HOME HELP CONTACT US SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kancheva, R. Articles by Stárka, L. Search for Related Content PubMed PubMed Citation Articles by Kancheva, R. Articles by Stárka, L.

Radmila Kancheva, Martin Hill, David Cibula¹, Helena Veláková, Lyudmila Kancheva, Jana Vrbíková, Tomá Fait¹, Antonín Paízek¹

Institute of Endocrinology, Národnítída 8, CZ 116 94 Prague 1, Czech Republic
¹ Department of Gynecology and Obstetrics, 1st Medical Faculty, Charles University, Apolináská 18, CZ 128 51 Prague 2, Czech Republic

(Requests for offprints should be addressed to M Hill; Email: mhill{at}endo.cz)

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pregnanolone isomers (PIs) and their polar conjugates (PICs) modulate ionotropic receptors such as -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 (P3 5 ), isopregnanolone (P3ß 5 ), pregnanolone (P3 5ß ), epipregnanolone (P3ß 5ß ), 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 5-PIC to 5-PI found in P indicate the more intensive conjugation of 5-PI during pregnancy. This mechanism probably provides for the elimination of neuroinhibitory P3 5 in the maternal compartment. Additionally, our result points to a limited sulfation capacity for neuroinhibitory P3 5ß in P. In contrast to the situation in M, F, and L where the P3 5ß C is the most abundant PIC, and P3 5ß is present in minor quantities compared with the P3 5 , P3 5ß may acquire physiological importance during pregnancy, contributing to the sustaining thereof. On the other hand, the declining formation of P3 5ß 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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reduced progesterone metabolites, including pregnanolone isomers (PIs) and their polar conjugates (PICs), are efficient neuromodulators. They are effective on neurotransmitter receptors influencing the permeability of ion channels (Majewska 1990). PIs with a hydroxy group in the 3-position shorten the period of paradoxical sleeping, attenuate the release of acetylcholine in the neocortex and hippocampus, suppress neurogenesis, and deteriorate spatial memory. These effects are provided via modulation of the receptors of type A -aminobutyric acid (GABA_A-R; Darnaudery et al. 1999). The 3ß-PI competes with 3-PI for the binding sites on GABA_A-R (Prince & Simmonds 1992, Wang et al. 2002, Lundgren et al. 2003). Conjugation counteracts the effect of 3-PI, and further amplifies the antagonistic effect of the 3ß-PI on GABA_A-R. For instance, the GABA_A-R inhibiting efficiency of 3ß-hydroxy-5-pregnane-20-one sulfate is comparable with the GABA_A-R activating effectiveness of allopregnanolone (P3 5 ). Some reports indicate that in addition to the brain’s in situ synthesis, circulating PIs penetrate the blood–brain barrier (Bixo et al. 1997, Corpechot et al. 1997). Although several studies have brought information to light regarding the levels of PI in humans, only a small number have included data regarding the respective polar conjugates (Mickan & Zander 1979, Hill et al. 2000, Havlikova et al. 2006).

In addition to the action on GABA_A-R, the 5ß-PI block type T calcium channels in rat peripheral neurons; these channels play a significant role in pain perception (Todorovic et al. 2004). The 5ß-PI also binds to nuclear progesterone receptors (Putnam et al. 1991). It has recently been reported that 5ß-PI may act chronically through a mechanism mediated by pregnane X receptors to regulate uterine contractility (Mitchell et al. 2005). The 5- and 5ß-PIC are effective as positive and negative modulators of N-methyl-D-aspartate receptors (NMDA-R) respectively (Park-Chung et al. 1997, Weaver et al. 2000). The activation of NMDA-R in hypothalamic magnocellular neuroendocrine cells of the supraoptic nucleus may induce oxytocin production (Pak & Curras-Collazo 2002), resulting in onset of parturition.

PIs originate from progesterone (Prog) through the action of the ubiquitous 5- and 5ß-reductase, which is most active in the liver (Ingelman-Sundberg 1976). Both enzymes show significant activity in the tissues associated with pregnancy (Lisboa & Holtermann 1976, Milewich et al. 1979, Sheehan et al. 2005); they catalyze the formation of 5- and 5ß-dihydroprogesterone (P5 and P5ß ) respectively. The subsequent metabolism of dihydroprogesterone epimers to individual PI is catalyzed by stereospecific 3- or 3ß-hydroxysteroid oxidoreductases (Okuda & Okuda 1984, Melcangi et al. 1994, Corpechot et al. 1997, Ottander et al. 2005). The latter enzyme may be identical to the 3ß-hydroxysteroid dehydrogenase, as indicated in the human placenta study (Dombroski et al. 1997).

PIs, and particularly their polar conjugates, are synthesized in large quantities in the human fetoplacental unit from Prog (Fig. 1; Milewich et al. 1979, Dombroski et al. 1997). As has been documented for 5-PI, the steroids are metabolized to respective 3-oxo-derivatives (P5 /ß ), which are then transported to the maternal compartment via the placenta –where they are again converted into PI (Dombroski et al. 1997). The levels of all PI in pregnancy are strikingly higher in comparison with the situation in non-pregnant women (Genazzani et al. 1998, Hill et al. 2000, Luisi et al. 2000, Pearson Murphy et al. 2001, Havlikova et al. 2006).

View larger version (14K): [in this window] [in a new window]   Figure 1 The biosynthesis of pregnanolone isomers.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
The circulating levels of all PICs, Preg and PregC were measured in all groups. The levels of all PIs and Prog were measured in F, L, and P. The quantification of P5 and P5ß was limited to P.

