Mol. Hum. Reprod. Advance Access originally published online on July 28, 2005
Molecular Human Reproduction 2005 11(7):495-501; doi:10.1093/molehr/gah201
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5ß-Dihydroprogesterone and steroid 5ß–reductase decrease in association with human parturition at term
1Pregnancy Research Centre and University of Melbourne Department of Obstetrics and Gynaecology, Royal Women’s Hospital, Carlton and 2Mercy Perinatal Research Centre, Mercy Hospital for Women, East Melbourne, Victoria, Australia
3 To whom correspondence should be addressed at: Pregnancy Research Centre, Royal Women’s Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia. E-mail: penny.sheehan{at}rwh.org.au
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Abstract |
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The role of progesterone withdrawal in human parturition continues to provoke controversy. One possible mechanism by which functional progesterone withdrawal may be achieved is by a decrease in the circulating concentration of its bioactive metabolites. The progesterone metabolite 5ß-dihydroprogesterone (5ßDHP) has been shown to be a potent tocolytic in vitro. We quantified plasma concentrations of 5ßDHP in association with the onset of spontaneous labour in women at term and steroid 5ß-reductase mRNA expression in placenta, myometrium, chorion and amnion in relation to parturition, using real time RT–PCR. Serial blood samples were obtained from patients late in pregnancy, before term labour, during term labour and within the first 24 h postpartum. Following organic solvent extraction, steroids including 5ßDHP were separated by high-performance liquid chromatography (HPLC) and then quantified by radioimmunoassay (RIA). 5ßDHP concentration decreased two-fold (P = 0.00001, n = 25) from 0.317 ± 0.039 nmol/ml to 0.178 ± 0.017nmol/ml in association with active labour. Tissue 5ß-reductase mRNA-relative abundance was determined in placenta, myometrium, chorion and amnion obtained from labouring and non-labouring women. In placenta and myometrium, relative expression decreased significantly in association with labour, by about two-fold and 10-fold, respectively. These data are consistent with a possible role for 5ßDHP in the onset of spontaneous human labour. Further studies exploring this hitherto unrecognized endocrinological pathway are indicated.
Key words: 5ß-dihydroprogesterone/5ß-reductase/human/parturition/progesterone metabolites
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Introduction |
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One of the unresolved issues in our understanding of human parturition is the role and mechanism of progesterone withdrawal. In many mammalian species, the onset of labour is preceded by a decrease in circulating progesterone concentration and an increase in estrogen concentration. For example, in sheep, a rise in fetal cortisol triggers a change in placental steroid 17,20-lyase activity that results in decreased progesterone secretion and increased estrogen secretion (Liggins et al., 1972
). In mice, increasing prostaglandin F2
(PGF2) concentrations induce luteolysis and a fall in the concentration of serum progesterone concentration (Muglia, 2000). Common to both species is the state of increased uterine contractility induced by the fall in progesterone concentration or bioactivity. This involves a number of mechanisms including reduction in direct antagonism of oxytocin receptor signalling, and induction of oxytocin receptors, contractile PG receptors and gap junctions (Muglia, 2000).
Since Csapo pronounced his ‘progesterone withdrawal theory’ of human parturition (Csapo, 1977), subsequent investigators have been unable to conclusively demonstrate a decrease in progesterone concentration in serum or reproductive tract tissues in association with human parturition (Okada et al., 1974; Antonipillai and Murphy, 1977; Arai and Yanaihara, 1977; Haning et al., 1978; Sippell et al., 1979; Mathur et al., 1980a,b; Kauppila and Jarvinen, 1985; Davidson et al., 1987; Ohana et al., 1996). Despite this, there is clinical evidence for a role for progesterone withdrawal in the endocrine mechanisms underlying human parturition. The progesterone antagonist RU486 (mifepristone) induces abortion in the luteal phase of human pregnancy. In primate studies of induction of labour at term, mifepristone administration induced prostaglandin F2a production by decidua, but not prostaglandin E2 production by amnion (Haluska et al., 1994). A number of trials have been conducted of induction of labour in human term pregnancies with mifepristone as compared to placebo. These indicate that mifepristone can induce both ripening of the cervix and labour. Compared to placebo, mifepristone-treated women were less likely to have an unfavourable cervix at 48 or 96 h, and were more likely to have delivered within 48 and 96 h of treatment than were placebo/untreated women (Neilson, 2000).
