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Original Studies |
Department of Reproductive Medicine and Child Development, Section of Gynecology and Obstetrics, University of Pisa, Pisa; the Department of Surgical Sciences, Chair of Obstetrics and Gynecology, University of Udine (F.P.), Udine; Endocrine Unit of the Italian Research Council, CNR (E.C., M.L.), Pisa; the Department of Obstetrics and Gynecology, University of Pavia (R.E.N.), Pavia; and the Department of Obstetrics and Gynecology, University of Catania (M.P.), Catania, Italy; and the Department of Psychiatry, Veterans Administration Hospital (R.H.P.), San Diego, California 92161
Address all correspondence and requests for reprints to: Prof. A. R. Genazzani, Department of Reproductive Medicine and Child Development, Section of Gynecology and Obstetrics, University of Pisa, Via Roma 35, 56100 Pisa, Italy.
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Abstract |
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The present study included 189 fertile women, 112 postmenopausal women, and 46 men. Serum steroid levels were determined after extraction, using specific RIAs. Allopregnanolone levels in fertile women in the follicular phase were similar to those in age-matched men; no significant difference was found between fertile women in the follicular phase and postmenopausal women. The highest levels were found in fertile women during the luteal phase (P < 0.01). An age-related decrease was observed in men (P < 0.01), but not in women. P and DHEA levels were significantly higher in women than in men and were higher in fertile women than in postmenopausal women (P < 0.01). Both P and DHEA showed an age-related decrease in men and women (P < 0.01).
Serum allopregnanolone and P, but not DHEA, significantly increased in response to a GnRH test, whereas corticotropin-releasing factor and ACTH tests elicited a significant increase in allopregnanolone, P, and DHEA levels (P < 0.01). The suppression of adrenal steroidogenesis by dexamethasone markedly reduced both allopregnanolone and DHEA serum levels (P < 0.01).
In conclusion, the present study demonstrated that although men show an age-related decrease, serum allopregnanolone levels in women do not change with age and correlate with P levels during the menstrual cycle and in response to endocrine tests. Ovary and adrenal cortex may be major sources of circulating allopregnanolone in fertile women.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxy-5-pregnan-20-one, is a ring A-reduced pregnane
derivative of progesterone (P), synthesized in the gonads, adrenal
cortex, and central nervous system (1, 2, 3, 4, 5, 6, 7). In rats, an ovarian origin
of circulating allopregnanolone has been demonstrated by 1) its
presence in ovarian tissue, 2) increased circulating levels at estrous,
and 3) decreased levels after ovariectomy (8). An adrenal origin is
suggested by the disappearance of plasma allopregnanolone in female
rats only after combined adrenalectomy and ovariectomy (8).
Experimental studies have shown an involvement of allopregnanolone in
stress, mood, and sexual behavior (9, 10, 11). The changes in brain
allopregnanolone concentration in female rats during the estrous cycle
(12) and the effect of intracerebroventricular injection of
allopregnanolone or its antiserum on ovulatory rat function (13, 14)
suggest a correlation between brain allopregnanolone and reproductive
function. The central effect of allopregnanolone is probably mediated
by its interaction with 
-aminobutyric acidA
(GABAA) receptors or through an increase in
GABAA receptor sensitivity to endogenous GABA (15, 16, 17).
Modifications of circulating allopregnanolone levels throughout the menstrual cycle in humans have been reported (18, 19). High levels have been observed during the luteal phase of the menstrual cycle, with controversial results on the possible involvement of allopregnanolone in the premenstrual syndrome (14, 18). An increase in serum allopregnanolone levels in women during the third trimester of pregnancy has been correlated with an increase in GABAA receptor sensitivity to GABA agonists and with alterations in mood (15).
