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Original Studies |
Department of Obstetrics and Gynecological Endocrinology, Université Catholique de Louvain, B-1200 Brussels, Belgium
Address all correspondence and requests for reprints to: Frédéric Debieve, M.D., Physiology of Human Reproduction Research Unit, Department of Obstetrics and Gynecologic Endocrinology, Faculty of Medicine, UCL 5330, Avenue Em. Mounier 53, B-1200 Brussels, Belgium. E-mail: debieve{at}obst.ucl.ac.be
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Abstract |
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LH and FSH were detected in midpregnancy male fetuses (4.4 ± 3.3 and 0.77 ± 0.49 mIU/mL, respectively), with higher levels in females (33.0 ± 23.2 and 54.4 ± 27.7 mIU/mL, respectively), and were suppressed at term. PRL did not exhibit sexual difference, but showed a higher level at term (322.4 ± 113.8 ng/mL) than at midpregnancy (33.0 ± 26.1 ng/mL). Comparison of inhibin B with FSH levels showed correlation coefficients of -0.565 at midpregnancy vs. +0.445 at term. Serum from fetuses with intrauterine growth retardation or trisomy 21 did not show any different hormonal profiles.
These data suggest that inhibin B is probably an additional factor in FSH inhibition at midpregnancy, whereas activin A is not associated with any change in the different studied populations. We speculate that inhibin A could be a method to detect maternal blood contamination in cord venipuncture.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsAssaysResultsDiscussionReferences |
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-subunit and one of two ß-subunits
(A and B) for inhibin and a combination of two ß-subunits for activin
(3). Initially, they were isolated from gonadal tissue, but recent
studies have shown the presence of different messenger ribonucleic acid
subunits in nongenital sites (4). During pregnancy, the placenta is now
recognized as the main source of inhibin production (5). In adult
reproductive life, inhibin and activin regulate FSH production by the
anterior pituitary; inhibin selectively suppresses FSH secretion,
whereas activin stimulates FSH release (6). They have been shown to
have paracrine effects on gonads and other tissues, and they could play
a role during fetal development (7, 8). Little is known about the origin of inhibin and activin during human fetal life at different gestational ages. In term fetuses, the fetal testis seems to be the production site for inhibin B, and activin is localized in fetal adrenal glands, enhancing ACTH-stimulated during in vitro steroidogenesis (9, 10).
Until recently, all inhibin immunoassays were unable to differentiate
between the two dimeric forms and had a strong cross-reactivity with
free -subunits (11). All studies of preterm fetal serum were
performed with those assays except activin A (12, 13, 14). The development
of sensitive and specific enzyme-linked immunosorbant assays (ELISAs)
for inhibin A (15), inhibin B (16), and activin A (17) has allowed us
to make a discriminating evaluation of these proteins during fetal
development. In this study, we compare inhibin A, inhibin B, and
activin A concentrations in human fetal serum from normal midterm and
term pregnancies with those of the pituitary hormones LH, FSH, and
PRL.
It is now well established that maternal serum inhibin A levels are higher in pregnancies associated with Down’s syndrome (18). Even if the placenta seems to be the main source of inhibin A during pregnancy (5), no data are available on Down’s syndrome fetal serum. Finally, as activin A could be implicated in fetal development (8), we also measured activin A levels in fetuses with intrauterine growth retardation (IUGR).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsAssaysResultsDiscussionReferences |
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Midpregnancy human fetal blood was obtained by in utero cord venipuncture in our prenatal diagnosis unit. Initially, samples were taken for prenatal diagnosis in case of maternal infection (toxoplasma, cytomegalovirus, rubella) or suspicion of fetal anemia, but we only took into account fetal serum with normal results confirmed at birth. All of these samples therefore issue from normal fetuses and pregnancies. Samples from trisomy 21 and IUGR groups were also obtained by in utero cord venipuncture. In each case, the fetal hemoglobin count, medium corpuscle red cell volume and Kleihauer test were performed to assess the fetal origin of the sample and the absence of contamination with maternal blood. The karyotype of each sample was subsequently reported. Term pregnancy fetal blood was obtained by direct cord venipuncture immediately after a normal vaginal delivery before placental expulsion. All were from normal pregnancies with a normal subsequent neonatal examination. After clotting at 4 C, the samples were centrifuged, and the serum was kept frozen at -20 C until the assays were performed. All samples were tested on the same day within one assay for LH, FSH, PRL, activin A, inhibin A, and inhibin B, respectively, to reduce any interassay error. This study was approved by our local ethical committee, and we only used surplus serum taken for diagnosis purpose.