Studies in which the selective 3ß-hydroxysteroid dehydrogenase inhibitor epostane was administered to rhesus monkeys at term resulted in a reduction of maternal and fetal circulating progesterone concentrations, increase in uterine activity and cervical ripening within 24 h and spontaneous vaginal delivery within 48 h. Supplemental progesterone prevented the epostane effects but supplemental progesterone alone did not prevent the onset of spontaneous labour (Haluska et al., 1997). This demonstrated that artificially induced progesterone withdrawal is able to induce labour in a primate species although supplemental progesterone does not prevent spontaneous parturition. Although these studies demonstrate a role for progesterone in maintaining uterine quiescence at a genomic level, they also suggest some intervening mechanism, rather than merely an effect of progesterone, may be involved. Although progesterone does not act as an acute tocolytic, recent studies have suggested that progesterone administration throughout gestation may reduce the risk of preterm labour in high-risk patients (da Fonseca et al., 2003; Meis et al., 2003; Sanchez-Ramos et al., 2005).
Further studies have sought to describe other mechanisms by which progesterone withdrawal may result in labour onset in the human. Notable amongst these, changes in tissue concentrations of progesterone (Lenz et al., 1989), changes in progesterone synthesis (Grimshaw et al., 1983; Khan-Dawood, 1987; Mitchell et al., 1987; Chibbar and Mitchell, 1990), increased progesterone metabolism (Milewich et al., 1975, 1977a,b, 1978; Diaz-Zagoya et al., 1979) and changes in ratio of progesterone A and B receptors (Pieber et al., 2001; Mesiano et al., 2002) have all been proposed as mechanisms by which progesterone withdrawal may occur, at least at a paracrine level. Despite such considerable work, this issue has not yet been clearly resolved.
One alternative mechanism by which functional progesterone withdrawal might be achieved is through the actions of a progesterone metabolite. One progesterone metabolite that has been shown to possess tocolytic activity in vitro is 5ß-dihydroprogesterone (5ßDHP). Kubli-Garfias et al. (1979) demonstrated that 5ßDHP was the most potent tocolytic steroid of ten progestins tested on isolated single myometrial cells in vitro, results confirmed by Thornton et al. (1999). In 1998, Grazzini et al. reported that progesterone affected rat myometrial activity by binding directly to the oxytocin receptor, thus preventing activation of the receptor by circulating oxytocin (Grazzini et al., 1998). The authors, however, were unable to demonstrate significant binding of progesterone to the human oxytocin receptor. Instead, it was observed that 5ßDHP displayed the greatest antagonist affinity for the human oxytocin receptor. This observation has been disputed by two subsequent studies (Burger et al., 1999; Astle et al., 2003) that did not confirm the blockage of oxytocin receptors by 5ßDHP. Burger et al. (1999), however, demonstrated that 5ßDHP had an ability to reduce ligand-induced calcium signalling in human myometrium, that was equivalent to the action of progesterone, both being more potent than other steroids such as pregnenolone, E2 and dihydroepiandrosterone. Thus, although evidence exists for a tocolytic effect of 5ßDHP, the mechanism is unlikely to be related to binding to the oxytocin receptor.
Although studies have identified 5ßDHP in human reproductive tract tissues (Pollow et al., 1975; Junkermann et al., 1977), there is a paucity of information detailing pregnancy and labour-associated changes in circulating concentrations of the 5ß-reduced progesterone metabolite.
The human 5ß-reductase enzyme catalyses the conversion of progesterone to 5ßDHP, which previously has been regarded as an inactivating step. In 2001, Charbonneau and The identified and sequenced the human 5ß-reductase gene (Charbonneau and The, 2001). Northern blot analysis of a standard panel of human tissues showed that the 5ß-reductase mRNA was strongly expressed in liver, colon and testis. The enzyme produced by cells transfected with the cloned mRNA acted on a range of steroids including progesterone and cortisol.