Although several studies have focused on the central effects of allopregnanolone, no study regarding physiological changes in serum levels in humans is available at present. Consequently, the aim of the present study was to assess the difference in allopregnanolone concentrations according to gender, menstrual cycle, age, and menopause in normal subjects. In addition, possible sources of allopregnanolone in human circulation were investigated by evaluating the response of allopregnanolone to functional endocrine tests activating ovary (GnRH test) or adrenal cortex [corticotropin-releasing factor (CRF) and ACTH tests]. In all specimens, simultaneous evaluation of serum P and dehydroepiandrosterone (DHEA) levels was also performed in the present study.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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After giving informed consent, 347 subjects participated in the present study; they were subdivided into the following 3 groups: 1) fertile women (n = 189; follicular phase, n = 81; luteal phase, n = 108), aged 16–39 yr; 2) postmenopausal women (n = 112), aged 40–72 yr; and 3) men (n = 46), aged 19–70 yr. Before entering the study each subject had medical history, physical examination, and routine laboratory tests performed, which disclosed no abnormalities. Subjects with a history of cancer were excluded. None of the subjects was taking psychoactive medications, hormonal drugs (including oral contraceptives), mineral or vitamin supplements, or antiinflammatory drugs. A diagnostic interview did not show current or recent (within the past 2 yr) medical or psychiatric illness, and no subject described pathological changes in mood or behavior in the recent years. All fertile women reported regular menstrual cycle lengths, with serum P values above 20 nmol/L. Informed consent was obtained from each subject after full description of the protocol, which had been approved by the local ethical committee.
Protocol
In each subject a blood sample was drawn between 0700–0900 h. Blood samples were centrifuged and serum stored at -20 C until assayed.
In some of the fertile women the following functional tests were performed: 1) GnRH test (n = 6 in the luteal phase), consisting of a bolus injection of 100 µg GnRH (Ferring, Kiel, Germany); 2) CRF test (n = 6 women in the follicular phase), consisting of an iv bolus injection of 100 µg human CRF (Clinalfa, Laufelfingen, Switzerland); 3) ACTH test (n = 6 in the follicular phase), consisting of an im injection of 0.50 mg ACTH (Synachten, Ciba-Geigy, Huningue, France); and 4) dexamethasone (DXM) test (n = 4 in follicular phase), consisting of the administration of DXM (0.5 mg Decadron, Merck, Sharp, and Dohme, Rome, Italy) at 6-h intervals for 48 h.
In all patients a catheter was inserted into the antecubital vein, and a slow infusion of 0.9% saline solution was begun. Blood samples were taken 0, 15, 30, 45, 60, 90, and 120 min after injection of GnRH or CRF and 0, 30, 60, and 90 min after ACTH injection; blood samples were taken at 0800 h and 24 and 48 h after the first administration of DXM.
Allopregnanolone, P, and DHEA were assayed by using a specific RIA.
Allopregnanolone, P, and DHEA assays
Analytical grade solvents were purchased from Merck (Darmstadt,
Germany); C18 Sep-Pak cartridges were obtained from Waters
Corp. (Milford, MA). Standard allopregnanolone was purchased from Sigma
Chemical Co. (St. Louis, MO), and
5-[9,11,12-N-3H]pregnan-3-ol-20-one (45 Ci/nmol)
was purchased from Amersham (Aylesbury, UK). The polyclonal antisera
raised in sheep against allopregnanolone carboxymethyl ether coupled to
BSA was provided by Dr. R. H. Purdy. Serum samples (1 mL) were
thawed. Standard solutions of allopregnanolone (0, 15.6, 31.2, 62.5,
125, 250, 500, and 1000 pg steroid/tube) in steroid-free serum (pooled
human serum, stripped from endogenous steroids by repeated treatment
with charcoal: 50 mg/mL), recovery standard of tritium-labeled steroid
was added (1200–1500 cpm), and the samples were extracted twice with 8
mL diethyl ether. The organic phases were removed and evaporated to
dryness in Universal Vacuum System Plus (Savant, Rome, Italy). The
extracts were eluted with assay buffer (20 mmol/L sodium phosphate in
saline, containing sodium azide and BSA, 0.1 g/L each) and divided into
two aliquots, each of 500 µL, corresponding to 500 µL serum. The
samples in duplicate were passed through a C18 Sep-Pak
cartridge, previously equilibrated with a solution of aqueous methanol
(50:50, vol/vol) containing 1% acetic acid. After washing the
cartridge with 50% aqueous methanol containing 1% acetic acid and
50% aqueous methanol, the elution of allopregnanolone was performed
with absolute methanol (10 mL). The dry extracts were then assayed by
RIA as following: the radioligand (7000 cpm) and the antibody (working
dilution, 1:4000, vol/vol; 100 µL each) dissolved in the assay buffer
were added to the tubes containing samples and standards, and the
volume was adjusted to 500 µL with buffer. The samples for
determination of nonspecific binding contained only the radioligand.
