Production of inhibin forms by the fetal membranes, decidua, placenta and fetus at parturition

Simon C. Riley1,4, Rosemary Leask1, Claire Balfour1, Janet E. Brennand2 and Nigel P. Groome3

1 Department of Obstetrics and Gynaecology, University of Edinburgh, 37 Chalmers Street, Edinburgh, 2 Department of Obstetrics and Gynaecology, University of Glasgow, Glasgow and 3 School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inhibins are regulators of paracrine and endocrine function during pregnancy, but their intrauterine sites of secretion are not well established. In amniotic fluid, inhibin A-, inhibin B- and inhibin pro-{alpha}C-containing isoforms were present in high concentrations, whereas in maternal serum, inhibin A and pro-{alpha}C forms were present in high amounts, with low concentrations of inhibin B. In fetal cord serum, inhibin pro-{alpha}C was present in all samples, inhibin B was detectable in male but not female fetuses, with no detectable inhibin A in either sex. From cultured explants, both inhibin A and B were secreted by chorion laeve, whereas only inhibin A was secreted by placenta, with both tissues secreting inhibin pro-{alpha}C. Only low concentrations of both dimeric inhibins and pro-{alpha}C forms were secreted by decidua parietalis and amnion. The dual perfused placental cotyledon secreted both inhibin A and pro-{alpha}C into maternal perfusate, but only inhibin pro-{alpha}C into the fetal circulation and less than to the maternal side. We conclude that trophoblast is the predominant source of dimeric inhibins, but with markedly different secretion depending on its intrauterine location. There was a significant decrease in inhibin A and pro-{alpha}C in amniotic fluid collected at term active labour compared to elective Caesarean section (P < 0.001). This may reflect a local change in inhibin/activin processing at labour, likely in chorion laeve trophoblast cells, which may be important in the paracrine control of the feto-maternal communication required to maintain pregnancy and initiate labour.

Key words: fetal membranes/inhibin/labour/placenta/pregnancy/trophoblast


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The inhibins are heterodimeric glycoprotein hormones of the transforming growth factor-ß (TGFß) superfamily which were initially described for their suppression of follicle stimulating hormone (FSH) secretion (Qu and Thomas, 1995Go; Petraglia, 1997Go). They are made up of a common {alpha}-subunit and one of two ß-subunits, ßA and ßB. These give rise to two mature 32 kDa dimeric inhibins, inhibin A ({alpha}A) and inhibin B ({alpha}B), while the activins are made of dimers of the ß-subunits. Other ß-subunits have been recently reported but the formation of novel inhibin forms and their roles are not well defined (Fang et al., 1996Go). Towards the end of human pregnancy, concentrations of both immunoreactive and bioactive inhibin are present in increasing amounts in maternal serum (Muttukrishna et al., 1995Go; Qu and Thomas, 1995Go; Petraglia, 1997Go; Fowler et al., 1998Go). All inhibin isoforms are present in extracts of term human placenta, with the placenta as the principal source of inhibin in the maternal circulation (de Kretser et al., 1994Go). At term, the placenta, fetal membranes and decidua express the mRNA and also the protein to the {alpha}-subunit and both the ßA- and ßB-subunit forms as determined by in-situ mRNA analysis and immunohistochemistry (Petraglia et al., 1991aGo, 1993Go). However, the likely sources and secretion of the specific inhibin isoforms into the different compartments of pregnancy, the amniotic fluid and fetal and maternal circulation, and their role in the regulation of placental and fetal membrane function are still not well defined (Petraglia, 1997Go).