In this study, we sought to examine the involvement of 5ßDHP in progesterone withdrawal pathways associated with the onset of human parturition. We hypothesized that a decrease in circulating 5ßDHP concentrations is associated with increased uterine contractility and a predisposition to labour. Furthermore, we hypothesized that 5ß-reductase mRNA expression is decreased in association with onset of spontaneous labour in the human. This would be consistent with a reduction in the conversion of progesterone to 5ßDHP, a decrease in circulating 5ßDHP concentration, and thus, a withdrawal of its tocolytic effect, with subsequent facilitation of the labour process.
The aims of this study were to compare plasma concentrations of 5ßDHP before, during and after spontaneous labour in women at term, and to relate these to studies of mRNA for the enzyme 5ß-reductase in reproductive tract tissues.
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Subjects and methods |
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Measurement of circulating 5ßDHP in serum
Subjects
The patients in this study had uncomplicated pregnancies. Complete sets of three blood samples consisting of ‘before labour’, ‘during labour’ and ‘post-partum’ samples were obtained from 25 patients. The initial blood sample was taken at recruitment, between 36 and 38 weeks’ gestation. A second blood sample was collected after a diagnosis of active, spontaneous-onset labour had been made on the basis of cervical dilatation of at least 3 cm and full effacement in the presence of active contractions but before delivery. Patients who had undergone induction of labour by any method including the use of prostaglandin gel were excluded from the study. Three of the patients were later augmented by syntocinon infusion after presentation in spontaneous labour and two patients were later delivered by caesarean section, one for fetal heart rate abnormalities and one for obstructed labour. The patients’ characteristics are summarized in Table I. The post-partum plasma sample was taken within 24 h after delivery. All subjects provided written informed consent to participate in the study, which was approved by the Research and Ethics Committees at the Royal Women’s Hospital, Melbourne, where the patients delivered.
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Samples
Blood was collected by venepuncture into 9 ml EDTA tubes and stored at 4°C until centrifuged at 1800 g for 10 min within 3 h of collection. Plasma was collected and stored as 1 ml aliquots at –40°C until assayed.
Studies of 5ß-reductase mRNA
Subjects
Permission to obtain tissue samples was obtained from the Royal Women’s Hospital Research and Ethics Committees before the commencement of the study. The tissues chosen for the study were placenta, myometrium, amnion and chorion obtained from term gestations. Placenta, amnion and chorion were obtained from patients undergoing elective caesarean section and from patients following onset of spontaneous labour and subsequent vaginal delivery. None of these were the same patients who gave blood samples for 5ßDHP concentration in plasma, as 5ß-reductase studies were contingent upon the earlier findings. All were from Caucasian patients with uncomplicated pregnancies. No patient contributed more than one tissue. Again, one group consisted of patients undergoing elective caesarean section before labour onset whereas the other caesarean group consisted of patients presenting in spontaneous labour, as diagnosed by the presence of regular uterine contractions and cervical dilatation of more than 3 cm and full effacement. Reasons for caesarean in labour included abnormal fetal heart rate pattern, obstructed labour and undiagnosed breech presentation.
None of the groups had any significant differences with respect to maternal age or birth weight. There were significant differences in two groups with regard to gestational age at delivery (Table II). Amongst the patients who contributed placenta or chorion, the ‘in labour’ group were statistically significantly more advanced with respect to gestation than the ‘not in labour’ group. This reflects the fact that elective sections are routinely performed at term but shortly before the expected onset of spontaneous labour. Thus, although there were these two statistical differences, they were unlikely to be of clinical or biological significance given all samples were from term gestations and the relevant variable was the presence or absence of labour.
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Samples
Myometrium samples consisted of a sample of tissue taken from the edge of the lower uterine segment incision at caesarean section. Placenta and membranes were delivered intact by controlled cord traction.