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Assays |
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TopAbstractIntroductionMaterials and MethodsAssaysResultsDiscussionReferences |
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Activin A, inhibin A, and inhibin B were tested with a two-monoclonal antibody, solid phase, sandwich, microtiter plate ELISA (Serotec, Oxford, UK) according to the manufacturer’s protocol. These assays standardly use diluted bovine (activin A) or human (inhibin A and B) follicular fluid that has been calibrated against a recombinant standard. Activin A, inhibin A, and inhibin B showed sensitivities of less than 100 pg/mL, 2 pg/mL, and 15 pg/mL, respectively. The inter- and intraplate coefficients of variation were less than 7% and 10%, respectively, for inhibin B, inhibin A, and activin A.
The assay for activin A had no detectable cross-reaction with inhibin
A, follistatin, activin B, or inhibin B; a small cross-reaction
(1–5%) was observed with activin AB. The assay for inhibin B
demonstrated nonsignificant cross-reactivity with the pro-C-subunit
or activins and a small cross-reaction (1%) with inhibin A. With
regard to inhibin A, the kit demonstrated nonsignificant
cross-reactivity with the pro-C-subunit, inhibin B, or activin.
LH, FSH, and PRL assays
LH, FSH, and PRL were tested with a solid phase, two-site chemiluminescent immunometric assay (Diagnostic Products Corporation, Belgium) according to the manufacturer’s protocol. The assays use standard international reference preparations of LH, FSH, and PRL in line with the WHO’s recommendations. The sensitivities were 0.5 ng/mL, 0.7 mIU/mL, and 0.1 mIU/mL for PRL, LH, and FSH, respectively. The intra- and interassay coefficients of variation were, respectively, 6.5% and 10.6% for LH, 7.7% and 8.1% for FSH, and 6.8% and 9.6% for PRL. The LH assay was highly specific, with 0.4% cross-reactivity to hCG and none to FSH or TSH. The FSH assay showed no cross-reactivity to hCG, TSH, or LH, and the PRL assay showed no cross-reactivity to FSH, hCG, human GH, human placental lactogen, LH, or TSH.
Statistics
Comparisons between female and male samples in the two gestational age groups and between the groups themselves were made using a two-way ANOVA (StatView 4.51, Abacus Concepts, Berkeley, CA). In the case of heterogeneous distributions (by ANOVA, P < 0.05), post-hoc tests (Scheffe, Bonferroni-Dunn, and Fisher’s protected least significant difference) were used to compare subgroups. Results are presented as the mean ± SD, with the ANOVA’s F value and the post-hoc test’s P value when comparisons are used. Correlation studies were performed with a correlation coefficient.
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Results |
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TopAbstractIntroductionMaterials and MethodsAssaysResultsDiscussionReferences |
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The karyotype of each sample was normal except for the Down’s syndrome group.
We did not encounter any inhibin A immunoreactivity in the serum tested, except in three midpregnancy male fetuses (3 of 14), which showed detectable inhibin A concentrations (131, 21, and 234 pg/mL) and undetectable or low inhibin B levels (0, 0, and 86 pg/mL, respectively), ruling out amniotic fluid contamination. Activin A levels were in the high range too (1709, 3348, and 713 pg/mL, respectively), but PRL, LH, and FSH in these samples were in the normal range for gestational age.