From in-vitro studies it appears that inhibins are involved in the paracrine regulation of prostaglandin, human chorionic gonadotrophin (HCG) and progesterone release (Petraglia et al., 1989Go; Petraglia et al., 1993Go; Qu and Thomas 1995Go), but how the different inhibin isoforms are involved in the maintenance of pregnancy and subsequent initiation and maintenance of labour is unclear. Measurement of inhibins has proved difficult due to the lack of selectivity of the {alpha}-subunit-directed immunoassays, which cannot measure the bioactive dimeric isoforms and are confounded by the presence of high concentrations of free {alpha}-subunit isoforms. Furthermore, both immuno- and bioassays can be affected by the presence of activins and the activin/inhibin binding protein follistatin. Specific assays have been developed to identify the two dimeric inhibins A and B (Groome et al., 1994Go, 1996Go) and inhibin isoforms containing pro and {alpha}C forms which are predominantly free {alpha}-subunit (Groome et al., 1995Go; Robertson et al., 1997Go). In this study we have measured the dimeric and {alpha}-subunit forms of inhibin in the different compartments of pregnancy at term before and at labour. We have identified using in-vitro techniques the principal tissues that secrete inhibins within the uterus. Our findings have revealed that inhibin production is tissue-dependent with directional secretion, resulting in the different bioactive isoforms secreted into different compartments in late gestation. These findings are important in elucidating the regulation and potential roles of the different inhibin isoforms for the paracrine and endocrine control of pregnancy and parturition.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collection of fluid and tissue samples
Matched samples of amniotic fluid and maternal and cord serum were obtained from women undergoing elective Caesarean section (n = 15; 38–40 weeks; dated from last menstrual period) and after spontaneous vaginal delivery (n = 15; 38–41 weeks; not associated with an intrauterine infection by clinical assessment, although no subsequent histology was performed) at term. Immediately after delivery by elective Caesarean section, samples of fetal urine (n = 8; only clear samples that were uncontaminated with blood were retained) at the first micturition and lung fluid (expired immediately after delivery; n = 4) were collected from the neonate. All samples were centrifuged (1000 g) and stored at –20°C prior to immunoassay for the different inhibin isoforms. For explant tissue culture, placentae with attached fetal membranes were collected from women undergoing elective Caesarean section at term (>37 weeks; not associated with labour). Decidual tissue was dissected from the myometrial aspect of the posterior uterine wall, from a site away from the placental bed, after delivery of the placenta. For placental perfusion, placentae were collected immediately after spontaneous vaginal delivery at term (n = 6). Ethical approval for the collection of all fluid and tissue samples was obtained from the Lothian Trust Ethical Committee with the informed and written consent of patients.

Explant tissue culture
Explants of tissues were cultured as described previously by this laboratory (Brennand et al., 1995Go). Discs of amnion (12 mm diameter; wet weight 10–20 mg) and chorion laeve with adherent decidua (9 mm diameter; 15–25 mg) were prepared using a cork borer, and pieces of villous placental tissue (collected from the middle of a central cotyledon; 20–30 mg) or decidua (20–30 mg) from the maternal aspect by curettage were placed on absorbent capillary matting. Tissues were maintained in culture medium (RPMI1640; Life Technologies Ltd, Paisley, UK) supplemented with 10% fetal calf serum (Life Technologies) plus antibiotics in a water saturated air/5% CO2 atmosphere at 37°C. After 24 h, culture medium was collected and stored at –20°C prior to immunoassay.

Dual perfusion of placental cotyledon
The in-vitro isolated dual perfused human placental cotyledon was used, as described previously (Schneider et al., 1972Go) with minor modifications (Benediktsson et al., 1997Go). Within 15 min of delivery, a peripheral intact cotyledon with parallel chorionic artery and vein and also the maternal lacunae were cannulated and perfused with Krebs' solution, which was supplemented with dextran (20 g/l; 74 kDa) in the fetal perfusate. To mimic partial pressures in vivo, the solutions used to perfuse the maternal and fetal circulations were gassed with 95% O2/5% CO2 and 95% N2/5% CO2, respectively. The cotyledon was perfused for 40 min to flush out residual blood and allow circulatory perfusion pressures to stabilize. Perfusates were not recycled. Samples (10 min) were collected after this equilibration period and stored at –20°C prior to assay. Viability and integrity of each preparation was assessed by establishing (i) the input perfusion rate in both fetal (6 ml/min) and maternal (10 ml/min) circuits equalled outflows, (ii) there was adequate exchange of O2 from the maternal to fetal circuits (step-up of pressure from <40 to >70 mmHg on fetal side), (iii) at the end of the experiment, the fetal vasculature responded to a bolus of noradrenaline (20 mg; Sigma), (iv) lactate concentrations remained within the normal low range (<2 mmol/l; Benediktsson et al., 1997) throughout the sampling period, and (v) there were no significant morphological changes on subsequent histological examination.

