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Inhibin A and Activin A in the First Trimester of Human Pregnancy

Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital (M.B., W.L., S.M.), Headington, Oxford, OX3 9DU; School of Biological and Molecular Sciences, Oxford Brookes University (N.G.), Oxford OX3 0BP; and The Fertility and Endocrinology Center, Lister Hospital (H.A.), London, United Kingdom

Address all correspondence and requests for reprints to: Shanthi Muttukrishna, Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom.

	Abstract

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Recent studies show that high concentrations of inhibin A and activin A are present in the maternal serum throughout human pregnancy. The aim of this study was to determine whether the corpus luteum produces significant quantities of inhibin A and activin A during the first trimester of pregnancy. This prospective study examined two groups of women who had blood samples taken from 5–12 weeks gestation. One group consisted of 14 women with donor egg pregnancies (8 singletons and 6 multiples) who did not have corpora lutea, and the other group consisted 5 women with spontaneous pregnancies who had corpora lutea. Inhibin A and activin A were measured at weekly intervals using specific enzyme immunoassays. All pregnancies progressed to term, with healthy babies being delivered.

Maternal serum concentrations of inhibin A significantly increased throughout the study period in the donor egg pregnancies (P < 0.001) and the control pregnancies (P < 0.001). Circulating concentrations of activin A also increased significantly in both the spontaneous and donor egg pregnancies (P < 0.001) during the study period. However, the concentrations of inhibin A and activin A in the first trimester of human pregnancy were not significantly different in the women with or without corpora lutea, suggesting a fetoplacental origin. Multiple donor egg pregnancies were found to have higher concentrations of inhibin A (P < 0.001) and activin A (P < 0.05) compared with singleton donor egg pregnancies, which also supports a placental source.

	Introduction

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INHIBIN consists of two glycoprotein subunits, and ß, which are linked by disulphide bonds. There are two different forms of ß subunit forming inhibin A (-ß_A) and inhibin B (-ß_B). Activin is composed of homodimers or heterodimers of the same ß subunits, thereby forming activin A (ß_A-ß_A), activin B (ß_B-ß_B), or activin AB (ß_A-ß_B).

Inhibin and activin are glycoprotein hormones involved in the regulation of pituitary FSH secretion. Inhibin decreases and activin increases FSH release by pituitary cells in vitro. Inhibin and activin were thought to act solely on the pituitary gland in a classical endocrine feedback loop, however these factors have since been described in a large number of other tissues, namely placenta, pituitary, adrenal, bone marrow, kidney, spinal cord, and brain, making it likely that they have more diverse biological actions (1).

The corpus luteum is the major site of inhibin production during the luteal phase of the human menstrual cycle (2, 3). There is conflicting evidence about the source of inhibin production in early pregnancy. Trophoblast has been shown to express inhibin and ß subunit messenger RNA using Northern blot analysis (4). Immunohistochemistry has demonstrated immunoreactive (ir) inhibin production by the trophoblast, and cell culture studies have also shown that placental cytotrophoblast produces ir-inhibin (5). In 1987, McLachlan et al. (6) reported that the levels of ir-inhibin in the maternal circulation were similar in women with functional ovaries and those on a donor egg program, and therefore concluded that inhibin production was predominantly from the placenta. A conflicting study published in 1991 (7) showed decreased levels of ir-inhibin in women conceiving on a donor egg program, compared with women conceiving spontaneously. Both of these studies used the Monash RIA for inhibin, which cannot differentiate between the free subunit monomer and dimeric inhibin forms, because the antibodies heavily cross-reacted with the monomer. Recent development of specific and sensitive two-site enzyme immunoassays for dimeric inhibin A (2, 3), dimeric inhibin B (8), pro- C (9), and total activin A (10) have allowed accurate measurements of various inhibin forms and activin A.

This study was designed to determine the source of inhibin A and activin A during early human pregnancy using a model that has no corpus luteum. This was accomplished by measuring inhibin A and activin A levels before conception and then at weekly intervals in women who became pregnant using donated eggs, and whose own ovarian function was suppressed with GnRH analogs and therefore have no corpora lutea. As a control group, five women conceiving spontaneously and who presumably had normal corpora lutea, were also studied by drawing blood samples at weekly intervals from 5–12 weeks gestation.