Subjects

The subjects in all groups were Caucasian. The M group consisted of 15 healthy men (16–62 years of age) participating in a study on iodine deficiency in the Czech Republic. The blood from non-pregnant women was collected on the 5th and 22nd days of the MC for the F (n = 15) and L (n = 16) groups respectively. Eleven women were followed in both phases. The 30 pregnant volunteers in the P group entered the study protocol according to the inclusion and exclusion criteria. The volunteers were enrolled into the study at a single institution between January 2004 and September 2005, according to the following inclusion criteria: age between 18 and 30 years, physiological pregnancy, cephalic presentation of the fetus. The exclusion criteria were as follows: manifestation of pre-eclampsia, chronic medication, anemia, any chronic disease or medication that might influence steroid levels or delivery, premature rupture of membranes, labor induction, or operative delivery in a previous pregnancy. The subjects in all groups used no hormonal treatment for at least 3 months prior to, and during, the trial. All patients enrolled in the study gave signed consent to participate in the study after a full explanation of the purpose and the nature of all the procedures used. The local Ethical Committees of the Institute of Endocrinology and the General Faculty Hospital (both in Prague, Czech Republic) approved the protocol for the study.

Sample collection

After signing written informed consent, the patients underwent blood sampling from the cubital vein. All blood samples were taken at the same time of day (0900–1200 h, at least 2 h after waking), under standard conditions (elimination of stress factors, after 15 min of resting).

Cooled plastic tubes containing 100 µ l of 5% EDTA and 50 pl aprotinin (Antilysin from Spofa, Prague, Czech Republic) were used for blood sampling. Plasma was obtained after centrifugation for 5 min at 2000 g at 0 ° C. The plasma samples were stored at – 20 ° C until analysis.

Steroids and chemicals

The steroids were purchased from Steraloids (Wilton, NH, USA). The solvents for the extraction and high performance liquid chromatography (HPLC) were of analytical grade, sourced from Merck. The derivatization agent Sylon BFT (bis(trimethylsilyl)-trifluoroacetamide 99% and trimethylchlorosilane) was purchased from Supelco (Bellefonte, PA, USA).

Instruments

The GC–MS system was supplied by Shimadzu (Kyoto, Japan). The system consisted of a GC 17A gas chromatograph equipped with automatic flow control, AOC-20 autosampler and for the MS a QP 5050A quadrupole electron-impact detector with a fixed electron voltage of 70eV. A Zebron ZB-50 medium polarity capillary column (50% phenyl/50% methylpolysiloxane) from Phenomenex (St Torrance, CA, USA) was used for the analysis. The length of the column was 15 m, the internal diameter was 0.25 mm, and the film thickness was 15 µ m.

Preparation of the plasma samples for GC–MS free steroids analysis

Frozen samples were thawed and 1 ml sample was spiked with 17-estradiol as an internal standard to attain a concentration of 1 µ g/ml. The spiked sample was extracted with 3 ml diethyl ether. The water phase was kept frozen in a mixture of solid carbon dioxide and ethanol, and the organic phase was decanted into glass tubes and evaporated to dryness. The dryorganic phase residue was used for the determination of free steroids. The dry residue was partitioned between 1 ml of 80% methanol with water and 1 ml n-pentane to eliminate the lipids and sterols. The n-pentane phase was discarded, while the methanol/water phase containing steroids for analysis was evaporated in a vacuum centrifuge. The samples were prepared twice for further processing using two different derivatization techniques. The first was used for the preparation of steroids with hydroxy groups modified to trimethylsilyl (TMS) derivatives and with intact oxo groups. The second technique produced derivatives with hydroxy groups modified as in the former case but, in addition, with the oxo groups modified by methoxylamine (MOX–TMS derivatives).

The first derivatization technique was used for the quantification of Preg and PIs. The second technique was applied for the measurement of Prog, P5 , and P5ß .

Sample preparation for the GC–MS analysis of steroid polar conjugates

The frozen water phase in glass tubes was thawed and mixed with 1 ml methanol. The tubes were centrifuged and the 1 ml aliquot of the supernatant was transferred into a glass tube and evaporated in a vacuum centrifuge. The steroid sulfates were hydrolyzed using a method described elsewhere (Dehennin et al. 1996). The hydrolyzed sample was evaporated in a vacuum centrifuge; the dry residue was spiked with 17-estradiol as an internal standard to attain a concentration of 1 µ g/ml and further processed in the same way as the free steroids.

Derivatization

The TMS derivatives of the steroids were prepared using the modified method of Hill et al.(2000), with some of the modifications reported recently (Havlikova et al. 2006). In summary, Sylon B (99% bis (trimethylsilyl)-trifluroacetamide (BTSFA) + 1% trimethylchlorosilane (TMCS); 50 µ l) was added to the dry residues from plasma, mixed briefly and heated at 90 ° C for 45 min. The derivatization agent was evaporated under a stream of nitrogen. The dry residue was rinsed down with isooctane (50 µ l) and the mixture was evaporated again. Finally, steroid derivatives were dissolved into 20 µ l isooctane, and 4 µ l portions were injected into the GC–MS system.

The MOX–TMS derivatives were prepared as follows: 50 µ l of 2% solution of MOX–hydrochloride in pyridine were added to the dry residues from plasma, mixed briefly, and heated at 60 ° C for 2 h. The mixture was then evaporated under a stream of nitrogen, before further TMS derivatization proceeded as described previously.