Immunoassays for inhibin isoforms
The inhibin assays used were two-site enzyme-linked immunosorbent assay (ELISA). These assays have been previously described, characterized and their specificities and cross-reactivities detailed (Groome et al., 1994Go, 1995Go, 1996Go) and were performed with minor modifications (Riley et al., 1996Go; Wallace et al., 1997Go). Immunoassay plates (96-well; Nunc Maxisort, Life Technologies Ltd) were coated passively with specific mouse monoclonal capture antibodies raised to peptide sequences of the inhibin ßA (antibody E4) and ßB (C5) subunits and the pro- portion of the inhibin-{alpha} subunit (INPRO) which conferred the assay specificity to inhibin A-, inhibin B- and pro- and {alpha}C-containing inhibin immunoreactivity. Plates were dried using a dry coating reagent and stored at 4°C (diluted 1:1 with H2O; Bionostics Ltd, Wyboston, UK). For the inhibin A and B assays samples and standards were diluted as appropriate with fetal calf serum, incubated in sodium dodecyl sulphate (2% final volume) for 3 min at 100°C which improves the signal and removes false positive results. Samples were then pretreated with H2O2 (1% final volume) for 30 min at 23°C, which increases antibody reactivity by modification of the ß-subunit epitopes (Knight and Muttukrishna, 1994Go), prior to adding to the appropriate ELISA plate. In the assay for pro-{alpha}C inhibin immunoreactivity all dilutions were performed in triton assay diluent and samples were applied directly to the plate without pretreatment (Groome et al., 1995Go).

The standard used for the inhibin A and inhibin B assay is a partially immunopurified inhibin standard from human follicular fluid calibrated against recombinant 32 kDa human inhibin A or inhibin B (Genentech, CA, USA) with results expressed in terms of this recombinant form. These assays have been validated for amniotic fluid (Riley et al., 1996Go; Wallace et al., 1997Go) and plasma samples (Groome et al., 1994Go; Robertson et al., 1997Go). For the assay for inhibin forms containing pro- and {alpha}C immunoreactivity the capture antibody (INPRO) was raised against a sequence of the pro-{alpha}C subunit, with a highly purified preparation of pro-{alpha}C inhibin as standard, as described previously (Groome et al., 1995Go). The pro-{alpha}C assay predominantly measures the 36 and 29 kDa forms (Robertson et al., 1997Go), although it may possess some cross-reactivity with larger forms of dimeric inhibin containing the {alpha}-subunit pro sequence as indicated by immunoblot (Groome et al., 1995Go). All assays utilized the same detection antibody (R1), the Fab fraction raised against the N-terminal region of the 20 kDa inhibin-{alpha} subunit which is conjugated to alkaline phosphatase. This alkaline phosphatase activity was detected in the inhibin A and pro-{alpha}C inhibin assays by addition of the substrate p-nitrophenylphosphate (Kirkegaard and Perry Laboratories, Gaithersburg, MA, USA), then measuring absorbancy at 405 nm using a microplate reader (Molecular Devices Corp., Menlo Park, CA, USA) with integrated software (Softmax; Molecular Devices), or for inhibin B using an amplification kit (Life Technologies Ltd) and measuring absorbancy at 490 nm. The assay sensitivity, and intra- and inter-plate coefficients of variation were: for inhibin A, 7 pg/ml and 6% and 8%; for inhibin B, 15 pg/ml and 8% and 10%; and for inhibin pro-{alpha}C, 3 pg/ml and 4% and 6%, respectively.

Data analysis
All sample sets had a normal distribution and statistical differences were assessed using Student's t-test. Differences were recognized as significant when P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amniotic fluid
Inhibin A, inhibin B and inhibin containing pro and {alpha}C immunoreactivity were all present in a similar range of concentrations in samples of amniotic fluid (Figure 1Go and Table IGo). There were significant decreases in forms containing pro-{alpha}C inhibin (P < 0.001) and inhibin A (P < 0.05) present at the time of delivery when compared to samples collected at elective Caesarean section. There was no significant change in inhibin B between these times.