Determination of 5ßDHP
Extraction
Plasma aliquots of 1 ml were extracted three times with 10 ml of hexane [HiPerSolv for high-performance liquid chromatography (HPLC), BDH, Quebec, Canada] in a 100 ml Pyrex extraction tube. The organic phase was removed using a glass pasteur pipette, ensuring that the lower aqueous phase was not disturbed, evaporated to dryness under nitrogen and resuspended in 500 µl acetonitrile (HiPerSolv for HPLC, BDH, Quebec, Canada).
HPLC
Using a Shimadzu LC10A High Performance Liquid Chromatograph, including two pumps and an autoinjector module, 100µl of extract was loaded onto a Supelcosil LC-18 ID Modular HPLC column (25 cm x 4.6 mm). The sample was eluted isocratically with acetonitrile and water (60:40; 1 ml/min) for 30 min at room temperature according to the method described by Walters et al. (1981). Fractions of 1 ml were collected in Eppendorf tubes by a Shimadzu FRC-10A fraction collector each minute between 10 and 20 min. Standard solutions of various progestins including progesterone and 5ßDHP, 1 mg/ml in acetonitrile were run after the samples. Steroids were obtained from Sigma (St Louis, MO, USA). The standard for 5ßDHP corresponded to fraction 6 (Figure 1). Mean retention times for various progestins are given in Table III.
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Radioimmunoassay
The fraction containing 5ßDHP was dried under nitrogen at room temperature and resuspended in phosphate buffer by vortexing for 60 s and incubating overnight at 4°C. A 100 µl aliquot of each fraction was assayed by radioimmunoassay (RIA) using a cross-reacting progesterone antibody obtained from Bioquest (NSW, Australia). The antibody was raised in sheep using progesterone–6 carboxymethyoxime : bovine serum albumin as antigen and was used at a final concentration of 1:1333 in phosphate buffer 0.1 M, pH 7.4 containing 0.1% sodium azide. Tubes were incubated overnight at 4°C with tritiated progesterone (72 kBq/tube) (Amersham Biosciences, Piscataway, NJ, USA) as tracer. Bound tracer was precipitated using 50 µl human immunoglobulin G (IgG) (1:38 000, CSL, Victoria, Australia) and 1 ml of 27% solution of polyethylene glycol (PEG 6000, Sigma). Precipitate was resuspended in 2 ml of scintillant (Starscint, Packard Bioscience, Boston, MA, USA) and radioactivity was quantified by scintillation spectrometry in a Wallac 1409 liquid scintillation counter. The standard curve was calculated and concentrations of individual samples were estimated by the MultiCalc on-line software. Details of cross-reactivity for key steroids progesterone, 5-dihydroprogesterone (5DHP) and 5ßDHP were checked under the described laboratory conditions and found to be 100, 62.5 and 25%, respectively (n = 3).
Intra-assay coefficient of variation (CV) was determined to be 5 ± 0.1% on five samples at a concentration in the midrange of the assay. (All results are given as mean ± SEM.) Inter-assay CV was estimated at 14 ± 0.8% on 10 assays. The effects of inter-assay variation were minimized by ensuring that samples for comparison were measured in the same assay.
Recovery
Recovery of steroids from the plasma sample was estimated by addition of 72 kBq/tube of tritiated progesterone tracer in 10 µl of phosphate buffer to the original plasma sample, which was then extracted, resuspended and eluted through the HPLC column as described above. The second fraction, corresponding to the progesterone standard peak was then dried and resuspended in buffer as described and a sample of this quantified by scintillation spectrometry. Using this method, mean recovery was estimated as 71.5% (n = 2).
Studies of 5ß-reductase mRNA
Total RNA extraction
Tissue samples were immediately placed on dry ice and stored at –80°C until RNA extraction. Total RNA was extracted from placenta, amnion and chorion samples using the Qiagen midi RNeasy kit (Qiagen, Valencia, CA, USA) according to the method described by the manufacturer and stored at –80°C until assay. The quantity of total RNA recovered was determined spectrophometrically by measuring absorbance at 260 nm and the integrity assessed by non-denaturing electrophoresis on a 1% agarose gel. Myometrial samples were obtained from patients as described above at caesarean section and placed immediately in 3 ml of RNA Later (Qiagen) and stored at 3°C until extracted within 7 days of collection. Extraction was then performed as previously described.