After vortex mixing and overnight incubation at 4 C, bound and free
steroids were separated by dextran-charcoal adsorption (0.25 g charcoal
and 0.025 g dextran-70 in 100 mL assay buffer, 0.5 mL) followed by
centrifugation at 4 C for 10 min. The radioactivity of supernatants was
measured in a liquid scintillation spectrometer equipped with a program
for counting disintegrations per min and RIA calculation. After
extraction with ether, the recovery of labeled allopregnanolone was
96.3 ± 4.8%; after extraction and chromatography, it was
87.5 ± 7.9%; and for the entire procedure, the recovery of
unlabeled standard allopregnanolone was 77.5 ± 9.8%. The
sensitivity of the assay, expressed as a minimal amount of
allopregnanolone distinguishable from the zero sample with 95%
probability, was 15–20 pg/tube, and the intra- and interassay
coefficients of variation were 7.2% and 9.1%, respectively. A
parallelism test was also performed: a sample containing a high
concentration of standard unlabeled allopregnanolone (
2000 pg/mL)
was diluted with 0 standard, producing the results reported in Table 1
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Serum samples for the determination of DHEA were extracted with ether, purified through a C18 Sep-Pak cartridge, and then assayed by RIA using trade kit (DSL, Webster, TX); the sensitivity was 15 pg/mL, and the intra- and interassay coefficients of variation were 3.1% and 6.9%, respectively.
Statistical analysis
Statistical analysis of the results was performed with a Macintosh personal computer using Abacus Concepts, Stat-View 4.0 program (Berkeley, CA). All results are reported as the mean ± SEM. Related measures ANOVA was used for comparison of hormone levels between times, and Scheffe’s F test was used to determine the locations of significant differences in mean values.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). Serum P levels in women during both
follicular (5.32 ± 2.10 nmol/L) and luteal (34.41 ± 7.80
nmol/L) phases were significantly higher than those in men (1.9 ±
0.08 nmol/L; P < 0.001; Fig. 2). Also, DHEA levels were higher in
women than in men (33.39 ± 3.89 nmol/L in the follicular phase
and 31.33 ± 6.06 nmol/L in the luteal phase vs.
16.33 ± 0.61 nmol/L in men; P < 0.01; Figs. 1 and 2).
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). Both P and DHEA
levels in women and men showed a significant decrease according to age
(P < 0.01; Fig. 2). When data were evaluated on the
basis of menopausal age, mean ± SEM serum
allopregnanolone levels in postmenopausal women (0.66 ± 0.34
nmol/L) resulted in the same range of values as those in age-matched
men and fertile women in the follicular phase (Fig. 2). P and DHEA
levels in postmenopausal women (1.59 ± 1.13 and 9.59 ± 4.43
nmol/L, respectively) were significantly lower than those in fertile
women (P < 0.01; Fig. 2).