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Figure 1. Concentrations (pg/ml; n = 15) of inhibin A (round symbols), inhibin B (square symbols) and inhibin isoforms containing pro and {alpha}C (triangular symbols) in amniotic fluid collected before the onset of labour at elective term Caesarean section (cs; open symbols) and at spontaneous delivery at term (svd; solid symbols). Significantly (*P < 0.05; **P < 0.001) lower at delivery compared to elective Caesarean section prior to the onset of labour.

 

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Table I. Concentrations of inhibin isoforms containing pro and {alpha}C in amniotic fluid and maternal serum
 
Maternal serum
Inhibin A was present in maternal plasma in much higher amounts than inhibin B (Table IGo). Inhibin containing pro and {alpha}C isoforms was also present in high concentrations in maternal serum. There were no significant differences in the concentrations of inhibin A or pro-{alpha}C inhibin between samples collected before the onset of labour at elective Caesarean section or after active labour and delivery. There were no differences in immunoreactive pro-{alpha}C inhibin or in inhibin A according to fetal sex.

Fetus: cord serum, fetal urine and expirated lung fluids
The concentrations of inhibin isoforms in cord serum are shown in Figure 2Go. There were no differences between concentrations of inhibins in cord serum from fetuses of the same sex collected before the onset of labour at elective Caesarean section and after labour, so these groups were combined (Figure 2Go). Immunoreactive pro-{alpha}C inhibin was present in cord serum from both male (2689 ± 276 pg/ml; mean ± SEM; n = 17) and female (2674 ± 379; n = 13) fetuses at similar concentrations with no significant differences between sexes. These concentrations in cord blood are similar to those present in maternal serum. Inhibin B was only detectable in cord serum from male fetuses (110 ± 11 pg/ml; mean ± SEM; n = 17), but was undetectable in serum collected from females. No dimeric inhibin A was detectable in cord serum from either sex. In fetal urine (n = 8), no inhibin A, inhibin B or pro-{alpha}C immunoreactivity were detectable. In fetal lung fluids (n = 4), inhibin A, inhibin B and pro and {alpha}C immunoreactive inhibin isoforms were present in similar amounts to those found in the respective matched amniotic fluid sample (data not shown). This is probably not surprising as this fluid is made up principally of amniotic fluid inhaled during fetal breathing movements.



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Figure 2. Concentrations (pg/ml) of inhibin A, inhibin B (square symbols) and inhibin isoforms containing pro and {alpha}C (triangular symbols) in umbilical cord blood collected at term (elective Caesarean section and spontaneous delivery samples combined) from male (open symbols; n = 17) and female fetuses (solid symbols; n = 13). Note different scales for dimeric inhibins and pro-{alpha}C-containing forms. nd represents none detected as below sensitivity of assay.

 
Secretion of inhibins by explants of placenta, amnion, chorion laeve and decidua parietalis
The placenta and chorion laeve were the tissue explants which secreted the greatest amounts of both dimeric and pro-{alpha}C subunit forms of inhibin into culture medium (Figure 3Go). Placental explants secreted only inhibin A in large concentrations with barely detectable concentrations of inhibin B, whereas explants of chorion laeve secreted inhibin A and also inhibin B in high concentrations. Both placenta and chorion laeve secreted large amounts of pro-{alpha}C inhibin forms. Decidua parietalis and amnion secreted only low amounts of inhibin A, inhibin B and pro-{alpha}C isoforms into culture medium.



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Figure 3. Secretion (output expressed as pg/mg wet weight tissue/24 h; mean ± SEM; n = 6 different placentae) of inhibin A (open bars), inhibin B (solid bars) and immunoreactive inhibin containing pro and {alpha}C (hatched bars) by explants of amnion, chorion laeve with adherent decidua parietalis, decidua parietalis (collected from the myometrial aspect) and villous placenta obtained at elective Caesarean section at term and maintained in culture for 24 h.

 
Secretion of inhibins by the dual perfused placental cotyledon
In the maternal perfusate, dimeric inhibin A was present, with no detectable dimeric inhibin B (Figure 4Go). In the perfusate collected from the fetal circulation, no dimeric inhibin A or inhibin B were detectable. Inhibin isoforms containing pro-{alpha}C immunoreactivity were secreted into both the maternal and fetal output, with a significantly (P < 0.05) greater output in the maternal compared to the fetal effluent.



