Abstract
Pregnanolone isomers (PIs) and their polar conjugates (PICs) modulate ionotropic receptors such as γ-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 (P3α 5α ), isopregnanolone (P3β 5α ), pregnanolone (P3α 5β ), epipregnanolone (P3β 5β ), 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 5α-PIC to 5α-PI found in P indicate the more intensive conjugation of 5α-PI during pregnancy. This mechanism probably provides for the elimination of neuroinhibitory P3α 5α in the maternal compartment. Additionally, our result points to a limited sulfation capacity for neuroinhibitory P3α 5β in P. In contrast to the situation in M, F, and L where the P3α 5β C is the most abundant PIC, and P3α 5β is present in minor quantities compared with the P3α 5α , P3α 5β may acquire physiological importance during pregnancy, contributing to the sustaining thereof. On the other hand, the declining formation of P3α 5β 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
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 3α-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 γ-aminobutyric acid (GABAA-R; Darnaudery et al. 1999). The 3β-PI competes with 3α-PI for the binding sites on GABAA-R (Prince & Simmonds 1992, Wang et al. 2002, Lundgren et al. 2003). Conjugation counteracts the effect of 3α-PI, and further amplifies the antagonistic effect of the 3β-PI on GABAA-R. For instance, the GABAA-R inhibiting efficiency of 3β-hydroxy-5α-pregnane-20-one sulfate is comparable with the GABAA-R activating effectiveness of allopregnanolone (P3α 5α ). 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 GABAA-R, the 5β-PI block type T calcium channels in rat peripheral neurons; these channels play a significant role in pain perception (Todorovic et al. 2004). The 5β-PI also binds to nuclear progesterone receptors (Putnam et al. 1991). It has recently been reported that 5β-PI may act chronically through a mechanism mediated by pregnane X receptors to regulate uterine contractility (Mitchell et al. 2005). The 5α- and 5β-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 5α- and 5β-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 5α- and 5β-dihydroprogesterone (P5α and P5β ) respectively. The subsequent metabolism of dihydroprogesterone epimers to individual PI is catalyzed by stereospecific 3α- or 3β-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 3β-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 5α-PI, the steroids are metabolized to respective 3-oxo-derivatives (P5α /β ), 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).
The aim of this study was to evaluate the physiological impact of endogenous neuroactive PI and PIC in humans, respecting the status of sex, menstrual cycle, and pregnancy. The evaluation was based on the steroid circulating levels and steroid neuromodulating activities reported in the literature. A further objective was to assess inter-group differences in the biosynthesis and metabolism of PI. For these reasons, we measured the circulating levels of all PIs, such as P3α 5α , isopregnanolone (P3β 5α , epiallopregnanolone, isoallopregnanolone), P3α 5β , epipregnanolone (P3β 5β ), and PIC as conjugates of P3α 5α (P3α 5α C), P3β 5α (P3β 5α C), P3α 5β (P3α 5β C), and P3β 5β (P3β 5β C) in adult men (M), in women in the follicular (F) and luteal phases (L) of the menstrual cycle (MC), and in women in the 36th week of gestation (P), using GC–MS. In addition, the levels of the respective precursors, free pregnenolone (Preg) and conjugated pregnenolone (PregC), Prog, P5α , and P5β were also quantified.
Materials and Methods
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 P5α and P5β 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 17α-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, P5α , and P5β .
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 17α-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.
Statistical analysis of the data
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
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 P3α 5α (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 5β-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).
Ratios of conjugates to free steroids
Both 5α-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 P3β 5α C to P3β 5α , however, the differences between M and the groups of non-pregnant women did not reach significance (Fig. 5B). In contrast to the situation in 5α-PI, the ratio of P3α 5β C to P3α 5β 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 P3β 5β C to P3β 5β did not exhibit any between-group difference (Fig. 5D).
Ratios of 3α- to 3β-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.
Ratios of the 5α- to 5β-isomers
The ratios of 5α-PI to 5β-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).
Correlations between conjugated and unconjugated steroids in women
In the F group, the only significant correlation between conjugated and free PI was found for the P3β 5β (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 P3α 5α significantly correlated with the P3α 5α C (r = 0.753, P = 0.001, n = 16) and P3β 5α with P3β 5α C (r = 0.585, P = 0.017, n = 16). The respective correlations for the P3α 5β (r = 0.409, P = 0.116, n = 16) and P3β 5β (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 P3β 5α (r = 0.528, P = 0.004, n = 28) and for the P3β 5β (r = 0.431, P = 0.022, n = 28).