Temperature and pressure gradients for the GC–MS analysis of TMS derivatives

For the GC–MS analysis of TMS derivatives, the temperature and pressure gradients used were as follows: 1-min high-pressure injection at 120 ° C and 100 kPa followed by a pressure release to 30 kPa and a rapid linear gradient of 40 ° C/min and 8.5 kPa/min up to 200 ° C and 49.3 kPa, then a slow linear gradient of 2.9 ° C/min and 0.5 kPa/min up to 220 ° C and 52.7 kPa, a medium linear gradient of 20 ° C/min and 8 kPa/min a up to 265 ° C and 70 kPa, and a rapid linear gradient of 40 ° C/min and 10.0 kPa/min up to 310 ° C and 80.7 kPa, followed by a 2-min delay. The overall time taken for the analysis was 15.3 min.

For the analysis of TMS–MOX derivatives, the following temperature and pressure gradient was used: one minute high-pressure injection at 120 ° C and 100 kPa followed by a pressure release to 30 kPa and a rapid linear gradient of 40 ° C/min and 8.5 kPa/min up to 220 ° C and 51.0 kPa, then a slow linear gradient of 2.9 ° C/min and 0.5 kPa/min up to 240 ° C and 54.5 kPa, and a rapid linear gradient of 40 ° C/min and 9.0 kPa/min up to 310 ° C and 70 kPa, followed by a 3-min delay. The overall time taken for the analysis was 15.2 min.

Retention times and effective masses used for the determination of steroids

To exploit the samples, the individual samples were applied three times in independent courses, in each case employing a part of the steroids under investigation. The choices of the steroids measured within the individual courses, as well as the effective masses used for the measurement, were optimized to attain maximum sensitivity at sufficient selectivity. The types of gradients, effective masses for determination and quantification, order numbers of injection, and retention times for the individual steroids are shown in Table 1.

The data showed mostly non-Gaussian distribution and non-constant variance. To evaluate changes in the steroid levels and steroid ratios, a robust Kruskal–Wallis ANOVA was used with group as the factor. Multiple testing was handled by Kruskal–Wallis multiple comparisons to evaluate differences between the individual groups. The relationships between the steroids were evaluated using Spearman’s correlations and partial Spearman’s correlations. Given the sufficient number of pairs, the partial Spearman’s correlations in pregnant women were adjusted to constant levels of all steroids included in a correlation matrix, except the pair under investigation. Statistical computations were performed using NCSS 2002 statistical software from Number Cruncher Statistical Systems (Kaysville, UT, USA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification and quantification of the steroids

The steroids separated well from each other and from the background. As demonstrated in Table 1, the selectivity and sensitivity were sufficient for quantification of all of the investigated steroids. In terms of reproducibility, the inter-assay coefficient of variance did not exceed 10% for any steroid.

Steroid levels

The levels of free and conjugated steroids are shown in Figs 2–4. The levels of P3 5 (Fig. 3A) increased in the sequence M, F, L, and P. All inter-group differences except the differences between M and F were significant. A similar situation was found in all PIs except the missing data for the M group in 5ß-PI. The PIC showed a similar trend. The levels in both PI and PIC in P were about two orders of magnitude higher than those found in F or M (Figs 3 and 4).

View larger version (27K): [in this window] [in a new window]   Figure 2 Levels of polar conjugates of pregnenolone (A), unconjugated pregnenolone (B), and progesterone (C) in 15 adult men (M), 15 women in follicular phase of menstrual cycle (F), 16 women in the luteal phase (L), and 30 women in the 36th week of gestation (P). Bars with error bars represent group means with S.D.s. Owing to skewed data distribution and non-constant variance, the between-group were evaluated using robust Kruskal–Wallis ANOVA followed by Kruskal–Wallis multiple comparisons. The significance of ANOVA model is shown in the first line of the embedded table, while significant inter-group differences as found by the multiple comparisons (P < 0.05) are shown in the further lines of this table (*P < 0.05, **P < 0.01, ***P < 0.001).

View larger version (17K): [in this window] [in a new window]   Figure 3 Levels of 5-pregnanolone isomers (A and B) and their polar conjugates (C and D), in 15 adult men (M) (only ten men were examined for conjugated steroids), 15 women in follicular phase of menstrual cycle (F), 16 women in the luteal phase (L), and 30 women in the 36th week of gestation (P). The drawings and symbols are the same as for Fig. 2.

View larger version (17K): [in this window] [in a new window]   Figure 4 Levels of 5ß-pregnanolone isomers (A and B) and their polar conjugates (C and D), in 15 adult men (M) (only ten men were examined for conjugated steroids), 15 women in follicular phase of menstrual cycle (F), 16 women in the luteal phase (L), and 30 women in the 36th week of gestation (P). The drawings and symbols are the same as for Fig. 2.

Both 5-PI exhibited higher conjugate to free steroid ratios for M and P (which did not differ from each other) when compared with F and L (Fig. 5A and B). In the ratios of P3ß 5 C to P3ß 5 , however, the differences between M and the groups of non-pregnant women did not reach significance (Fig. 5B). In contrast to the situation in 5-PI, the ratio of P3 5ß C to P3 5ß showed decreasing trend in the sequence F, L, and P. Significantly lower value of the ratio was found in P when compared with the values in F and L (Fig. 5C). The ratios of P3ß 5ß C to P3ß 5ß did not exhibit any between-group difference (Fig. 5D).

View larger version (14K): [in this window] [in a new window]   Figure 5 Ratios of polar conjugates to unconjugated steroids for allopregnanolone (P3 5; A), isopregnanolone (P3ß 5; B), pregnanolone (P3 5; C), and epipregnanolone (P3ß 5ß; D), in the groups of 15 women in the follicular (F) and 16 women in the luteal (L) phases of the menstrual cycle, and 30 women in the 36th week of gestation (P). The letter C at the end of the aforementioned abbreviations denotes conjugated steroids. The remaining drawings and symbols are the same as for Fig. 2.