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Figure 4. Secretion (pg/min/cotyledon; mean ± SEM; n = 6 different placentae) of inhibin A (A; open bars), inhibin B (B; solid bars) and inhibin isoforms containing pro and {alpha}C (pro-{alpha}C; hatched bars) into the maternal and fetal circulations of the in-vitro isolated dual perfused placental cotyledon preparation. nd represents none detected as below sensitivity of assay.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study has demonstrated that there are several different sites of secretion of bioactive dimeric inhibin A and inhibin B isoforms, and inhibin containing pro and {alpha}C isoforms, with specific patterns of secretion in the amniotic fluid and maternal and fetal circulations. Furthermore, inhibin A and inhibin pro and {alpha}C decrease in amniotic fluid with the onset of labour. Both dimeric inhibin forms are present in similar concentrations in amniotic fluid whereas only inhibin A is the predominant form in the maternal circulation. Trophoblast tissues are the likely main source of dimeric inhibins. Placental trophoblast secretes inhibin A, while trophoblast of the chorion layer of the fetal membranes secretes inhibin A and inhibin B in similar amounts, apparently unidirectionally into the amniotic fluid. In the fetus, secretion of dimeric inhibin B into the fetal circulation is sex specific with the testis as the likely source. The source of inhibin pro-{alpha}C-containing forms in the fetal circulation may be due, at least in part, to secretion by the placenta. No inhibin A is present in the fetal circulation.

The predominant source of the inhibin A and inhibin B in amniotic fluid is likely trophoblast of chorion laeve of the fetal membranes. The main sources of amniotic fluid production are via the fetal membranes and fetal urine, with some contribution from the fetal lung (Gilbert and Brace, 1993Go). Chorion explants secrete similar amounts of inhibin A and B and this is the same pattern of secretion in amniotic fluid. Although no attempt was made to separate adherent decidua from the chorion explants, this presence of trophoblast cells would seem to be the determinant of secretion of dimeric inhibins. This is because decidua parietalis obtained from the myometrial aspect, which contains little contamination by trophoblast cells, only secreted low concentrations of inhibin isoforms. In decidua parietalis, mRNA to the ß-subunit isoforms is expressed and this probably results in mostly activin secretion (Petraglia et al., 1990Go, 1997Go). There is no secretion of inhibins via the fetal urine. The fetal lung cannot be discounted as a potential source, but lung fluids obtained at first expiration after delivery contained similar concentrations to those found in the amniotic fluid, as would be anticipated, as it largely contains amniotic fluid. The source of immunoreactive pro-{alpha}C inhibin isoforms is less easy to define and may be secreted from several sites, including the amnion, chorion laeve and decidua parietalis. The fetus may also be a site of production of pro-{alpha}C inhibin, which is present in the fetal circulation although the mechanism of secretion into amniotic fluid is unclear but would probably be via the lung. These results are consistent with findings in the first trimester (Riley et al., 1996Go, 1998Go) where dimeric inhibins A and B and pro-{alpha}C isoforms and activin A are present in extra-embryonic coelomic fluid, with pro-{alpha}C but no dimeric inhibin in amniotic fluid. This indicates that the trophoblast layer is the principal site of dimeric inhibin production at this stage. It is only when the amnion fuses to the chorion laeve with the loss of the extra-embryonic coelom, and the amniotic cavity becomes the sole intrauterine compartment, that dimeric inhibins are present in the amniotic fluid and increase with gestation (Wallace et al., 1997Go). This unidirectional secretion of inhibin B secretion into the amniotic fluid but not into maternal serum is similar to that of prolactin, which is produced by decidua parietalis but is only present in amniotic fluid (Rosenberg et al., 1980Go), demonstrating that protein hormones can be directed into amniotic fluid.