Correlations between 3α- and 3β-isomers
Significant correlations between 3α- and 3β-PI were mostly found within all groups for both 5α- and 5β-PI, except for the missing data for 5β-PI in men. P3α 5α and P3β 5α 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 P3α 5α C and P3β 5α 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). P3α 5β and P3β 5β 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, P3α 5β C and P3β 5β 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 P3α 5α 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). P3β 5α 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 P3α 5β 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 P3α 5β , P3β 5β 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 5α-PI in men, attaining significance for P3α 5α (r = − 0.709, P < 0.022, n = 10) but not for P3β 5α (r = − 0.503, P < 0.14, n = 10). The subsequent partial correlations confirmed a negative borderline association between PregC and P3α 5α (r = − 0.633, P < 0.1, n = 10, adjusted to constant Preg and P3β 5α ), but showed no direct relationship between PregC and P3β 5α (r = 0.260, P = 0.504, n = 10, adjusted to constant Preg and P3α 5α ). Both pair correlations and partial correlations identified a strong positive relationship between P3α 5α and P3β 5α (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
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, 5α-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 3α-PI, resulting in a decreased affinity of GABAA-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 GABAA-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 P3α 5α . 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 P3α 5α but showed no relationship between PregC and P3β 5α . As expected, both pair and partial correlations found a strong positive relationship between P3α 5α and P3β 5α . Given the aforementioned results, the major metabolic pathway in adult men may be expected in the sequence PregC→ Preg→ Prog→ P5α → P3α 5α → P3β 5α .
Women in the luteal phase and early pregnancy produce the bulk of the PI in the corpus luteum (Ottander et al. 2005) PI (Fig. 8C).
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 P5α /β , in sequence. The P5α and possibly also the P5β penetrate into the maternal compartment, being further metabolized to PI and PIC (Dombroski et al. 1997).
While the 5α-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 P3β 5β (Fig. 5D) and showed a significantly decreasing trend for P3α 5β 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 P3α 5β . In these subjects, the P3α 5β may be rapidly metabolized to its conjugates. Alternatively, in L and P groups, the conjugation capacity for P3α 5β appears to be limited. The increased conjugation of 5α-PI probably diminishes the difference between the P3α 5α and P3α 5β levels in pregnant women and may also regulate the proportions between neuroinhibiting P3α 5α and counter-acting 5α-PIC. On the GABAA-R, the P3β 5α C exerts about 10% efficiency when compared with the effect of P3α 5α . However, P3β 5α 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 3α-PI positively modulating GABAA-R and 3β-PI being inactive but competing with the former PI on the receptors, the ratios of the sum of the 3α-PI to the sum of 3β-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 GABAA-R modulators are more manifestly prevalent in pregnancy over their inactive competitors on the GABAA-R. The respective differences in conjugates were much less pronounced (Fig. 6B).
Strong correlations between 3α- and 3β-PI that were found within all groups illustrate an uncomplicated, reversible oxidoreductive switch between the 3α- and 3β-PI via the 3-oxo-intermediate product in all groups. These metabolic steps converting neuroinhibiting 3α-PI to 3β-PI which competes with the 3α-PI on GABAA-R, as well as the sulfoconjugation of the PI-forming products operating on the GABAA-R in an opposite way to free 3α-PI, may be of great importance. It is known that P3α 5α dosage in animals or humans usually results in a weaker response than expected in consideration of the results of in vitro experiments. P3α 5α dosage eventuates in a bell-shaped response. At lower concentrations, P3α 5α 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, P3α 5β is an unstable substance with a short half-life in the human organism (Carl et al. 1994). P3α 5β 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 P3α 5β 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 GABAA-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 5β-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 5β-PI demonstrated in Fig. 7A allows speculation regarding the pregnancy sustaining role of pregnanolone. The weakening activity of 5β-reductase (Sheehan et al. 2005) as well as the declining levels of reduced 5β-pregnane steroids near term, further supports this hypothesis (Gilbert Evans et al. 2005). The results obtained in this study suggest that the sulfation of P3β 5α 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, P3β 5α C in pregnant women negatively correlated with Prog (r = − 0.512, P = 0.043, n = 27). The negative partial correlation between Prog and P3α 5β (r = − 0.548, P = 0.019, n = 29) indicates that a significant amount of Prog may be also metabolized via the 5β-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 3α-PI in the circulations of all groups. Significantly higher ratios of 5α-PIC to 5α-PI found in P compared with F and L groups indicate the more intensive conjugation of 5α-PI in pregnancy, including that of the most abundant neuro-inhibiting steroid P3α 5α . This mechanism probably provides for the elimination of excessive amounts of neuroinhibiting P3α 5α in the maternal compartment. The opposite situation was observed in P3α 5β . 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 P3α 5β C is the most abundant PIC but P3α 5β is a minor substance compared with the remaining PI, P3α 5β may acquire physiological importance in pregnancy, contributing to the sustaining thereof. Quite the opposite effect, the declining formation of P3α 5β 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).