Figures 1
and 2
show the inhibin B and activin A levels in female and male human fetal
serum. Inhibin B was secreted in midpregnancy fetuses with a 10-fold
greater mean value in males than in females (167.6 ± 67.1
vs. 16.4 ± 12.0 pg/mL; F = 79.97;
P < 0.0001); in term fetuses, inhibin B was only
present in males, at a lower mean value (125.3 ± 32.8 pg/mL),
which was not statistically significant different from that at
midpregnancy. The activin A level profile did not show any significant
statistical difference between males and females or between
midpregnancy and term groups (mean, 612.5 ± 120.8 pg/mL).
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, 4, and 5. Both gonadotropins were present in
midpregnancy fetuses, with a clear difference between males and
females; the latter had much higher levels [33.0 ± 23.2
vs. 4.4 ± 3.3 mIU/mL for LH (F = 9.78;
P = 0.005) and 54.4 ± 27.7 vs.
0.77 ± 0.49 mIU/mL for FSH (F = 23.97; P <
0.0001)]. Only very low FSH levels (0.33 ± 0.21 mIU/mL; F =
23.97; P < 0.0001) and no LH were seen in term male
fetuses. In term female fetuses, both LH and FSH were undetectable. The
PRL level was not different between males and females, but was higher
in term pregnancies compared to midpregnancy (322.4 ± 113.8
vs. 33.0 ± 26.1 ng/mL; F = 38.76;
P < 0.0001).
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We also tested these hormones in six cases of IUGR (25–36 weeks gestation) diagnosed by ultrasound and confirmed by weight at birth. We did not find any significant difference from normal fetuses in the level of activin or other hormones studied.
Our trisomy 21 group included six male fetuses with gestational ages varying from 22–35 weeks of pregnancy. The values for all of the parameters tested were in the normal range, except in two cases. A 35-week-old fetus showed high values of inhibin A (679.8 pg/mL), inhibin B (2539.4 pg/mL), and activin A (7761.9 pg/mL). Pituitary hormones were in the normal range for FSH (0.28 mIU/mL), but were higher for LH (14 mIU/mL). A 31-week-old fetus showed a low level of inhibin A (6.4 pg/mL), with inhibin B (158.6 pg/mL) and activin A (1213.3 pg/mL) in the normal range. Gonadotropins were undetectable. Contamination with maternal blood was excluded with classic methods.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsAssaysResultsDiscussionReferences |
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After a recent report on term pregnancy cord serum (19), our study
confirms that neither inhibin A nor inhibin B is present in cord serum
from term female fetuses, but inhibin B is detected in males.
Midpregnancies show the same sexual discordance in fetal inhibin B
levels; higher inhibin B levels are found in males, and a lower, but
measurable, inhibin B concentration is present in females. As molecular
biology studies localized -subunit messenger ribonucleic acid tissue
expression only in adrenals and testes (8), one could postulate that
inhibin B has a fetal male gonadal origin. Where does the inhibin B in
midpregnancy females come from? One hypothesis is that there could be a
small adrenal contribution during midpregnancy fetal life. Another
hypothesis suggests an ovarian origin, because the occurrence of this
low inhibin B level coincides with the ovarian germ cell population
lifetime peak level (
7 million) and the development of the first
primordial follicles (20). During the latter half of pregnancy, this
population declines to about 2 million by atresia, explaining the
inhibin B disappearance.
Wallace et al. (19) describe inhibin B levels in amniotic fluid from 14–20 weeks of pregnancy, showing no difference between males and females, disputing any significant fetal contribution to the amniotic fluid inhibin B content. In our study, the inhibin B level found in male midpregnancy fetuses was much lower than their 50th percentile level in amniotic fluid, supporting the hypothesis of Wallace et al.