Real time RT–PCR
A 20 ng aliquot of total RNA from each sample was reverse-transcribed using superscript II according to the manufacturer’s manual (Invitrogen, USA). Two microlitres of cDNA were amplified by real time PCR with 2x Taqman universal PCR mastermix (Applied Biosystems, Foster City, CA, USA). A 5ß-reductase Assay-on-Demand primer and probe kit was used (ID number Hs00195594_m1; Applied Biosystems). The Taqman probe sequence was TGGAAAGCTATGGGCTACAAATC. The probe used in this study crossed the boundary between exon 1 and exon 2, spanning intron 1, the largest of eight introns with length 13 kb (Charbonneau and The, 2001). For glyceraldehyde-3-phosphatedehydrogenase (GAPDH) housekeeping gene, a predetermined assay was obtained containing both primers and probe (Assays-on-Demand catalogue number Hs9999905_m1; Applied Biosystems). Each sample was analysed in duplicate 20 µl reaction under universal thermal cycling parameters as described by the manufacturer and using the ABI PRISM 7700 (Applied Biosystems).
Steps consisted of uracil-DNA glycosylase (UNG) digestion of amplified DNA at 50°C for 2 min, followed by denaturation at 95°C for 10 min. The thermocycling profile consisted of a denaturation step at 95°C for 15 s, followed by an anneal-extension step at 60°C for 1 min for 40 cycles.
Relative quantitation was performed using the comparative CT method for separate tubes as described by the manufacturer (Applied Biosystems User Bulletin #2, Relative Quantitation of Gene Expression, 1997) which has been discussed in the literature (Pfaffl, 2001). A validation experiment was performed to demonstrate equal efficacy of target and reference. Under these conditions, the amount of target, normalized to an endogenous reference and relative to a calibrator, is given by 2–
C
. The calculation of CT involves subtraction by the CT for the calibrator tissue where CT is the threshold cycle for target amplification and CT is determined by subtracting the average endogenous control CT from the average target CT.
Liver was chosen as the calibrator tissue for this experiment. A commercially available preparation of cDNA was obtained (Stratagene, La Jolla, CA, USA) which consisted of cDNA generated from poly (A) RNA. The tissue source was two human adult females aged 60 and 67 years.
Statistical analysis
‘Not in labour’ and ‘in labour’ groups were compared using an unpaired t-test for groups with unequal variance.
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Results |
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Measurement of circulating 5ßDHP in serum
Quantification of immunoreactive steroid concentration in plasma obtained from patients in spontaneous labour at term is shown in Figure 2. The data were analyzed using the nonparametric Mann–Whitney two sample statistic to test the groups in pairs. This technique was chosen because data from the three groups were not normally distributed, nor was it possible to perform a single uniform transformation to produce a normal distribution in all three groups. Using this technique, statistically significant differences were identified between the ‘before labour’ group and the ‘during labour’ group and between the ‘before labour’ group and the ‘post-partum’ group. There was no statistically significant difference between the ‘during labour’ and the ‘post-partum’ groups.
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Studies of 5ß-reductase
Comparative quantitation of mRNA in the four reproductive tract tissues and the effect of spontaneous labour are shown in Table IV. The results are expressed normalized to the housekeeping gene GAPDH, and relative to the calibrator tissue, liver. There were no significant differences in GAPDH CT values in the various tissues with labour states. Liver was used as a calibrator tissue as 5ß-reductase is known to be highly expressed in that tissue.
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5ß-Reductase mRNA was most highly expressed in placenta by two orders of magnitude in comparison to the next most abundant tissue, myometrium. 5ß-Reductase mRNA expression was lowest in amnion, being undetectable in a number of samples from both the ‘in labour’ and ‘not in labour’ groups.
Significant decreases were noted in 5ß-reductase mRNA expression between ‘in labour’ and ‘not in labour’ groups for placenta (P = 0.031) and myometrium (P = 0.004). The latter in particular decreased to only approximately one tenth the level found in tissues taken from women before labour onset.