Ratios of 3- to 3ß-isomers

As demonstrated in Fig. 6A, the F and L groups showed more than two times lower values for the ratio than was the case for the P group. The respective differences in the conjugates were much less pronounced (Fig. 6B); nevertheless, the ratios for P still exhibited the highest values.

View larger version (31K): [in this window] [in a new window]   Figure 6 Ratios of the sum of unconjugated pregnenolone isomers with a hydroxy group in the 3-position (3-PI/3ß-PI; A) to the sum of unconjugated pregnenolone isomers with a hydroxy group in the 3ß-position and the ratio of the steroid polar conjugates (3-PIC/3ß-PIC; B), in the groups of adult men (M), women in the follicular (F) and luteal (L) phases of the menstrual cycle, and women in the 36th week of gestation (P). The drawing and symbols are the same as for Fig. 2.

Ratios of the 5- to 5ß-isomers

The ratios of 5-PI to 5ß-PI showed higher values for F and L groups when compared with P group (Fig. 7A). The situation was different in PIC, where the ratios were significantly higher in L and even more elevated in the P group when compared with the values found in M and F groups that did not differ from each other (Fig. 7B).

View larger version (27K): [in this window] [in a new window]   Figure 7 Ratios of the sum of unconjugated pregnenolone isomers with hydrogen in the 5-position (5-PI/5ß-PI; A) to the sum of unconjugated pregnenolone isomers with hydrogen in the 5ß-position and the ratio of the steroid polar conjugates (5-PIC/5ß-PIC; B), in the groups of adult men (M), women in the follicular (F) and luteal (L) phases of the menstrual cycle, and women in the 36th week of gestation (P). The drawings and symbols are the same as for Fig. 2.

In the F group, the only significant correlation between conjugated and free PI was found for the P3ß 5ß (r = 0.536, P = 0.040, n = 15). The symbols r, P, and n symbolize the Spearman’s correlation coefficient, its statistical significance, and number of pairs under investigation respectively. In L group, the P3 5 significantly correlated with the P3 5 C (r = 0.753, P = 0.001, n = 16) and P3ß 5 with P3ß 5 C (r = 0.585, P = 0.017, n = 16). The respective correlations for the P3 5ß (r = 0.409, P = 0.116, n = 16) and P3ß 5ß (r = 0.338, P = 0.200, n = 16) did not reach significance. In P group, the correlations between conjugated and free PI reached significance for the P3ß 5 (r = 0.528, P = 0.004, n = 28) and for the P3ß 5ß (r = 0.431, P = 0.022, n = 28).

Correlations between 3- and 3ß-isomers

Significant correlations between 3- and 3ß-PI were mostly found within all groups for both 5- and 5ß-PI, except for the missing data for 5ß-PI in men. P3 5 and P3ß 5 significantly correlated in F (r = 0.693, P = 0.040, n = 15), L (r = 0.965, P < 0.001, n = 16), and P (r = 0.695, P < 0.001, n = 30). The correlation between the P3 5 C and P3ß 5 C did not reach significance in the F group (r = 0.275, P = 0.321, n = 15) but strongly correlated in L (r = 0.918, P < 0.001, n = 16) and P (r = 0.660, P < 0.001, n = 28). P3 5ß and P3ß 5ß significantly correlated in F (r = 0.764, P = 0.001, n = 15), L (r = 0.594, P = 0.015, n = 16), and P (r = 0.649, P < 0.001, n = 30). Similarly, P3 5ß C and P3ß 5ß C significantly correlated in F (r = 0.543, P = 0.037, n = 15), L (r = 0.600, P = 0.014, n = 16), and P (r = 0.812, P < 0.001, n = 28).

Correlations between PI and progesterone

The correlation between P3 5 and Prog did not reach significance in F (r = 0.434, P = 0.106, n = 15) but the steroids strongly correlated in L (r = 0.744, P < 0.001, n = 16) and P (r = 0.771, P < 0.001, n = 29). P3ß 5 and Prog significantly correlated in F (r = 0.523, P = 0.045, n = 15), L (r = 0.788, P < 0.001, n = 16), and a weaker but still significant correlation was found in P (r = 0.399, P = 0.032, n = 16). The P3 5ß and Prog strongly correlated in L (r = 0.818, P < 0.001, n = 16) but not in F (r = – 0.183, P = 0.514, n = 15) and P (r = 0.348, P = 0.434, n = 29). Like the P3 5ß , P3ß 5ß did not correlate with Prog in F (r = – 0.219, P = 0.065, n = 15), but the significant correlations were found in L (r = 0.500, P = 0.049, n = 16) and P (r = 0.710, P < 0.001, n = 29).

