Academic Unit of Obstetrics and Gynaecology, University Department of Reproductive and Developmental Medicine, The Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK
1 To whom correspondence should be addressed. e-mail: W.Ledger{at}Sheffield.ac.uk
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Abstract |
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Key words: inhibin A/pro- C/pregnancy/medical termination
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Introduction |
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Inhibin A is the major circulating bioactive inhibin found in early pregnancy. Inhibin B is not detectable in early pregnancy in the human (Illingworth et al., 1996; Fowler et al., 1998
). Circulating levels of inhibin A and pro-
C have been implicated in the process of implantation and early pregnancy development (Muttukrishna et al., 1995
). They have also been proposed as markers of fetal viability (Norman et al., 1993
).
In the non-pregnant female, inhibins are secreted and synthesized by both the developing Graafian follicle and corpus luteum (Groome et al., 1994; Muttukrishna et al., 1994
). The corpus luteum has been shown to be the major site of inhibin production during the luteal phase in a normal menstrual cycle (Illingworth et al., 1991
) and production of inhibin A continues within the corpus luteum as pregnancy is established (Illingworth et al., 1996
; Rombauts et al., 1996
). During early pregnancy, mRNA for
,
A and
B have been demonstrated in the corpus luteum (Roberts et al., 1993
). However, inhibins are also synthesized and secreted by the developing human placenta (McLachlan et al., 1986
). Both
and
A-subunit mRNAs (Meunier et al., 1988
; Baird and Smith et al., 1993
) and proteins (Petraglia et al., 1991
) have been localized in the human placenta, the major expression being from the syncytiotrophoblast. The local actions during placental growth and differentiation are mirrored by changes in the circulating levels of dimeric inhibins and inhibin pro-
C as pregnancy progresses (Ledger, 2001
).
Concentrations of inhibin A in the circulation increase progressively in early pregnancy (Illingworth et al., 1996). There is a transient fall in circulating concentration at approximately 12 weeks gestation, followed by further increases in concentration from mid-gestation onwards (Muttukrishna et al., 1995
). Studies demonstrating lower levels of inhibin A in failing pregnancies have implicated inhibin A in the processes of successful implantation and early pregnancy development (Florio et al., 1995
). Inhibin A may also have a role as a marker of fetal viability in IVF pregnancies (Lockwood et al., 1997
).
There is, however, little information about the possible functions of the inhibins during early pregnancy. Such information will follow from studies aiming to identify the source of inhibin in early pregnancy, and the effects of pregnancy disruption on inhibin production. To date, studies of early pregnancy in patients undergoing IVF with donor oocytes in whom the corpus luteum was either inactive following treatment with a GnRH analogue, or after early menopause, showed increases in circulating inhibin A (Birdsall et al., 1997; Lockwood et al., 1997
) indicating the placenta as a source of inhibin A. However, although concentrations of inhibin A are increased in both normal pregnancies and in women pregnant after donor oocyte IVF, concentrations were higher in pregnancies in women with functional ovaries (Treetampinich et al., 2000
), suggesting an ovarian origin for inhibin A in addition to the placenta in early pregnancy. In order to explore the relative contribution of the corpus luteum and fetoplacental unit to the circulating pool of inhibin A in early pregnancy, changing concentrations of inhibin A and B during surgical termination of pregnancy have been studied. This study showed rapid decline in concentrations of inhibins following surgical evacuation of the uterus, despite maintenance of luteal progesterone secretion (Muttukrishna et al., 1997a
). Circulating concentrations of inhibin A declined to
30% of pre-termination levels by 24 h after surgery. This background concentration of inhibin A is thought to represent the contribution of the corpus luteum to the overall pool.
The present study was designed to investigate the production of the corpus luteum and feto-placental unit in early pregnancy using medical termination of pregnancy as a means of dissociating luteal function from that of the feto-placental unit. Medical termination of pregnancy involves consecutive use of two agents, mifepristone and misoprostol. Mifepristone acts predominantly on steroidogenic activity within the corpus luteum. Misoprostol, a prostaglandin analogue, produces uterine contractions, interrupts placental blood flow and causes expulsion of the feto-placental unit. Observation of the effects of the two agents on concentrations of inhibins allows an assessment of the relative importance of the two sources of inhibin A in early pregnancy.
