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Original Studies |
Department of Gynecology, Obstetrics and Pediatric Sciences, University of Modena (F.P., A.V., S.B.), Modena; Department of Obstetrics and Gynecology, University of Pisa (S.L., P.F., E.C., A.R.G.); and Department of Obstetrics and Gynecology, University of Torino (C.B., M.Z.), Italy
Address all correspondence and requests for reprints to: Felice Petraglia, Department Obstetrics Gynecology, University of Modena, Via del Pozzo 71, 41100 Modena, Italy. E-mail: Petraglia{at}unimo.it
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Abstract |
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The protocol included various groups of pregnant women. A longitudinal evaluation of serum inhibin B levels was done at specific intervals (8–12, 13–18, 19–24, 25–28, 29–33, and 34–40 weeks) in the following groups: 1) healthy pregnant women (n = 13); 2) women at risk of hypertension who did not develop hypertension (n = 8); and 3) women with chronic hypertension (n = 13). In women in group 1, a blood sample was also obtained in the postpartum period (12, 24, and 48 h after delivery). Other pregnant women with abnormal bleeding in the first trimester were studied; they were subdivided into women with ongoing pregnancy (n = 12); and women with miscarriage (n = 22); a control group of healthy pregnant women at the same gestational age was also included (n = 18). A final group of women with gestational diseases (n = 34) was included in the study and included women with: 1) pregnancy-induced hypertension (n = 10); 2) preeclampsia (n = 17); and 3) intrauterine fetal growth retardation (n = 7). A group of healthy nonpregnant women (n = 9) was used as controls, and a blood specimen was collected during both the early- to midfollicular and midluteal phases of the menstrual cycle. Serum dimeric inhibin B levels were measured by using a double-antibody enzyme-linked immunoadsorbent assay.
Early gestation inhibin B levels were similar to those of nonpregnant controls and showed a significant rise during the third trimester (P < 0.01). The highest maternal serum inhibin B levels were found at term (P < 0.01). Values significantly returned to control levels within 12–48 h (P < 0.01) after placental delivery. Women at risk of hypertension showed a similar gestational-related increase of inhibin B levels during the third trimester, without any significant difference when compared with healthy women. Women with chronic hypertension showed significantly lower levels at term (P < 0.01). Women with pregnancy-induced hypertension or preeclampsia, or who were carrying a fetus with intrauterine growth retardation showed serum inhibin B levels during the third trimester of gestation consistently lower than in control healthy women at the same gestational age (P < 0.001, mean ± SEM). Maternal serum inhibin B levels in women with early pregnancy bleeding or miscarriage were similar to those of healthy pregnant women at the same gestational age, independent from the outcome of gestation.
The present study showed that maternal serum inhibin B levels increase in the last trimester of normal pregnancy, with low levels in women with hypertensive disturbances or intrauterine growth retardation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ßA) and inhibin B (ßB) (1). Human placental cells
express inhibin , ßA, and ßB subunit messenger RNAs (mRNAs) (2, 3) and contain the two forms of immunoreactive inhibins (4, 5). By
using a nonspecific RIA, several studies showed that large amount of
immunoreactive inhibin is measurable in maternal serum, and that it was
highest at term (6, 7, 8). The rapid decrease following placental delivery
(6), and the detectable levels in women with premature ovarian failure
who become pregnant following oocyte donation (9), suggested that
placenta is the major source for maternal immunoreactive inhibin. High
levels of immunoactive inhibin were shown in pregnant women with
preeclampsia, intrauterine growth retardation, or hydatidiform mole
(10, 11). However, the assay used in these studies did not distinguish
between inhibin A and B and detected precursor molecules, as well as
free inhibin subunits. When a specific assay for the dimeric form of
inhibin A was developed (12), the progressive increase of inhibin A
through gestation was confirmed, showing a considerable amount of
inhibin pro-C-related immunoreactivity in early gestation (13). For
inhibin B levels, the validation of a double antibody enzyme-linked
immunoadsorbent assay (ELISA) enabled researchers to establish high
levels of inhibin B throughout the follicular phase of the menstrual
cycle, a short-lived peak 2–3 days after ovulation, and very low
levels during the luteal phase (13) and during the establishment of
pregnancy (14, 15) until 20 weeks of gestation (16). Because no data are available on the changes of inhibin B throughout the entire course of pregnancy, the present study aimed to evaluate, by using a specific ELISA, inhibin B levels in maternal circulation of healthy pregnant women followed longitudinally throughout gestation. The same longitudinal protocol was used to evaluate serum inhibin B levels in two groups of women who were at risk for hypertention or who had chronic hypertension. In addition, inhibin B levels were measured in women with abnormal early pregnancy or with gestational diseases (pregnancy-induced hypertension, preeclampsia, and intrauterine growth retardation).