Relationships between steroids

PregC negatively correlated with the 5-PI in men, attaining significance for P3 5 (r = – 0.709, P < 0.022, n = 10) but not for P3ß 5 (r = – 0.503, P < 0.14, n = 10). The subsequent partial correlations confirmed a negative borderline association between PregC and P3 5 (r = – 0.633, P < 0.1, n = 10, adjusted to constant Preg and P3ß 5 ), but showed no direct relationship between PregC and P3ß 5 (r = 0.260, P = 0.504, n = 10, adjusted to constant Preg and P3 5 ). Both pair correlations and partial correlations identified a strong positive relationship between P3 5 and P3ß 5 (r = 0.707, P < 0.003, n = 15) and (r = 0.758, P < , P < 0.003, n = 15, adjusted to constant PregC and Preg).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The levels of Preg, PregC, all PIs, and PICs in women in the 36th week of gestation and in non-pregnant women in both phases of the MC were quantified. In addition, the levels of the Preg, PregC, 5-PI as well as the concentrations of all PICs were measured in men (Figs 2–4). The results clearly showed the need to differentiate among M, F, L, and P groups when evaluating changes of circulating PI in connection with various pathologies. The distinctive effects of PI are to be expected in pregnancy due to persistently elevated levels of 3-PI, resulting in a decreased affinity of GABA_A-R for these neuroactive steroids either due to the changed expression of the receptor subunits and/or as a result of the changed phosphorylation status of the specific sites on the GABA_A-R (Leng & Russell 1999, Brussaard et al. 2000, Koksma et al. 2003).

In men and to a large extent in women in the follicular phase, the most important role of adrenal activity and in situ brain synthesis of PI can be expected in the overall balance of PI (Fig. 8A and B). Our data suggest that the production of PI in men may depend on sulfatase activity. In M group, PregC correlated with P3 5 . This finding, like the increased ratios of PIC to PI, may indicate that lower sulfatase activity could be associated with a lower amount of Preg as the primary substrate for enzymes catalyzing the formation of Prog and PI. The partial correlations confirmed the negative borderline association between PregC and P3 5 but showed no relationship between PregC and P3ß 5 . As expected, both pair and partial correlations found a strong positive relationship between P3 5 and P3ß 5 . Given the aforementioned results, the major metabolic pathway in adult men may be expected in the sequence PregC Preg Prog P5 P3 5 P3ß 5 .

View larger version (9K): [in this window] [in a new window]   Figure 8 Biosynthesis of pregnanolone isomers in men (A), women in the follicular and luteal phase of menstrual cycle respectively (B and C respectively) and in pregnant women (D).

After the luteo-placental shift, the primary precursor of the PI, PregS, originates almost entirely from the fetal zone of the fetal adrenal (Fig. 8D). PregS is transported into the placenta by circulation, being converted to Preg, Prog, and P5 /ß , in sequence. The P5 and possibly also the P5ß penetrate into the maternal compartment, being further metabolized to PI and PIC (Dombroski et al. 1997).

While the 5-PI showed higher ratios of conjugates to the free steroids (Fig. 5A and B) in P group, the respective ratios were similar in F, L, and P groups for P3ß 5ß (Fig. 5D) and showed a significantly decreasing trend for P3 5ß in sequence F, L, and P (Fig. 5C) groups. It is evident that the groups with low Prog production exhibited very low concentrations of unconjugated P3 5ß . In these subjects, the P3 5ß may be rapidly metabolized to its conjugates. Alternatively, in L and P groups, the conjugation capacity for P3 5ß appears to be limited. The increased conjugation of 5-PI probably diminishes the difference between the P3 5 and P3 5ß levels in pregnant women and may also regulate the proportions between neuroinhibiting P3 5 and counter-acting 5-PIC. On the GABA_A-R, the P3ß 5 C exerts about 10% efficiency when compared with the effect of P3 5 . However, P3ß 5 C operates in the opposite way (Park-Chung et al. 1999). In all groups, the levels of all PICs exceed the levels of PI from one to more than two orders of magnitude. The situation in the proximity of the active sites may not necessarily reflect the conditions in the circulation, however. The conjugation may also hinder the transport of PI across the blood–brain barrier, but in the periphery, it probably facilitates the transport of significant amounts of neuroactive steroids by circulation to the sites where they take effect.

To evaluate the inter-group differences in the relative amount of 3-PI positively modulating GABA_A-R and 3ß-PI being inactive but competing with the former PI on the receptors, the ratios of the sum of the 3-PI to the sum of 3ß-PI were evaluated. As demonstrated in Fig. 6A, the F and L groups displayed more than two times lower values for the ratio than the P group. This means that as regards the free steroids, the circulating positive GABA_A-R modulators are more manifestly prevalent in pregnancy over their inactive competitors on the GABA_A-R. The respective differences in conjugates were much less pronounced (Fig. 6B).

Strong correlations between 3- and 3ß-PI that were found within all groups illustrate an uncomplicated, reversible oxidoreductive switch between the 3- and 3ß-PI via the 3-oxo-intermediate product in all groups. These metabolic steps converting neuroinhibiting 3-PI to 3ß-PI which competes with the 3-PI on GABA_A-R, as well as the sulfoconjugation of the PI-forming products operating on the GABA_A-R in an opposite way to free 3-PI, may be of great importance. It is known that P3 5 dosage in animals or humans usually results in a weaker response than expected in consideration of the results of in vitro experiments. P3 5 dosage eventuates in a bell-shaped response. At lower concentrations, P3 5 paradoxically acts as neurostimulant showing a neuroinhibiting effect as late as at levels comparable with the concentrations common in pregnancy (Backstrom et al. 2003). Moreover, one of the neuroinhibiting PIs, P3 5ß is an unstable substance with a short half-life in the human organism (Carl et al. 1994). P3 5ß is identical to a short-term central anesthetic eltanolone that has previously been tested in pharmacological studies (Hering et al. 1996). Given the data from this study, it seems likely that the instability of P3 5ß is strongly associated with the reversible metabolic steps outlined above.