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Materials and methods |
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Medical termination of pregnancy was initiated by oral administration of mifepristone (200 mg) followed 48 h later by the administration of vaginal misoprostol (200 µg) at 4 h intervals until expulsion of the fetus. Only the women who did not exhibit significant vaginal bleeding after mifepristone alone were included in the study.
During the procedure, three blood samples were collected: sample A, immediately before administration of mifepristone: sample B, immediately before administration of misoprostol; and sample C, soon after the expulsion of the products of conception. Blood was collected in EDTA and plasma was separated and stored at 40°C for hormone assay.
Measurement of inhibin A and pro- C in serum
Quantification of inhibin A and pro- C concentrations was performed by using commercial specific two-site enzyme immunoassay kits developed from the assay described by Groome et al. (1995
) (dimeric inhibin A ELISA MCA1273KZZ, pro-
-C ELISA MCA1254KZZ; Oxford Bio Innovation Ltd, Upper Heyford, Oxfordshire, UK).
Samples (100 µl) were treated and assayed in duplicate for dimeric inhibin A content according to the manufacturers instructions. The kit was supplied with lyophilised inhibin A purified from human follicular fluid, which has been calibrated against recombinant human 32 kDa inhibin. This was used as the assay standard by reconstitution followed by serial dilution with fetal calf serum to achieve a concentration range of 3.9500 pg/ml. Quality control samples (20, 120 and 300 pg/ml) were prepared by dilution of the stock inhibin A preparation, and were employed in each assay performed. The assay sensitivity was 3.9 pg/ml and the intra- and inter-assay coefficients of variation were <5.3 and <9.7% respectively.
In the pro- C assay, samples were diluted 1:10 in assay buffer provided within the kit prior to assay. Samples were analysed in duplicate according to the manufacturers instructions. The reference preparation for the assay was human follicular fluid-derived pro-
C supplied with the kit. This was reconstituted and diluted with assay diluent to produce the assay standard curve ranging from 1.56200 pg/ml. The assay employed three quality control samples (40, 80 and 160 pg/ml) and these were included in each assay performed. The sensitivity of the assay was 1.56 pg/ml and the intra- and inter-assay coefficients of variation were <2.3 and <5.4% respectively (Table I).
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Statistical analysis
The data were analysed using non-parametric analyses. To test for differences in each hormone between the three gestation groups at each of the sample collection times, the Kruskal-Wallis test was used. As the patterns of clearance were similar in each of the three gestation groups, further analysis was performed on the three groups combined. Friedman tests were performed to determine differences over all three time-points. As these proved significant, Wilcoxon Signed Rank tests were then carried out. To determine whether there were any associations between the patterns of the hormones, Spearmans rank correlation coefficients were calculated.
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Results |
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Changing concentration of inhibin A
Concentrations of dimeric inhibin A were not affected by the administration of the progesterone receptor antagonist mifepristone, concentrations being maintained at a similar level at the end of the 48 h observation period after mifepristone. Levels of inhibin fell significantly (P < 0.001) after vaginal misoprostol and expulsion of the fetoplacental unit. Concentrations of inhibin A fell to levels comparable with those observed in the late luteal phase in non-pregnant women (Illingworth et al., 1996). The final concentration reached of inhibin A was also similar between groups, irrespective of the gestation of the pregnancy (Figure 1A).
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Changing concentrations of other hormones
Progesterone
Peripheral concentrations of progesterone continued to rise in group C after the administration of mifepristone but remained steady in the other two groups. However, the levels were noted to fall significantly (P < 0.001) after the administration of prostaglandins and expulsion of the pregnancy. The rate of fall was comparable in groups A and B but was more rapid in group C (Figure 1C).
hCG
Levels of circulating hCG were not affected by the administration of mifepristone. The levels were seen to fall after the vaginal misoprostol and expulsion of the fetus. However it was noted that although the rate of fall was significant, (P < 0.01) the rate of clearance from the circulation was much lower than that seen for progesterone, pro- C and inhibin and reflects the long half life of hCG in the circulation (Figure 1C).