There was no significant difference in 5ß-reductase mRNA expression in chorion or amnion from women before or after labour onset.
It was noted in the examination of the patient characteristics that gestational age of patients in the ‘in labour’ group for placenta and chorion was significantly advanced over the ‘not in labour’ group. Although it is possible that the difference in 5ß-reductase mRNA seen in the comparison of the two placenta groups may be due to gestational age rather than labour state, given there was no difference in 5ß-reductase mRNA quantitation between the two chorion groups, this suggests that the 5ß-reductase decrease detected in the ‘in labour’ placenta group is biologically more relevant to the onset of labour than to gestational age at term.
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Discussion |
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The data obtained in the study demonstrate that plasma concentrations of 5ßDHP decrease significantly in association with the active phase of the first stage of human labour, as diagnosed by the presence of regular contractions in addition to cervical changes. These data are consistent with the decrease occurring in association with the onset of parturition and support the involvement of 5ßDHP in the cessation of uterine quiescence at the end of pregnancy.
Furthermore we demonstrated that mRNA expression for the enzyme that converts progesterone to 5ßDHP also decreases in association with labour in both placenta and myometrium. To our knowledge, this is the first report measuring 5ß-reductase mRNA in reproductive tract tissues in association with labour. 5ß-reductase mRNA was detected in all four reproductive tissues studied. Expression in amnion and chorion relative to liver were very low at term gestation. The highest levels were observed in placenta. The change with labour status of the greatest magnitude, however, occurred in myometrium (about 10-fold).
Although 5ßDHP has been identified amongst other progesterone metabolites in the urine of pregnant women (Milewich et al., 1979), this is the first report of its direct measurement in plasma in association with human parturition. A recent publication described the measurement of 5ßDHP as well as four other progesterone metabolites in plasma of pregnant women throughout pregnancy and postpartum in association with depression (Pearson Murphy et al., 2001). The investigators demonstrated that 5ßDHP increased 16-fold during pregnancy, reaching a plateau at about 30 weeks’ gestation, and then decreasing to approximately a quarter of the pregnant level by the end of the first week postpartum. Concentrations were not studied in relation to parturition. Our current study more specifically demonstrates that 5ßDHP concentration has already decreased by the time of the active phase of labour and that little further decrease takes place in the first 24 h post-partum.
Our studies were limited to the last few weeks of pregnancy only. There is evidence that an increase in myometrial responsiveness to contractile stimuli occurs before this time. The process of myometrial activation includes changes in the resting membrane potential of the myometrial cell, expression of the receptors for oxytocin and prostaglandins and the enhancement of post-receptor coupling mechanisms. Most of these events are presumed to be related to the action of estrogen although prostaglandins may also contribute to this process. It may be that 5ßDHP action allows these processes to occur in preparation for labour while maintaining uterine quiescence.
Previous in vitro studies have suggested that a decrease in 5ßDHP concentration may be mechanistically important for the onset of human parturition (Kubli-Garfias et al., 1979). This change could be the result of a decrease either in 5ß-reductase enzyme amount or enzyme activity. The data obtained in this study are consistent with a reduction in enzyme content contributing to decrease conversion of progesterone to 5ßDHP. In their work on the gene sequence for 5ß-reductase, Charbonneau and The (2001) commented that human 5ß-reductase has a long-3'-noncoding adenine-thymine (AT)-rich region that is known to be present in mRNA species that are rapidly degraded. This is consistent with our hypothesis that acute changes in 5ß-reductase mRNA expression may take place in association with the spontaneous onset of human parturition, resulting in a decrease in 5ßDHP concentrations. The pronounced decrease in 5ß-reductase mRNA in myometrium may indicate that a more significant decrease in 5ßDHP occurs in this tissue than can be detected by measurement of circulating concentrations. This is consistent with a possible paracrine effect of 5ßDHP in this tissue. The progesterone metabolite 5ßDHP is known to be a ligand for the nuclear receptor pregnane X receptor (PXR) and studies in PXR knockout mice have suggested that 5ßDHP may act via the PXR to increase inducible nitric oxide synthase, which produces the potent smooth muscle relaxant nitric oxide. (Mitchell et al., 2005).