	Materials and Methods

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Subject selection and hormonal preparation

Donor egg recipients (group 1). The donor egg recipients comprised eight premenopausal (regular menstrual cycles + FSH <20 IU) and six menopausal women (no periods + FSH >20 IU), with a median age of 37 yr (26–46 yr). The premenopausal women were treated with a long-acting GnRH agonist, leuprorelin acetate (Wyeth, Maidenhead, Berkshire, UK) for at least 1 month before fresh embryo transfer. The last injection of analog was given 2 weeks before embryo transfer. A trial cycle of exogenous estrogen and progesterone was commenced during the period of ovarian down-regulation. This consisted of 14 days of estradiol valerate 2 mg three times daily (Progynova, Schering, Sussex, UK) and micronized vaginal progesterone 100 mg three times daily from days 15–28 (Uterogestan, Laboratories Besins-Iscovesco, Paris, France). Estrogen supplementation recommenced when the donor began injecting her gonadotropins. The recipient commenced additional progesterone on the day after the donor had her human CG injection. Embryo transfer was performed 2 days after egg collection. Estrogen and progesterone replacement were continued until a pregnancy test was carried out 13 days after embryo transfer. If positive, hormonal supplementation continued until serum progesterone level exceeded 120 mmol/L, establishing placental competency. A clinical pregnancy was confirmed by the presence of fetal heart activity at 6 weeks gestation (day 0 of gestation was the day of fertilization).

All the pregnancies in this study progressed well with healthy live-born babies. There were six multiple pregnancies and eight singleton pregnancies.

Spontaneous pregnancies (group 2). A control group of five women who conceived spontaneously and progressed to deliver healthy term singleton babies was also included in this study. These women had weekly blood samples drawn from 5–12 weeks gestation.

Hormone assays

Inhibin A. Serum inhibin A was measured in duplicate 20-µL aliquots, using a two-site enzyme linked immunosorbent assay as described elsewhere (2). The intra- and interplate assay variations were 4.3% and 5.1%, respectively. The minimum detection limit of the assay for human recombinant inhibin A [code 91/624, 5 µg = 150,000 IU provided by National Institute for Biological Standards and Controls, UK (11)] was 5 pg/mL.

Total activin A. Plasma concentrations of dimeric activin A were measured using a two-site enzyme immunoassay that has also been described previously (10). Recombinant human activin A (a gift from Genentech, San Francisco, CA) was used as standard, and the minimum detection limit of the assay was 50 pg/mL. The intra- and interassay coefficients of variation were 6.5% and 7.7%, respectively.

Statistical analysis

The data were found to be normally distributed and one-way ANOVA was used to determine whether the concentration of each hormone varied significantly at different time points throughout the study period. Two-way ANOVA was used to determine whether the concentrations of the individual hormones differed between the different time points and the different types of pregnancy (donor egg vs. spontaneous conceptions). Unpaired Student’s t tests were carried out to investigate whether there was any difference at particular time points between the donor egg and spontaneous singleton pregnancies. All statistical analysis was carried out using Statswork statistical package (Cricket Graph Inc, Philadelphia, PA) using 95% confidence interval limit.

	Results

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Inhibin A

Inhibin A was undetectable (<5 pg/mL) in the serum of women 28 days following pituitary suppression with a long-acting GnRH analog and after exogenous estrogen or estrogen and progesterone supplementation in down-regulated or menopausal women. Inhibin A was detectable from 5 weeks gestation and 20 days following embryo transfer, and the concentration of inhibin A increased steadily, reaching a peak at 10 weeks gestation, then declined somewhat by 12 weeks (Fig. 1). Higher inhibin A levels were observed in the multiple pregnancies (Fig. 2). There was no statistical difference in inhibin A concentrations in the singleton pregnancies with or without corpora lutea throughout the first trimester of pregnancy (two-way ANOVA). The multiple donor egg pregnancies had increased concentrations of inhibin A compared with the singleton donor egg pregnancies (P < 0.001). There was a significant increase in inhibin A concentrations throughout the study period in both the donor egg and control pregnancies (P < 0.001). Unpaired Student’s t tests show that there was no significant difference in inhibin A concentrations in donor egg and spontaneous pregnancies at any time point throughout the study period.

View larger version (13K): [in this window] [in a new window]   Figure 1. Mean ± SEM concentrations of inhibin A in singleton donor egg (, n = 8, P < 0.001, ANOVA) and spontaneous pregnancies (•, n = 5, P < 0.001, ANOVA)) in peripheral serum during first trimester of human pregnancy.

View larger version (17K): [in this window] [in a new window]   Figure 2. Mean ± SEM concentrations of inhibin A in donor egg singleton (, n = 8) and donor egg multiple (, n = 6) in peripheral serum during first trimester of human pregnancy. Donor egg multiple pregnancies had significantly increased concentrations of inhibin A compared with singleton donor egg and spontaneous pregnancies (P = 0.001; two-way ANOVA).

Total activin A was detected at the time of ovarian down-regulation and during the trial cycle of exogenous estrogen and progesterone in the women on the donor egg program. However, there was no significant difference in total activin A concentrations throughout the trial cycle (ANOVA).