The initiation of human parturition represents a complex system involving various factors and multiple, interconnected feedback loops (Nathanielsz 1998). Several mechanisms have been suggested to explain the primary impulse for parturition. Some of them involve significantly decreasing the placental synthesis of Prog, while at the same time increasing estradiol production before the onset of parturition (Nathanielsz 1998, Wu et al. 2004). In contrast to non-primate species, progesterone levels in human maternal serum do not change markedly around parturition, while estradiol levels escalate up to labor, as in other mammals (Mathur et al. 1980). Despite the number of mechanism suggested (McLean & Smith 2001, Patel et al. 2003, Condon et al. 2004, Mesiano 2004), our knowledge regarding the primary impulse for the human parturition remains inadequate. The role of neuro-active reduced progesterone metabolites – and particularly the most abundant of them, PIs – in the timing of human parturition is still unclear, despite studies reporting their major effects in other mammals (Brussaard et al. 1997, 2000). In rats, decreasing allopregnanolone levels just before labor induce a positive feedback loop in oxytocin production, resulting in a rapid delivery (Brussaard et al. 1997, 2000). The latter substance brings the GABA_A-R from a neurosteroid-sensitive mode toward a condition in which the receptors are not sensitive (Koksma et al. 2003), via a shift in the balance between the activities of endogenous Ser/Thr phosphatase and protein kinase C (Koksma et al. 2003). Given all the foregoing, it seems likely that changing the biosynthesis of PIs could influence the onset of labor. Moreover, unconjugated 5ß-pregnane steroids cause rapid uterine relaxation through a pregnane receptor X-mediated mechanism (Mitchell et al. 2005). In this respect, the increased proportion of the 5ß-PI demonstrated in Fig. 7A allows speculation regarding the pregnancy sustaining role of pregnanolone. The weakening activity of 5ß-reductase (Sheehan et al. 2005) as well as the declining levels of reduced 5ß-pregnane steroids near term, further supports this hypothesis (Gilbert Evans et al. 2005). The results obtained in this study suggest that the sulfation of P3ß 5 is an important metabolic step contributing to progesterone catabolism in the maternal compartment. Using partial correlations with adjustment to constant levels of all steroids in the correlation matrix, except for the pair under investigation, P3ß 5 C in pregnant women negatively correlated with Prog (r = – 0.512, P = 0.043, n = 27). The negative partial correlation between Prog and P3 5ß (r = – 0.548, P = 0.019, n = 29) indicates that a significant amount of Prog may be also metabolized via the 5ß-pathway.

In conclusion, the circulating levels of PI and PIC showed similar values for the M and F, while significantly higher levels of the steroids were found in L, and prominently higher concentrations were observed in P. The neuroactivating PICs were strikingly prevalent over the neuroinhibitory 3-PI in the circulations of all groups. Significantly higher ratios of 5-PIC to 5-PI found in P compared with F and L groups indicate the more intensive conjugation of 5-PI in pregnancy, including that of the most abundant neuro-inhibiting steroid P3 5 . This mechanism probably provides for the elimination of excessive amounts of neuroinhibiting P3 5 in the maternal compartment. The opposite situation was observed in P3 5ß . This result points to a limited sulfation capacity for pregnanolone in pregnant women. In contrast to the situation in men and non-pregnant women, where the P3 5ß C is the most abundant PIC but P3 5ß is a minor substance compared with the remaining PI, P3 5ß may acquire physiological importance in pregnancy, contributing to the sustaining thereof. Quite the opposite effect, the declining formation of P3 5ß as reported recently (Gilbert Evans et al. 2005, Sheehan et al. 2005), may participate in the initiation of human parturition, given relative abundance of the steroid, its potency to suppress the activity of oxytocin-producing cells (Leng & Russell 1999, Brussaard et al. 2000, Koksma et al. 2003) and its effectiveness in uterine relaxation (Mitchell et al. 2005).

	Acknowledgements

 
The excellent technical assistance of Mrs Ivona Králová and Mrs Marta Velíková is gratefully acknowledged. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Funding

This study was supported by Grant Agency of the Czech Republic grant no. 303/04/1086.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
Backstrom T, Andersson A, Andree L, Birzniece V, Bixo M, Bjorn I, Haage D, Isaksson M, Johansson IM, Lindblad C et al. 2003 Pathogenesis in menstrual cycle-linked CNS disorders. Annals of New York Academy of Sciences 1007 42–53.[CrossRef][Web of Science][Medline]

Bixo M, Andersson A, Winblad B, Purdy RH & Backstrom T 1997 Progesterone, 5alpha-pregnane-3,20-dione and 3alpha-hydroxy-5alpha-pregnane-20-one in specific regions of the human female brain in different endocrine states. Brain Research 764 173–178.[CrossRef][Web of Science][Medline]

Brussaard AB, Kits KS, Baker RE, Willems WP, Leyting-Vermeulen JW, Voorn P, Smit AB, Bicknell RJ & Herbison AE 1997 Plasticity in fast synaptic inhibition of adult oxytocin neurons caused by switch in GABA(A) receptor subunit expression. Neuron 19 1103–1114.[CrossRef][Web of Science][Medline]

Brussaard AB, Wossink J, Lodder JC & Kits KS 2000 Progesterone-metabolite prevents protein kinase C-dependent modulation of gamma-aminobutyric acid type A receptors in oxytocin neurons. PNAS 97 3625–3630.[Abstract/Free Full Text]

Carl P, Hogskilde S, Lang-Jensen T, Bach V, Jacobsen J, Sorensen MB, Gralls M & Widlund L 1994 Pharmacokinetics and pharmacodynamics of eltanolone (pregnanolone), a new steroid intravenous anaesthetic, in humans. Acta Anaesthesiologica Scandinavica 38 734–741.[Web of Science][Medline]