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Discussion |
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The present study seeks to dissociate production of inhibin A by the feto-placental unit from production by an ovarian source. This is done by determining the impact of treatment with mifepristone on circulating concentrations of inhibin A and inhibin pro- C. This study describes changes in circulating concentrations of inhibin A, inhibin pro-
C, progesterone and hCG after induction of medical termination of pregnancy with mifepristone and misoprostol.
The proportional rise in the baseline concentrations of inhibin A with advancing gestation suggests a connection with the increasing mass of the feto-placental unit. Administration of mifepristone appeared to have little effect on concentrations of inhibin A at all the gestations studied. We were unable to detect any significant reduction in levels of inhibin A until after administration of misoprostol and expulsion of the conceptus. We then observed a reduction in circulating levels of inhibin A, although it was still detectable in the circulation. Given the fairly rapid clearance of inhibin A from the circulation in early pregnancy, as observed previously (Muttukrishna et al., 1997a), it seems likely that the final values observed for inhibin A after medical termination of pregnancy were a reflection of continued secretion from the corpus luteum. The levels of hCG and progesterone either did not show any change or showed a small insignificant rise following mifepristone. Following misoprostol, levels of both hormones fell significantly. A similar pattern of fall in hCG and progesterone was also noted by another study of early pregnancy termination (Honkanen et al., 2002
).
In contrast to an apparent lack of impact of mifepristone on concentrations of inhibin A, mifepristone alone produced a significant fall in concentrations of inhibin pro- C. Irrespective of gestation (between 6 and 9 weeks), concentrations of inhibin pro-
C declined in the first 48 h after the administration of misoprostol and continued to fall throughout the study period. These findings are consistent with a predominantly ovarian source for inhibins containing the pro-
C region and suggest a relationship between inhibin subunits and ovarian steroidogenesis. In-vitro studies on cultured human granulosa and marmoset luteal cells have identified a possible role for inhibin free-
-subunit in maintaining luteal progesterone output (Webley et al., 1994
). The present study suggests that this effect may form one arm of a paracrine feedback loop within the corpus luteum of pregnancy, with disruption of progesterone synthesis by mifepristone resulting in a reduction in intra-ovarian synthesis of inhibin subunits. This effect was observed in the absence of significant reductions in circulating hCG or progesterone, suggesting a direct linkage between steroidogenesis and inhibin subunit production within the luteinized granulosa cell rather than an hCG-mediated effect.
There is increasing evidence to implicate the participation of inhibins and activins in the development of a healthy placenta. Early onset pre-eclampsia is associated with the appearance of elevated concentrations of inhibin A in circulation (Muttukrishna et al., 1997b; Muttukrishna et al., 2000
), and concentrations have also been found to be lower in the second trimester in Downs syndrome (Spencer et al., 1993
; Wallace et al., 1997
), a chromosomal disorder associated with relative placental immaturity. The lack of impact of mifepristone on inhibin A concentrations in early human pregnancy supports the hypothesis that this glycoprotein is mainly of placental origin in early pregnancy and suggests the existence of different pathways for regulation of inhibin synthesis within the ovary and placenta.
Our study demonstrates evidence of differential secretion of inhibin A and pro- C in early pregnancy. This is the first study to show the pattern of change in plasma levels of inhibin A and pro-
C during medical termination of pregnancy.
These results indicate that treatment with a competitive inhibitor of progesterone receptor (mifepristone) does not affect the function of the fetoplacental unit in any of the gestations studied. However, mifepristone treatment does reduce levels of inhibin pro- C, suggesting that inhibin pro-
C is a product mainly of the corpus luteum.
Further studies are required to provide more information about the source and the role of these hormones in normal and abnormal pregnancies.
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Acknowledgements |
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References |
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Submitted on May 7, 2002; resubmitted on November 18, 2002; accepted on January 9, 2003.