treatment methods, the antithrombin III and fibrinogen levels decreased, but within their physiological ranges. Conclusions: Oral administration of estrogen statistically significantly reduced the tissue factor pathway inhibitor and plasminogen activator inhibitor-1 levels when compared with the transdermal route. These changes cannot be unambiguously considered risky, and the zero change of D-dimers suggests that there was no activation of the coagulation cascade. We consider the neutral effect of the transdermal therapy more beneficial.

The distribution of major lymphocyte subsets in cord blood is associated with its pH.

OBJECTIVES To assess in venous cord blood the distribution of major lymphocyte subsets according to pH and medications used during labor. DESIGN AND METHODS Venous cord blood was sampled immediately after labor from 70 newborns (35 males and 35 females) delivered vaginally. Lymphocytes were immunophenotyped by flow cytometry and pH was measured using the AVL 900 automated blood gas analysis system. Data on birth weight, gestational age at delivery, length of labor, presence of stained amniotic fluid, medications used during labor, maternal risk factors, age and parity were collected. RESULTS The percentage of T lymphocytes decreased while the percentage of NK lymphocytes increased with decreasing pH over the whole range of pH values. The proportions of T and NK lymphocytes were associated with the administration of neuroplegics, spasmolytics or dihydroergotoxin in the first stage of labor. CONCLUSIONS Cord blood pH and labor-associated variables should be taken into account to adequately interpret the profile of major lymphocyte subsets as a marker of the effect of different prenatal factors on the immune system of neonates.

Association of MTHFR genetic variants C677T and A1298C on predisposition to spontaneous abortion in Slavonic population

AIM Up to 20% of pregnancies end in the first trimester by spontaneous abortion but the cause of a large proportion remains unexplained. The aim of this study was to investigate the role of two common variants (rs1801133, C677T and rs1801131, A1298C) within the MTHFR gene in the genetic determination of spontaneous abortions. METHODS DNA from 464 tissue samples of spontaneous abortions and population sample of adults (N=2,486) were genotyped for both MTHFR polymorphisms of interest. RESULTS The frequencies of the MTHFR polymorphisms in tissues from spontaneous abortions did not differ from the population cohort. However, when combined, carriers of at least three rs1801133 and/or rs1801131 alleles were more common in the spontaneous abortions (61.4%) than in controls (55.4%) and this combination was associated with higher risk of abortion (OR 1.28; 95% CI 1.05-1.57; P=0.017). In contrast, carriers of at least three minor alleles (T677 and C1298) of these polymorphisms were very rare in both groups (0.8% and 0.9% respectively). CONCLUSIONS Our study suggests that distinct combinations of the MTHFR polymorphisms could be associated with higher risk of spontaneous abortions in Caucasians.