Surprisingly, we encountered three midpregnancy fetuses with measurable inhibin A levels. All of our samples were tested to assess their fetal origin. Moreover, other hormones detected in the same sample dispute any amniotic contamination [inhibin B, 0 pg/mL; amniotic fluid contained inhibin B, 216-1078 pg/mL (19)] or significant maternal contamination (presence of LH or FSH, which are suppressed during pregnancy), but the presence of inhibin A is in association with a high level of activin A. There are two possible hypotheses to explain this. Either there was fetal secretion by the testes or adrenals only in three cases, or maternal blood contamination occurred either by spontaneous feto-maternal hemorrhage or in utero cord venipuncture, especially transplacental, with possible placental intervillous chamber or retroplacental blood contamination. In two cases where the inhibin A level was high (>100 pg/mL), the cord venipuncture was transplacental, but the fetal hemoglobin count, medium corpuscle red cell volume, and Kleihauer test confirmed the fetal origin of the sample. Inhibin A could therefore be a very sensitive marker of slight maternal contamination even with the usual markers in the normal range. This is important in prenatal diagnosis when using sensitive methods such as PCR in fetal blood sampling.
Our results for gonadotropin levels are in agreement with those of other studies (20, 21, 22); we observed elevated FSH and LH levels in midpregnancy, with higher levels in females and almost undetectable levels at term, but Massa et al. (12) failed to detect any sexual difference in fetal LH levels. This midpregnancy gonadotropin peak coincides with high testosterone levels (20, 21) compared to those at term in both sexes and with the first ovarian follicle or seminiferous tubule maturation. Most studies of gonadotropins in preterm fetuses explain the FSH and LH sex difference as resulting from feedback inhibition by the higher concentration of testosterone at this time (20, 21, 22). This is probably the main factor for both LH and FSH down-regulation, but our study shows that FSH inhibition is more acute than that of LH. Moreover, we found a negative correlation coefficient at midpregnancy, higher for FSH than for LH. Inhibin is probably an additional factor that could account for the more pronounced FSH inhibition, but it is certainly secondary, because no effect on LH secretion could be attributed. However, in term pregnancies, the FSH and LH levels are low in both sexes, and correlation coefficients are positive. Inhibin is probably not the main factor involved, but maturation of the feedback inhibition mechanism by placental estrogens at this time seems to offer a plausible explanation (20). This hypothesis could be investigated by detection of testosterone, estrogen, and activin/inhibin receptors in pituitary extracts from both term and preterm fetuses.
PRL levels were found to be in the range reported by other studies performed on preterm fetuses after delivery (23, 24). Our two groups can be compared because there is no association between the presence of labor or the route of delivery and cord serum levels of PRL (25). Higher levels in term pregnancies can be related to the increase in placental estrogens, stimulating fetal hypophysis (20). We found no correlation between inhibin and PRL levels. The role of PRL secretion is unclear; it may be involved in lung surfactant synthesis (20, 24).
Our trisomy 21 group shows no difference in inhibin secretion, except for two isolated cases that show the level of inhibin A in association with that of activin A in the high range. Even if the classic parameters were tested to ensure a fetal origin of the sample and extraplacental cord venipuncture, contamination by micro-feto-maternal hemorrhage is our main hypothesis. Indeed, these cases were isolated, and we found measurable inhibin A levels in three normal pregnancies also.
In conclusion, our study compares inhibin A, inhibin B, and activin A in human midpregnancy and term pregnancy in relation to gonadotropin and PRL levels. Inhibin B is probably an additional, but not main, factor in FSH inhibition in midpregnancy. We speculate that measurement of inhibin A levels could be a very sensitive method to detect maternal blood contamination in cord venipuncture. Activin A levels are present in the fetus, but are not associated with any major changes during pregnancy. The abnormal fetal conditions studied (intrauterine growth retardation and trisomy 21) are not linked to abnormal hormonal profiles.
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Acknowledgments |
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Footnotes |
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Received May 18, 1999.
Revised August 12, 1999.
Accepted August 31, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsAssaysResultsDiscussionReferences |
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, ßA and ßB
subunits in various tissues predicts diverse functions. Proc Natl Acad
Sci USA. 85:247–251.-subunit(s). J Clin Endocrinol Metab. 80:3173–3179.[Abstract]
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