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Original Studies |
Department of Reproductive Medicine and Child Development, Section of Gynecology and Obstetrics (S.L., F.B., A.R.G.), University of Pisa, Pisa; Department of Surgical Sciences, Chair of Obstetrics and Gynecology (F.P., M.F., F.M.R.), University of Udine, 33100 Udine; Department of Obstetrics and Gynecology (C.B.), University of Turin, Turin; Department of Obstetrics and Gynecology (R.E.N.), University of Pavia, Pavia; and Endocrine Research Unit, Consiglio Nazionale delle Ricerche (M.L.), Pisa, Italy
Address correspondence and requests for reprints to: Felice Petraglia, M.D., Department of Surgical Sciences, Chair of Obstetrics and Gynecology, University of Udine, piazzale S. Maria della Misericordia, 33100 Udine, Italy. E-mail: felice.petraglia{at}dsc.uniud.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase activity before spontaneous labor (6), whereas a
decrease of maternal serum 5-dihydroprogesterone level during the
last 10 days preceding spontaneous delivery (2) has been also
shown.
The inhibiting action of progesterone on the uterine contractile
activity during pregnancy could be mediated via the metabolite
3-hydroxy-5pregnan-20-one (allopregnanolone), which augments the
myorelaxant function of GABA-A uterine receptors (7, 8, 9). This hormone
is measurable in systemic circulation of pregnant and nonpregnant
women, and because it is also neuroactive, allopregnanolone may
contribute to the psychological adaptation associated with pregnancy
and puerperium (10). In addition, because progesterone influences the
vascular adaptation throughout gestation, a possible role of
progesterone in hypertensive disturbances of pregnant women has been
hypothesized (11, 12, 13). However, the measurement of maternal serum
progesterone levels did not result in a powerful index of placental
function, and neither serum progesterone nor 5-dihydroprogesterone
levels identified women at risk of developing pregnancy-induced
hypertension or other maternal pathological conditions (14, 15).
Following the development of a new assay to measure serum allopregnanolone levels, it has been shown that changes in healthy women are not fully dependent on progesterone changes (16); therefore, the present study aimed to evaluate: 1) the changes of maternal serum allopregnanolone levels and its relationship with progesterone throughout gestation in healthy women; 2) whether serum and umbilical cord serum allopregnanolone and progesterone levels vary according to the different modes of parturition; and 3) the changes of maternal serum allopregnanolone and progesterone levels in women with chronic hypertension, with or without superimposed preeclampsia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study included nulliparous pregnant women (n = 66; 23–35 yr of age), who were subdivided in the following groups:
Group 1.This group comprised healthy pregnant women (n = 14) longitudinally studied throughout gestation (blood sample collected every 5 weeks, starting from 5–6 weeks until 39–40 weeks).
Group 2.The different modes of parturition (between 38 and 41 weeks of gestation) were studied in pregnant women who had a physiological spontaneous labor (n = 40), in women who underwent elective (breech presentation) cesarean section (n = 13) or emergency (fetal distress) cesarean section (n = 13).
Group 3.In this group, women with chronic hypertension
(n = 12) matched to controls for maternal age were longitudinally
studied throughout gestation. They were distinguished into two study
populations: 1) women (n = 5) who remained with chronic
hypertension, without superimposed preeclampsia; and 2) patients
(n = 7) who developed severe preeclampsia [according to the WHO
criteria, Study Group for the Hypertensive Disorders of Pregnancy:
persistent elevation of blood pressure 
160 mm Hg (systolic) or 110
mm Hg (diastolic) and proteinuria >300 mg/dL, after the 20th week of
gestation) (17). All women were screened for gestational diabetes with
Carpenter’s test. None of the women included in the study developed
gestational diabetes. The study was approved by a local institutional
Ethical Committee, and all participants provided informed consent.
Blood sampling
Maternal cubital venous blood was collected throughout gestation
(groups 1 and 3), during spontaneous labor (cervical dilatation, 2
cm), and at vaginal delivery (group 2) and before and at the time of
cesarean delivery (group 2). Mixed umbilical cord blood was collected
immediately after birth in group 2.
In each subject a blood sample was drawn between 0700 and 0900 h. Blood samples were centrifuged, and serum was stored at -20 C until assayed.