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Materials and Methods |
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Pregnant women (age range 20–35 yr) were included in the study following informed consent. The entire study was approved by the local ethical committee.
Some patients were studied longitudinally throughout gestation. They were subdivided into the following three groups. Group 1, healthy normotensive pregnant women (n = 13). Group 2, women at risk of hypertension who did not develop hypertension (n = 8) classified using the following clinical criteria: a history of nephropaty with intact kidney functionality, previous preeclampsia, or intrauterine growth retardation (17). Group 3, pregnant women with chronic hypertension (n = 13), i.e. essential hypertension antedating pregnancy (diastolic blood pressure = 90 mmHg) treated by using nifedipine (40–60 mg/day) (Bayer AG, Leverkusen, Germany) (18).
These patients underwent a clinical examination and blood sampling during the course of gestation (at 8–12, 13–18, 19–24, 25–28, 29–33, and 34–40 weeks of gestation). In addition, in group 1 women blood specimens were collected in the postpartum period (12, 24, and 48 h after placental delivery).
A group of women with abnormal early pregnancy (between 8 and 18 weeks of gestational age) was included in the study at the time of diagnosis of bleeding (n = 34). According to clinical investigation (ultrasound) and pregnancy viability, patients were subdivided into two groups. Group 1, women with ongoing pregnancy (n = 12) in which the ultrasound showed intrauterine sac with fetal cardiac activity. Group 2, women with incomplete miscarriage (n = 22), in which the ultrasound showed a nonviable intrauterine pregnancy.
A group of healthy pregnant women (n = 18) at the same gestational age served as controls. All subjects had plasma CG levels ranging between 3,000 and 30,000 IU/L.
Last, three groups of patients with gestational diseases (n = 34)
were also studied: group 1, pregnancy-induced hypertension (n =
10); group 2, preeclampsia (n = 17); and group 3, intrauterine
fetal growth retardation (n = 7). The diagnosis of chronic
hypertension, pregnancy-induced hypertension, or preeclampsia was made
according to the criteria proposed by Davey and MacGillivray (18).
Pregnancy-induced hypertension was diagnosed during the last trimester
of pregnancy as a diastolic blood pressure 
90 mm Hg, and when
it was associated with proteinuria (>300 mg in one 24-h urine
collection) it was defined as preeclampsia (18). The diagnosis of
intrauterine growth retardation was made according to the criteria
proposed by Pollack and Divon (19); all patients showed a symmetric
type I form of intrauterine growth retardation.
In patients with abnormal early pregnancy or gestational disease, a blood specimen was collected within 24 h after hospitalization before starting any treatment. Peripheral blood samples were drawn from the anticubital vein with a polypropylene syringe and a butterfly needle, transferred to chilled tubes, and then immediately centrifuged at 4 C (3000 x g for 10 min). Serum samples were kept at -80 C until assay.