In the maternal circulation, high concentrations of inhibin pro and {alpha}C and inhibin A are present, with low concentrations of inhibin B. This is in the same range of concentrations as reported previously (Fowler et al., 1998Go). The likely site of secretion of inhibin A and inhibin pro-{alpha}C is the syncytiotrophoblast of the placenta. Protein and mRNA to {alpha} and ßA subunits are localized at this site (Petraglia et al., 1992Go; Petraglia, 1997Go) and placental trophoblast cells secrete inhibin A (Keelan et al., 1994Go; Qu and Thomas, 1995Go). This secretion of inhibin A by placenta is unidirectional into maternal blood, whereas inhibin pro-{alpha}C is secreted in both directions. The ovary may also remain a source of circulating inhibins at term (Kettel et al., 1991Go; Illingworth et al., 1996bGo), and also the adrenal gland (Spencer et al., 1992Go; Munro et al., 1999Go). The chorion laeve of the fetal membranes may contribute to the low concentrations of inhibin B found in the maternal circulation at term.

In the fetal circulation dimeric inhibin B, which is the important isoform in the male (Illingworth et al., 1996aGo), was detected in cord blood from male but not female fetuses, confirming our previous findings (Wallace et al., 1997Go). Immunoreactive inhibin containing pro-{alpha}C was present in similar amounts in cord serum from both fetuses of both sexes, although no inhibin A was detectable in cord serum from fetuses of either sex. Our current findings are in agreement with the reports using the non-selective assays for free {alpha}-subunit which demonstrated the presence of inhibins in cord serum (Massa et al., 1992Go; Billiar et al., 1995Go; Rombauts et al., 1996Go). Billiar et al. (1995) reported that there was no dimeric inhibin present although their assay was not able to detect inhibin B as detected in this and previous studies (Wallace et al., 1997Go). In the fetus, only the steroidogenic tissues of the testis and adrenal gland express the {alpha}-subunit mRNA, whereas the mRNA to both the ßA and ßB subunits is expressed in multiple organ systems (Rabinovici et al., 1991Go; Jaffe et al., 1993Go; Tuuri et al., 1994Go), possibly reflecting activin secretion. Therefore the fetal testis, not the adrenal gland, is the likely source of dimeric inhibin B as it is specific to the male fetus. Immunoreactive pro-{alpha}C inhibin detected in the fetal circulation may be due, at least in part, to placental secretion as shown by output into this circuit in the perfused placental cotyledon. The fetal adrenal gland also expresses mRNA to inhibin {alpha} (Munro et al., 1999Go) and may be a source of pro-{alpha}C inhibin in cord serum, irrespective of fetal sex.

These studies provide evidence for a decrease in local {alpha}-subunit production within the fetal membranes at parturition, with the reduction of both inhibin pro-{alpha}C and also inhibin A in amniotic fluid at spontaneous delivery. The likely site for this control is the trophoblast cells of chorion laeve, which secrete both bioactive dimeric inhibin isoforms. The placenta secretes only inhibin A in significant amounts into the maternal circulation with no changes at labour. However, the role of inhibins in the regulation of labour is not well defined. They are probably diverse due to their secretion into both maternal circulation with potential for an endocrine role, as well as being local regulators at the feto-maternal interface. Inhibins have a diverse range of functions which are important in the paracrine signalling to initiate labour including control of steroidogenesis (Petraglia et al., 1987Go) and peptide hormone and prostaglandin secretion (Petraglia et al., 1993Go; Qu and Thomas, 1995Go) and monocyte chemotaxis (Petraglia et al., 1991bGo). The presence of the novel inhibin receptors (Hertan et al., 1999Go) in the uterus remains to be defined, but inhibins may modulate activin action at the level of activin receptors (Martens et al., 1997Go) which are expressed by placenta, fetal membranes and decidua (Petraglia et al., 1997Go). Local decreases in inhibin A and {alpha}-subunit production altering the balance of inhibins and activins may modulate paracrine mechanisms involved in the timing of the onset and for maintaining the cascade of stimuli required for progression to delivery.


    Acknowledgments
 
The authors wish to thank Dr David C.Howe for collecting the decidual samples for culture, Dr Rafn Benediktsson and Mr Alistair Greystoke for help with the placental perfusion and Mr Tom McFetters for help with the illustrations.


    Notes
 
4 To whom correspondence should be addressed Back


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 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on August 23, 1999; accepted on November 19, 1999.