Analytical criteria of the method for the multi-component quantification of neuroactive pregnanolone isomers and related steroids
| Steroid | Form | Gradient/derivatization | Injection | Retention time (min) | Effective mass (m/z) | Detection limit (nmol/l) (mean ± S.E.M., n = 5) | |
|---|---|---|---|---|---|---|---|
| *F, free steroid, C, conjugated steroid; †S, trimethylsilyl derivatives, MS, methoxyamine-trimethylsilyl derivatives. | |||||||
| No | |||||||
| 1 | Pregnenolone | *F, C | †S | 3 | 11.592 | 298, 388 | 0.058 ± 0.006 |
| 2 | Progesterone | F | MS | 1 | 11.250, 11.392 | 100, 341, 372 | 0.033 ± 0.004 |
| 3 | 3α-Dihydroprogesterone (P3α ) | F | MS | 1 | 10.975, 11.008 | 288, 343 | 0.020 ± 0.002 |
| 4 | 3β-Dihydroprogesterone (P3β ) | F | MS | 1 | 10.396, 10.475 | 288, 343 | 0.054 ± 0.010 |
| 5 | Allopregnanolone (P3α 5α ) | F, C | S | 1 | 10.758 | 285, 300, 375 | 0.028 ± 0.004 |
| 6 | Isopregnanolone (P3β 5α ) | F, C | S | 1 | 11.563 | 285, 300, 375 | 0.030 ± 0.004 |
| 7 | Pregnanolone (P3α 5β ) | F, C | S | 1 | 10.950 | 285, 300, 375 | 0.043 ± 0.006 |
| 8 | Epipregnanolone (P3α 5α ) | F, C | S | 1 | 10.550 | 285, 300, 375 | 0.032 ± 0.005 |
| 9 | 17α-Estradiol (internal standard) | – | S, (MS) | 1–4 | 9.863 | 285, 416 | 0.026 ± 0.003 |
The biosynthesis of pregnanolone isomers.
Citation: Journal of Endocrinology 195, 1; 10.1677/JOE-06-0192
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).
Citation: Journal of Endocrinology 195, 1; 10.1677/JOE-06-0192
Levels of 5α-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.
Citation: Journal of Endocrinology 195, 1; 10.1677/JOE-06-0192
Levels of 5β-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.
Citation: Journal of Endocrinology 195, 1; 10.1677/JOE-06-0192
Ratios of polar conjugates to unconjugated steroids for allopregnanolone (P3α 5α; A), isopregnanolone (P3β 5α; B), pregnanolone (P3α 5α; C), and epipregnanolone (P3β 5β; 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.
Citation: Journal of Endocrinology 195, 1; 10.1677/JOE-06-0192
Ratios of the sum of unconjugated pregnenolone isomers with a hydroxy group in the 3α-position (3α-PI/3β-PI; A) to the sum of unconjugated pregnenolone isomers with a hydroxy group in the 3β-position and the ratio of the steroid polar conjugates (3α-PIC/3β-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.
Citation: Journal of Endocrinology 195, 1; 10.1677/JOE-06-0192
Ratios of the sum of unconjugated pregnenolone isomers with hydrogen in the 5α-position (5α-PI/5β-PI; A) to the sum of unconjugated pregnenolone isomers with hydrogen in the 5β-position and the ratio of the steroid polar conjugates (5α-PIC/5β-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.
Citation: Journal of Endocrinology 195, 1; 10.1677/JOE-06-0192
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).
Citation: Journal of Endocrinology 195, 1; 10.1677/JOE-06-0192
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.
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