Inhibin B assay
Serum inhibin B levels were measured using a two-site enzyme immunoassay ELISA, as previously described (14) (Serotec, Oxford, UK). Briefly, standard and samples were diluted as appropriate and then mixed with an equal volume of distilled water containing 10% SDS. After 3 min at 100 C, tubes were cooled before adding freshly prepared hydrogen peroxide solution. After an additional 30-min incubation at room temperature, duplicate aliquots of denatured and oxidized samples/standards were transferred to antibody-coated microtiter plates. Plates were incubated with agitation overnight at room temperature. After washing with [0.1 mol/L Tris-HCl, 0.15 mol/L NaCl, 10% (wt/vol) BSA, 5% (vol/vol) Triton X-100, and 0.1% (wt/vol) sodium oxide, pH 7.5)] wash buffer, 50 µL alkaline phosphatase-conjugated extravidin was added, and plates were incubated for 3 h. Plates were washed, and bound alkaline phosphatase was quantitated using a commercially available enzyme immunoassay amplification kit (Immuno Select ELISA Amplification System, Dako, Milan, Italy), which was used according to the supplier’s instructions. The plates were read at 490 nm on an automated ELISA plate reader (BRIO: Basil Radim Immunoassay Operator, Radim spa, Pometie, Italy).
The assay detection limit for inhibin B was <10 pg/mL. Within- and between-plate coefficents of variation were 6% and 8%, respectively. Cross-reactions for the assay with the various proteins of the inhibin-related family were less than 0.5%.
Statistical analysis
The statistical significance of inhibin B serum variations in the women throughout gestation was calculated using the ANOVA for multiple comparisons. Statistical comparisons among the various groups were performed by using the nonparametric Mann-Whitney U test.
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Results |
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).
Median ± SD values evaluated according to the three
trimesters of gestation showed that serum inhibin B levels during the
first (27.50 ± 2.72 pg/mL) and second trimester (38.00 ±
9.06 pg/mL) were significantly lower than at the third trimester
(115.5 ± 28.19 pg/mL) (P < 0.001). A significant
decrease was observed from 12–48 h after placental delivery (12 h
= 26.57 ± 0.55 pg/mL; 24 h = 31.22 ± 8.66 pg/mL;
48 h = 46.02 ± 7.35 pg/mL) (P < 0.001)
(Fig. 2). Values at term were
significantly higher than in control nonpregnant women during the early
follicular (55.33 + 7.90 pg/mL) and early luteal (35.11 + 5.91 pg/mL)
phases of the menstrual cycle (P < 0.01).
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). Although until 25–28 weeks no significant
difference was recorded between healthy controls and patients with
chronic hypertension, at 29–33 and at 34–40 weeks of gestation serum
inhibin B levels in patients with chronic hypertension were
significantly lower than in controls (P > 0.01) (Fig. 2).
In the group of women with pregnancy-induced hypertension, median
± SD serum inhibin B levels (82.00 ± 11.98 pg/mL)
were consistently lower than in control women at the same gestational
age (123.00 ± 24.06 pg/mL) (P < 0.01) (Fig. 3). Serum inhibin B levels in women with
preeclampsia at 29–33 weeks (66.00 ± 15.02 pg/mL) and at 34–40
weeks (84.00 ± 13.03 pg/mL) were significantly lower than control
values at the same gestational age (29–33 weeks: 90.00 ± 23.10
pg/mL) (P < 0.01) (Fig. 3). Patients with intrauterine
fetal growth retardation showed inhibin B levels at 29–33 weeks
(41.67 ± 5.62 pg/mL) that were lower than controls
(P < 0.01) but not at 25–28 weeks (33.00 ±
38.03 pg/mL) (Fig. 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ßB subunit mRNA expression in trophoblast tissue (2).
In association with the evidence that inhibin B levels at term are
higher than in nonpregnant women, and that inhibin B levels
significantly decrease in the postpartum period, our study suggests
that placenta and the other gestational intrauterine tissues are
sources of inhibin B in maternal circulation. In particular, the
hypothesis of a contribution from maternal decidua is supported by the
evidence of a higher expression of ßB mRNA in decidual cells at term
(20). Our finding of low serum inhibin B levels in early gestation confirms previous observations showing levels of serum inhibin B in the low range of the assay detection limit (14, 15, 16), which was recently further improved. The evidence of amniotic fluid inhibin B concentrations at midgestation that were higher than in maternal serum, with a progressive rise till 20 weeks of gestation, supports the idea of a major contribution from fetal membranes (16).