Allopregnanolone and progesterone assays
Analytic grade solvents were purchased from Merck & Co., Inc.(Darmstadt, Germany); C-18 Sep-Pak cartridge was
obtained from Waters Corp. (Milford, MA). Standard
allopregnanolone was purchased from Sigma (St. Louis, MO),
and pregna-3-ol-20-one, 5-[9,11,12,–3H(N)] (45 Ci/nmol) was
obtained from Amersham Pharmacia Biotech (Little Chalfont,
UK). The polyclonal antisera raised in sheep against allopregnanolone
carboxymethyl ether coupled with BSA was kindly provided by Dr. R.
H. Purdy (Department of Psych., Veterans Administration
Hospital, San Diego, CA). Serum samples (1 mL) were thawed. Standard
solutions of allopregnanolone (0, 15.6, 31.2, 62.5, 125.0, 250.0, 500,
and 1000 pg steroid per tube) were prepared 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 phase was
removed and evaporated to dryness in Universal Vacuum System Plus
(Savant, Rome, Italy).
The extracts were eluted with assay buffer (20 mM 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 v/v) containing 1% acetic acid and 50% aqueous methanol, and
the elution of allopregnanolone was performed with absolute methanol
(10 mL) (16). The dry extracts were then assayed by RIA: 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, 0.025 dextran 70 in 100
mL of assay buffer, 0.5 mL), followed by centrifugation at 4 C for 10
min. Radioactivity of supernatants was measured in a liquid
scintillator spectrometer equipped with a program for cpm counting and
RIA calculation (16). 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)
has been diluted with zero standard, showing the results reported in a
previous publication (16).
Serum progesterone was determined, after ether extraction and chromatographic partition on C18 Sep-Pak cartridge, by RIA using a commercially available kit (Radim SpA, Pomezia, Italy); the sensitivity of the assay was 50 pg/tube, and the intra- and interassay coefficients of variation were 6.5% and 8.7%, respectively.
Statistical analysis
Data are shown as means ± SEM. Data were analyzed either by one-way ANOVA (mode of delivery) or by ANOVA with repeated measures (longitudinal study), followed by Duncan’s post hoc multiple comparisons. Linear correlation coefficients were calculated to assess a possible correlation between serum allopregnanolone, serum progesterone, and blood pressure. Significance was set at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In healthy pregnant women, serum allopregnanolone and progesterone
levels showed a significant increase during pregnancy, with highest
levels at term (P < 0.01, repeated measures ANOVA,
Fig. 1
). Starting from 20 weeks of
gestational age, serum progesterone levels were significantly higher
than allopregnanolone levels (P < 0.01, Fig. 1). No
significant correlation between allopregnanolone and progesterone
levels was found in healthy pregnant women at any gestational age.
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Patients with chronic hypertension, with or without severe
preeclampsia, both during the 2nd and 3rd trimester of gestation,
showed mean serum allopregnanolone levels significantly higher
than healthy pregnant women (Fig. 2). In
detail, the group with chronic hypertension without
preeclampsia had higher allopregnanolone levels with respect to control
subjects from 20 weeks of gestation (36.9 ± 0.6 vs.
30.1 ± 1.2 ng/mL, P < 0.05) until 40 weeks
(57.0 ± 1.0 vs. 50.8 ± 1.1 ng/mL,
P < 0.05). The group of hypertensive women who later
developed preeclampsia had still higher levels of allopregnanolone
compared with the remaining groups throughout gestation
(P < 0.01, Fig. 2). Maternal serum progesterone levels
displayed a high variability between hypertensive subjects, without
reaching any statistical difference in comparison with healthy pregnant
women (Fig. 2). Serum allopregnanolone levels did not correlate with
progesterone levels or with blood pressure at any gestational age in
pregnant women with hypertension.
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Maternal serum allopregnanolone and progesterone levels observed
during normal spontaneous labor and at delivery were not result
significantly different from values present at term (Fig. 3). Progesterone levels were
significantly higher in umbilical cord than in maternal serum
(P < 0.01 vs. early labor and delivery),
whereas allopregnanolone levels in the two compartments were not
significantly different (Fig. 3). No significant correlation between
allopregnanolone and progesterone levels was found in the maternal
circulation or in umbilical cord serum.