Condon JC, Jeyasuria P, Faust JM & Mendelson CR 2004 Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition. PNAS 101 4978–4983.[Abstract/Free Full Text]

Corpechot C, Collins BE, Carey MP, Tsouros A, Robel P & Fry JP 1997 Brain neurosteroids during the mouse oestrous cycle. Brain Research 766 276–280.[CrossRef][Web of Science][Medline]

Darnaudery M, Pallares M, Bouyer JJ, Le Moal M & Mayo W 1999 Infusion of neurosteroids into the rat nucleus basalis affects paradoxical sleep in accordance with their memory modulating properties. Neuroscience 92 583–588.[CrossRef][Web of Science][Medline]

Dehennin L, Lafarge P, Dailly P, Bailloux D & Lafarge JP 1996 Combined profile of androgen glucuro- and sulfoconjugates in post-competition urine of sportsmen: a simple screening procedure using gas chromatography-mass spectrometry. Journal of Chromatography. B, Biomedical Applications 687 85–91.[CrossRef]

Dombroski RA, Casey ML & MacDonald PC 1997 5-Alpha-dihydropro-gesterone formation in human placenta from 5alpha-pregnan-3beta/alpha-ol-20-ones and 5-pregnan-3beta-yl-20-one sulfate. Journal of Steroid Biochemical Molecular Biology 63 155–163.[CrossRef]

Genazzani AR, Petraglia F, Bernardi F, Casarosa E, Salvestroni C, Tonetti A, Nappi RE, Luisi S, Palumbo M, Purdy RH et al. 1998 Circulating levels of allopregnanolone in humans: gender, age, and endocrine influences. Journal of Clinical Endocrinology and Metabolism 83 2099–2103.[Abstract/Free Full Text]

Gilbert Evans SE, Ross LE, Sellers EM, Purdy RH & Romach MK 2005 3Alpha-reduced neuroactive steroids and their precursors during pregnancy and the postpartum period. Gynecological Endocrinology 21 268–279.[CrossRef][Web of Science][Medline]

Havlikova H, Hill M, Kancheva L, Vrbikova J, Pouzar V, Cerny I, Kancheva R & Starka L 2006 Serum profiles of free and conjugated neuroactive pregnanolone isomers in non-pregnant women of fertile age. Journal of Clinical Endocrinology 91 3092–3099.[CrossRef]

Hering WJ, Ihmsen H, Langer H, Uhrlau C, Dinkel M, Geisslinger G & Schuttler J 1996 Pharmacokinetic-pharmacodynamic modeling of the new steroid hypnotic eltanolone in healthy volunteers. Anesthesiology 85 1290–1299.[CrossRef][Web of Science][Medline]

Hill M, Parizek A, Bicikova M, Havlikova H, Klak J, Fait T, Cibula D, Hampl R, Cegan A, Sulcova J et al. 2000 Neuroactive steroids, their precursors, and polar conjugates during parturition and postpartum in maternal and umbilical blood: 1. Identification and simultaneous determination of pregnanolone isomers. Journal of Steroid Biochemical Molecular Biology 75 237–244.[CrossRef]

Ingelman-Sundberg M 1976 Specific reductive metabolism of steroid sulphates in rat liver. Biochimica et Biophysic Acta 431 592–602.

Koksma JJ, van Kesteren RE, Rosahl TW, Zwart R, Smit AB, Luddens H & Brussaard AB 2003 Oxytocin regulates neurosteroid modulation of GABA(A) receptors in supraoptic nucleus around parturition. Journal of Neuroscience 23 788–797.[Abstract/Free Full Text]

Leng G & Russell JA 1999 Coming to term with GABA. Journal of Physiology 516 (Pt 2) VI.[CrossRef][Web of Science]

Lisboa BP & Holtermann M 1976 Metabolism of 20beta-hydroxy-4-pregnen-3-one in uterine tissue of non-pregnant rats in vitro. Acta Endocrinologica 83 604–620.[Abstract/Free Full Text]

Luisi S, Petraglia F, Benedetto C, Nappi RE, Bernardi F, Fadalti M, Reis FM, Luisi M & Genazzani AR 2000 Serum allopregnanolone levels in pregnant women: changes during pregnancy, at delivery, and in hypertensive patients. Journal of Clinical Endocrinology and Metabolism 85 2429–2433.[Abstract/Free Full Text]

Lundgren P, Stromberg J, Backstrom T & Wang M 2003 Allopregnanolone-stimulated GABA-mediated chloride ion flux is inhibited by 3beta-hydroxy-5alpha-pregnan-20-one (isoallopregnanolone). Brain Research 982 45–53.[CrossRef][Web of Science][Medline]

Majewska MD 1990 Steroid regulation of the GABAA receptor: ligand binding, chloride transport and behaviour. Ciba Foundation Symposia 153 83–97.[Medline]

Mathur RS, Landgrebe S & Williamson HO 1980 Progesterone, 17-hydroxyprogesterone, estradiol, and estriol in late pregnancy and labor. American Journal of Obstetrics and Gynaecology 136 25–27.

McLean M & Smith R 2001 Corticotrophin-releasing hormone and human parturition. Reproduction 121 493–501.[Abstract]

Melcangi RC, Celotti F & Martini L 1994 Progesterone 5-alpha-reduction in neuronal and in different types of glial cell cultures: type 1 and 2 astrocytes and oligodendrocytes. Brain Research 639 202–206.[CrossRef][Web of Science][Medline]

Mesiano S 2004 Myometrial progesterone responsiveness and the control of human parturition. Journal of the Society for Gynecologic Investigation 11 193–202.[CrossRef][Web of Science][Medline]

Mickan H & Zander J 1979 Pregnanolones, pregnenolone and progesterone in the human feto-placental circulation at term of pregnancy. Journal of Steroid Biochemistry 11 1461–1466.[CrossRef][Web of Science][Medline]

Milewich L, Gant NF, Schwarz BE, Chen GT & MacDonald PC 1979 5 alpha-Reductase activity in human placenta. American Journal of Obstetrics and Gynaecology 133 611–617.