The most abundant progesterone metabolites in human pregnancy are known to be 20DHP and 5DHP (Milewich et al., 1977a). Progesterone metabolism has been described in human placenta (Milewich et al., 1978, 1979), fetal membranes (Milewich et al., 1977b) and myometrium (Mickan, 1976; Junkermann et al., 1977). Both of these metabolites have been investigated in various tissues in relation to parturition and pregnancy. In the placenta, progesterone is mostly metabolized to 20DHP, with 5DHP as the second most abundant metabolite. Tissue 20DHP concentration has been shown to increase in placenta after labour mechanisms have been activated, either spontaneously or by artificial means (Diaz-Zagoya et al., 1979; Mitchell and Wong, 1993), the authors suggesting this as a possible reflection of progesterone withdrawal in the absence of a significant decrease in circulating progesterone concentrations. A similar pattern of progesterone metabolism has been shown in fetal membranes although amnion appears to have higher 5-reductase activity than chorion (Milewich et al., 1977b). In contrast to placenta, progesterone metabolism by 20 hydroxysteroid dehydrogenase (20HSD) in the fetal membranes may decrease with advancing gestation (Milewich et al., 1977b). Human myometrium has a different pattern of progesterone metabolism with a relatively greater importance of the 5-reductase pathway (Mickan, 1976). Other metabolites have been demonstrated to be produced by human myometrium, including 5ßDHP (Mickan, 1976; Junkermann et al., 1977).
Initially, investigators thought the liver to be the main site of 5ß-reductase activity, followed immediately by 3-hydroxysteroid reductase activity and subsequent urinary excretion (Atherden, 1959). All of these reactions were considered to be completed in the liver before the nonconjugated 5ß-reduced intermediates could be released into the blood (MacDonald et al., 1991). This suggests that circulating 5ßDHP originates in extrahepatic progesterone metabolism. The presence of 5ß-reductase mRNA in reproductive tract tissues identified for the first time in this study suggests a contribution from these tissues.
Characterization of human 20HSD has been recently completed (Nishizawa et al., 2000). An analysis of 20HSD gene expression in reproductive tract tissues would aid in our understanding of the complex pathways of progesterone metabolism in pregnancy and parturition.
Two tissues showed a significant change in 5ß-reductase mRNA in our study. In myometrium, the change in magnitude was particularly marked. This result is of interest because myometrium would be the expected target tissue for the maintenance of uterine quiescence by the progesterone metabolite, 5ßDHP. This hypothesis is supported by the fact that the presence of 5ß-reductase enzyme activity has been reported previously in myometrium (Mickan, 1976; Junkermann et al., 1977), although not in pregnancy. Although earlier studies have not identified 5ß-reductase activity in other human reproductive tract tissues (Milewich et al., 1977a,b, 1978), a more recent study has found evidence of 5ß-reductase activity in human placenta, chorio-decidua and amnion. (Mitchell et al., 2005). The earlier studies were limited by the in vitro conditions under which they were conducted which may be very different to those in vivo and also by the sensitivity of the method of product detection. It may be that 5ß-reduced progesterone metabolites were present in some of these studies but not detected.
In conclusion, our findings are consistent with a decrease in circulating concentration of 5ßDHP, contributing to increased myometrial activity at the onset of human parturition. This decrease in 5ßDHP is at least partly mediated by a decreased expression of 5ß-reductase in reproductive tract tissues as indicated by decreased mRNA expression. Further exploration of the 5ß-reductase pathway and the actions of metabolites produced is indicated in the investigation of the endocrinology of human parturition.
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Acknowledgements |
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The authors thank Vicki Doherty, May Grigurovic and Mahtab Riazati-Keshi for technical assistance. This work was supported by the Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG) Research Foundation and the Arthur Wilson Memorial Scholarship and also a research grant from the Royal Women’s Hospital Research Foundation.
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References |
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Submitted on June 2, 2005; accepted on June 7, 2005.
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