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). At emergency cesarean section, immediately before
surgery, maternal allopregnanolone concentrations were
significantly higher than at vaginal delivery (P <
0.01 vs. spontaneous labor, P < 0.05
vs. elective cesarean section), and a significant decrease
was found at delivery (P < 0.01 vs. early
labor, Fig. 3). Umbilical cord serum allopregnanolone and progesterone
levels after emergency cesarean section were both significantly lower
than at vaginal delivery (P < 0.001 vs.
spontaneous labor, P < 0.01 vs. elective
cesarean section). No correlation was found between allopregnanolone
and progesterone in maternal circulation or umbilical cord serum during
elective or emergency cesarean section. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study indicates for the first time that allopregnanolone is
a circulating steroid hormone whose serum levels increase during
pregnancy, in parallel with serum progesterone levels, suggesting a
common origin. Even though other different sources may contribute to
the two hormones, ovary and/or placenta are the most putative sources.
A previous study suggested that an increased production rate of
progesterone during pregnancy provides the intrauterine tissues with
more substrate for the formation of 5-dihydroprogesterone and
allopregnanolone (18). However, some discrepancy is observed at
delivery and in hypertensive patients, indicating that some
physiological or pathological events reveal another source(s) for
allopregnanolone. The lack of correlation between maternal and cord
serum progesterone and allopregnanolone levels at the time of delivery,
independently from the mode of parturition, supports a different
source(s) for allopregnanolone in both compartments. Indeed, a maternal
adrenal cortex or pituitary origin has been recently shown in healthy
nonpregnant women (16).
At emergency cesarean section both maternal and umbilical serum allopregnanolone and progesterone concentrations behaved differently from the other modes of parturition, and these changes may be explained by fetal distress. The significant decrease of maternal serum and umbilical cord serum allopregnanolone levels observed during emergency cesarean section cannot be exclusively explained by the surgery because it was absent at elective cesarean section. The present finding did not confirm the previous observation of higher maternal plasma progesterone levels at vaginal delivery than at elective cesarean section (4), and this was probably due to the different experimental design of the two studies.
Still uncertain is the role of allopregnanolone in pregnancy. A potential effect of allopregnanolone in uterine kinetics has been suggested (7, 8, 9), even though an in vitro study showed that progesterone metabolites are not crucial for uterine contractions (6). A current hypothesis is the possible correlation between maternal anxiety state and serum allopregnanolone levels, given the potent anxiolytic effect of this neuroactive steroid (19, 20).
The evidence of increased levels of serum allopregnanolone in hypertensive pregnant women is of great interest in view of the possible role of progesterone in vascular tension during pregnancy. Recent evidence suggests that the control of systemic blood pressure during pregnancy may be modulated by steroid hormones (4, 6, 21, 22). Indeed, progesterone modulates the antihypertensive effect of calcitonine gene-related peptide postpartum in the rat (21). Allopregnanolone may contribute to the adaptation of baroflex control of sympathetic outflow and heart rate in pregnant rats (22). Likely, in pregnant women with chronic hypertension, the elevation of allopregnanolone levels may be compensatory to this condition and represent an attempt on behalf of the maternal or fetal organism to lower maternal blood pressure levels. Moreover, direct vascular effects of estradiol and progesterone on isolated omental artery from normotensive and preeclamptic pregnant women supported the idea of a direct in vitro vasodilator activity of progesterone, partially dependent on the endothelium (23). However, plasma progesterone and its metabolite levels did not show any significant difference in women at risk of developing pregnancy-induced hypertension (1, 14). On the other hand, a significant high amniotic fluid progesterone concentration has been reported in pregnant women who later developed preeclampsia (24).
In conclusion, our present results demonstrated a significant increase of maternal serum allopregnanolone levels during pregnancy, with high levels in chronic hypertension and low maternal and umbilical allopregnanolone and progesterone levels in women at emergency cesarean section. The lack of correlation between progesterone and allopregnanolone over parturition and in hypertensive patients may be due to an extraplacental source and/or to a different role of allopregnanolone in the behavioral and cardiovascular adaptive response during pregnancy and delivery.
Received July 7, 1999.
Revised December 29, 1999.
Accepted January 30, 2000.
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Clin Endocrinol. 89:39–47.[Medline]This article has been cited by other articles:
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B. E. P. Murphy Allopregnanolone Assays J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1425 - 1425. [Full Text]
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F. Petraglia Author's Response: Allopregnanolone Assays J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1425a - 1425. [Full Text]
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) are compared with those who delivered by
elective (
) or emergency (
) cesarean section. a,
P < 0.01 vs. early labor and
delivery. b, P < 0.01 vs.
spontaneous labor. c, P < 0.01
vs. spontaneous labor and P < 0.05
vs. elective cesarean section. d, P
< 0.01 vs. early labor. e, P <
0.001 vs. spontaneous labor and
P < 0.01 vs. elective cesarean
section.