Serum allopregnanolone and P showed a significant increase in response
to GnRH test [peak values, 4.11 ± 1.03 nmol/L at 45 min
(P < 0.01) and 40.7 ± 6.6 nmol/L at 120 min
(P < 0.01), respectively], whereas there were no
significant changes in DHEA (Fig. 3).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The responses of allopregnanolone to GnRH test, CRF test, and ACTH test suggest that both ovary and adrenal cortex are possible sources of allopregnanolone. In fact, the GnRH test activating ovarian function as well as the CRF or ACTH test stimulating adrenal cortex function induce a significant increase in allopregnanolone levels. Therefore, ovary and adrenal cortex may both contribute to circulating allopregnanolone. The delayed response of allopregnanolone levels to CRF compared to ACTH is probably related to the time required for CRF to elicit pituitary hormone secretion. However, although the response of P to CRF is similar to its response to ACTH, the allopregnanolone response to CRF is significantly higher than its response to ACTH. The unexpectedly high response of allopregnanolone to the CRF test compared with that to the ACTH test may be a consequence of a direct and/or indirect stimulation of allopregnanolone synthesis by CRF, independent from the ACTH-mediated stimulation of adrenal steroidogenesis. The observation of a marked reduction in allopregnanolone levels after the DXM test supports the hypothesis that the adrenal cortex largely contributes to its circulating levels, directly or via the synthesis of a precursor converted peripherally in allopregnanolone.
On the other hand, the evidence that the GnRH test determines a significant increase in serum allopregnanolone levels and a limited and delayed increase in serum P levels suggests that steroid secretion from the ovary in response to GnRH is more prone toward allopregnanolone release than toward P release.
In men, allopregnanolone levels show a decrease correlated with age, whereas in women, this decrease is not observed; a GnRH-induced gonadotropin increase may determine an increase in allopregnanolone secretion from the ovary in postmenopausal women. Likewise, the high levels of gonadotropins observed in these subjects may explain the lack of an age-related decrease in allopregnanolone levels, as would be predicted on the basis of age-related P decrease. The age-related decreases in P and DHEA are consistent with previous findings (22, 23).
In conclusion, the present study demonstrated that serum allopregnanolone levels in men decrease with age, whereas no changes are observed in women either after menopause or with advancing age. In fertile women, allopregnanolone levels correlate with P levels during the luteal phase of the menstrual cycle and partially in the follicular phase in response to endocrine tests. The significant increase in serum allopregnanolone in response to CRF and ACTH tests and the reduction in plasma allopregnanolone after DXM-mediated suppression indicate that the adrenal cortex is a site of production of circulating allopregnanolone, directly or indirectly via the synthesis of a precursor. It seems unlikely that P is the precursor of allopregnanolone in men and in nonluteal phase women; therefore, it would be of interest to evaluate pregnenolone sulfate levels in these subjects in correlation with circulating levels of allopregnanolone. In addition, the increase in serum allopregnanolone in response to the GnRH test and the presence of a large amount of serum allopregnanolone not suppressed by DXM indicate that in women, the ovary significantly contributes to circulating allopregnanolone. Therefore, these results indicate that in man, the adrenal cortex is an important site of production of circulating allopregnanolone, and the age-related decrease in steroid levels might be related to an impairment of the adrenal steroidogenic activity. In women, an ovarian contribution to circulating allopregnanolone seems to be present in both fertile women and postmenopausal subjects. The real impact that these preliminary results may have on the physiopathology of female reproductive function has yet to be elucidated.
Received September 23, 1997.
Revised March 13, 1998.
Accepted March 18, 1998.
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References |
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-hydroxy-5-pregnan-20-one, a potent anesthetic steroid by the
adrenal gland of the rat. J Steroid Biochem. 22:97–102.[CrossRef][Medline]
-hydroxy-5-pregnan-20-one and its precursor in
the brain, plasma, and steroidogenic glands of male and female rats. Endocrinology. 133:1003–1009.[Abstract]
-pregnan-3,20-dione and 3-hydroxy-5-pregnan-20-one. J
Clin Endocrinol Metab. 81:1076–1082.[Abstract]
-hydroxy-5-pregnan-20-one in rat and in
human plasma. Steroids. 55:290–295.[CrossRef][Medline]
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), and DHEA
(
) levels according to the phase of the menstrual cycle.