Placenta and related tissues are also suggested as sources for the
other inhibin-related proteins measured in maternal circulation,
i.e. inhibin A, activin A, and follistatin (21). In healthy
pregnant women, serum levels of these proteins increase throughout the
three trimesters of pregnancy, with the highest values at term
(22, 23, 24). However, the present study showed a clear quantitative
difference between inhibin B and the other related protein level
changes. Although inhibin B levels are higher than in nonpregnant women
only at term, the other inhibin-related protein levels reach very high
values from first trimester of pregnancy, and at term are 5- to 10-fold
higher than in nonpregnant women (12, 22, 23). Indeed, inhibin B levels
in pregnancy never reach very high values and peak values at term are
in the same range of peak midcycle values a few days after ovulation
(13), suggesting a different secretory and/or metabolic rate for
inhibin B during pregnancy in comparison with the other inhibin-related
proteins. The higher levels of inhibin A and activin A are consistent
with the higher expression of and ßA mRNA in placenta (2). The
elucidation of the mechanisms regulating the posttranslational
processing of inhibin subunits will clarify this point. Anyway, the
present data suggest that inhibin B is not the predominant form of
inhibin in maternal circulation. A possible explanation for these low
levels of inhibin B may be related to methodological problems,
i.e. binding to follistatin or others forms of
inhibin-related proteins (25). However, because the present assay
detects total inhibin B, the amount of circulating inhibin B bound to
follistatin is also included. In addition, the 32-kilodalton inhibin B
used as standard is the only standard available, but the other size
variants present in human circulation differ only in the degree of
glycosylation and, even though with a reduced affinity, they might be
detected by the present assay (26).
The levels of inhibin B in patients with abnormal early gestation
remain unchanged independent from the pregnancy viability. In fact, in
patients with either miscarriage or ongoing pregnancy, inhibin B levels
did not show significant difference compared with healthy controls, and
were comparable with and in the same range of values of healthy
pregnant women at the same gestational stage. Therefore, the
measurement of inhibin B in maternal serum has no clinical value for
screening of early gestational problems, although previous studies in
women with miscarriage who showed reduced immunoreactive inhibin (27, 28) or inhibin pro-C-related immunoreactivity (14), suggested the
usefulness of their measurement in abnormal early pregnancy.
Pregnant women with chronic hypertension, pregnancy-induced hypertension, or preeclampsia showed maternal serum inhibin B levels lower than healthy controls. This finding suggests that secretion of inhibin B is impaired independently from the gravity of the hypertensive disturbances in pregnancy. The evidence that reduced inhibin B levels in women with chronic hypertension are significant only after 29–33 weeks of pregnancy, indicates that the secretory changes are not dependent on the high blood pressure but are also related to the pathological effect on the secretory compartments. Previous studies indicated that immunoreactive inhibin (10) or activin A (29) levels are increased in preeclamptic patients, which is the worst stage of the hypertensive disturbances in pregnant women.
Therefore, low inhibin B levels better reflect the existence of an hypertensive state in pregnancy. Women carrying intrauterine growth-retarded fetuses also showed reduced serum inhibin B levels. Because of the sequence homology with transforming growth factor-ß, inhibins have been studied as putative growth factors, and effects on growth and differentation of several organs have been shown (30, 31). The hypothesis of a reduced production of growth factor in women with intrauterine growth retardation is under investigation (32). In particular, the low insulin-like growth factor-I levels in cord serum of pregnant women with intrauterine growth retardation is the best representation of the impaired insulin-like growth factor axis in utero (33, 34, 35). However, a decrease of maternal serum hPL levels in these patients has been described (36), suggesting that maternal compartment may also reflects a reduced secretion of growth factors. According to the complex network of interactions occurring within fetoplacental unit (37), it has been speculated that a deranged secretory activity of regulatory factors from fetoplacental membranes may lead to a gestational disease and, vice versa, the occurrence of a gestational disease may affect secretory activities. The elucidation of inhibin B’s target function will explain the significance of the decreased levels in women with gestational diseases.
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Footnotes |
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Received March 14, 1997.
Revised May 1, 1997.
Revised June 4, 1997.
Accepted June 17, 1997.
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) (n = 13), pregnant women at
risk of hypertension (•) (n = 8), and pregnant women with
chronic hypertension (
) (n = 13) followed longitudinally
throughout pregnancy. *, P < 0.01
vs. controls.