Mitchell BF, Mitchell JM, Chowdhury J, Tougas M, Engelen SM, Senff N, Heijnen I, Moore JT, Goodwin B, Wong S et al. 2005 Metabolites of progesterone and the pregnane X receptor: a novel pathway regulating uterine contractility in pregnancy? American Journal of Obstetrics and Gynaecology 192 1304–1313 (discussion 1313–1305).[CrossRef]

Nathanielsz PW 1998 Comparative studies on the initiation of labor. European Journal of Obstetrics and Gynaecology and Reproductive Biology 78 127–132.[CrossRef]

Okuda A & Okuda K 1984 Purification and characterization of delta 4-3-ketosteroid 5 beta-reductase. Journal Biological Chemistry 259 7519–7524.[Abstract/Free Full Text]

Ottander U, Poromaa IS, Bjurulf E, Skytt A, Backstrom T & Olofsson JI 2005 Allopregnanolone and pregnanolone are produced by the human corpus luteum. Molecular and Cell Biology 239 37–44.

Pak CW & Curras-Collazo MC 2002 Expression and plasticity of glutamate receptors in the supraoptic nucleus of the hypothalamus. Microscopic Research and Technique 56 92–100.[CrossRef]

Park-Chung M, Wu FS, Purdy RH, Malayev AA, Gibbs TT & Farb DH 1997 Distinct sites for inverse modulation of N-methyl-D-aspartate receptors by sulfated steroids. Molecular Pharmacology 52 1113–1123.[Abstract/Free Full Text]

Park-Chung M, Malayev A, Purdy RH, Gibbs TT & Farb DH 1999 Sulfated and unsulfated steroids modulate gamma-aminobutyric acidA receptor function through distinct sites. Brain Research 830 72–87.[CrossRef][Web of Science][Medline]

Patel FA, Funder JW & Challis JR 2003 Mechanism of cortisol/progesterone antagonism in the regulation of 15-hydroxyprostaglandin dehydrogenase activity and messenger ribonucleic acid levels in human chorion and placental trophoblast cells at term. Journal of Clinical Endocrinology and Metabolism 88 2922–2933.[Abstract/Free Full Text]

Pearson Murphy BE, Steinberg SI, Hu FY & Allison CM 2001 Neuroactive ring A-reduced metabolites of progesterone in human plasma during pregnancy: elevated levels of 5 alpha-dihydroprogesterone in depressed patients during the latter half of pregnancy. Journal of Clinical Endocrinology and Metabolism 86 5981–5987.[Abstract/Free Full Text]

Prince RJ & Simmonds MA 1992 5 Beta-pregnan-3 beta-ol-20-one, a specific antagonist at the neurosteroid site of the GABAA receptor-complex. Neuroscience Letters 135 273–275.[CrossRef][Web of Science][Medline]

Putnam CD, Brann DW, Kolbeck RC & Mahesh VB 1991 Inhibition of uterine contractility by progesterone and progesterone metabolites: mediation by progesterone and gamma amino butyric acidA receptor systems. Biological Reproduction 45 266–272.[CrossRef]

Sheehan PM, Rice GE, Moses EK & Brennecke SP 2005 5 Beta-dihydroprogesterone and steroid 5 beta-reductase decrease in association with human parturition at term. Molecular Human Repoduction 11 495–501.[CrossRef]

Todorovic SM, Pathirathna S, Brimelow BC, Jagodic MM, Ko SH, Jiang X, Nilsson KR, Zorumski CF, Covey DF & Jevtovic-Todorovic V 2004 5 Beta-reduced neuroactive steroids are novel voltage-dependent blockers of T-type Ca²⁺ channels in rat sensory neurons in vitro and potent peripheral analgesics in vivo. Molecular Pharmacology 66 1223–1235.[Abstract/Free Full Text]

Wang M, He Y, Eisenman LN, Fields C, Zeng CM, Mathews J, Benz A, Fu T, Zorumski E, Steinbach JH et al. 2002 3 Beta -hydroxypregnane steroids are pregnenolone sulfate-like GABA(A) receptor antagonists. Journal of Neuroscience 22 3366–3375.[Abstract/Free Full Text]

Weaver CE, Land MB, Purdy RH, Richards KG, Gibbs TT & Farb DH 2000 Geometry and charge determine pharmacological effects of steroids on N-methyl-D-aspartate receptor-induced Ca(2 + ) accumulation and cell death. Journal of Pharmacological and Experimental Therapeutics 293 747–754.[Abstract/Free Full Text]

Wu WX, Ma XH, Coksaygan T, Chakrabarty K, Collins V, Rose J & Nathanielsz PW 2004 Prostaglandin mediates premature delivery in pregnant sheep induced by estradiol at 121 days of gestational age. Endocrinology 145 1444–1452.[Abstract/Free Full Text]

Received in final form 27 June 2007
Accepted 10 July 2007
Made available online as an Accepted Preprint 11 July 2007

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kancheva, R. Articles by Stárka, L. Search for Related Content PubMed PubMed Citation Articles by Kancheva, R. Articles by Stárka, L.