The concentration of total activin A increased during the first trimester of egg donation pregnancies, once again peaking at 10 weeks gestation and then declining slightly at 12 weeks (Fig. 3). There was a significant increase in concentrations of total activin A during pregnancy compared with the nonpregnant trial cycle (P < 0.001, ANOVA). Total activin A concentrations were significantly higher (P < 0.05) in the multiple donor egg pregnancies (n = 6) compared with the singleton donor egg pregnancies (Fig. 4).

View larger version (14K): [in this window] [in a new window]   Figure 3. Mean ± SEM concentrations of activin A in singleton donor egg (, n = 8, P < 0.001, ANOVA) and spontaneous pregnancies (•, n = 5, P < 0.001, ANOVA) in peripheral serum during first trimester of human pregnancy. **, P < 0.01; unpaired Student’s t Test.

View larger version (13K): [in this window] [in a new window]   Figure 4. Mean ± SEM concentrations of activin A in donor egg singleton (, n = 8) and donor egg multiple (, n = 6) in peripheral serum during first trimester of human pregnancy. Donor egg multiple pregnancies had significantly increased concentrations of activin A compared with singleton donor egg and spontaneous pregnancies (P < 0.05; two-way ANOVA).

	Discussion

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This study demonstrates that inhibin A and activin A are derived from the fetoplacental unit in the first trimester of human pregnancy, because these polypeptides are detected in similar concentrations in pregnant women with or without corpora lutea. Inhibin A and activin A levels were higher in women with multiple donor egg pregnancies, also supporting a fetoplacental source.

The presence of detectable levels of total activin A during the time of ovarian down-regulation and during exogenous estrogen and progesterone supplementation strongly suggests extraovarian source(s) of this hormone, such as bone marrow, pituitary, or adrenal, all of which have been shown to express inhibin/activin subunits messenger RNAs (1). The relative contributions of these different tissues to the overall concentration of total activin A in the circulation remain undetermined.

The findings of this study are consistent with other recent studies, such as the study reporting the rapid reduction in the concentration of inhibin A and activin A following surgical termination of pregnancy in the first trimester (12), as well as the concentrations of inhibin A and total activin A observed throughout spontaneous pregnancies from 8–38 weeks gestation (13, 14). The study by Illingworth et al. (15) showed that inhibin A levels peaked at 8 weeks gestation and then declined until measurements ceased at 11 weeks gestation in spontaneous pregnancies. Although similar observations were made in this study, because both spontaneous and donor egg pregnancies were being studied it is possible to reach the conclusion that inhibin A production is from the placental unit. The two previous studies that used the donor egg model to determine whether ir-inhibin is being produced by the placenta, corpus luteum, or both, used the Monash RIA (16), which cannot differentiate between the biologically inactive free subunit and the dimeric inhibin forms. The primate corpus luteum is shown to produce high concentrations of free subunits (17), and therefore the assay used would show decreased inhibin levels in the women without corpora lutea. In the present study the measurements were made using the specific two-site enzyme immunoassays for dimeric inhibin A (2), and the data shows that inhibin A is predominantly a fetoplacental product in early pregnancy.

The biological role(s) of total activin A and inhibin A in early pregnancy is unknown. Both inhibins and activins have been shown to regulate GnRH, human CG, and progesterone secretion from human placental cells in vitro (18). Thus the function of activin A and probably inhibin A during pregnancy relates more to their association with the transforming growth factor-ß family. Biological functions are therefore more likely to involve cellular differentiation and embryogenesis in an autocrine/paracrine manner.

Inhibin A concentrations rapidly increase until 9 or 10 weeks gestation, and then fall and gradually rise again throughout the third trimester. This suggests that inhibin A may have differing functions throughout pregnancy. Inhibin A may prove to be a marker for fetal or placental abnormalities in pregnancy and has already been shown to be useful in screening for fetal karyotypic abnormalities. Women who conceive babies with Down’s syndrome have recently been found to have significantly elevated serum levels of inhibin A during the second trimester (19).

Activin AB and activin B have not been shown to be present in term placenta (20) making activin A the major form of activin produced by the placenta. The role of activin A and the activin-binding proteins during pregnancy is unknown at present.

In summary, the recent development of a new generation of specific and sensitive enzyme immunoassays for inhibins and activin A has enabled us to carry out this study and also to reexplore the potential functions of these peptides in human. This study clearly shows that inhibin A and activin A in early pregnancy is of fetoplacental origin. The biological function of these raised levels of inhibin A and activin A in early pregnancy is yet to be determined.

	Acknowledgments

 
We thank the National Institute of Biological Standards and Controls for providing rh-inhibin A standard, Genentech for providing rh-activin A standard, and Prof. C.W.G. Redman for providing some of the samples.

	Footnotes

 
¹ Recipient of a Nuffield Dominion Fellowship.

Received October 16, 1996.

Revised January 21, 1997.

Accepted January 26, 1997.

	References

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