Mol. Hum. Reprod. Advance Access originally published online on September 10, 2004
Molecular Human Reproduction 2004 10(11):793-798; doi:10.1093/molehr/gah110
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Inhibin, activin, follistatin, activin receptors and ß-glycan gene expression in the villous tissue of miscarriage patients
Department of Obstetrics and Gynaecology, Royal Free University College Medical School, 86–96 Chenies Mews, London WC1E 6HX, UK
1 To whom correspondence should be addressed. Email: s.muttukrishna{at}ucl.ac.uk
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Abstract |
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Maternal circulating levels of inhibin A are significantly lower in patients with clinical symptoms of miscarriage. The objective of this study was to quantify relative expression of inhibin
, inhibin/activin ßA, ßB, ßC, follistatin, activin receptors and ß-glycan genes and content of inhibin A, activin A and follistatin protein in villous tissue of first trimester miscarriages and gestation-matched normal pregnancies. Twelve women with clinical symptoms of miscarriage were matched with 12 normal pregnancies for gestational age. Total RNA was isolated from placental samples. Complementary DNA produced by reverse transcription was used in the real-time PCR to quantify the expression of the genes. The ratio between the target and rRNA 18S was calculated to provide relative gene expression. Villous tissue homogenates were used for the determination of the content of inhibin A, activin A and follistatin protein. Maternal serum was assayed for inhibin A, activin A and follistatin. All villous samples expressed inhibin , inhibin/activin ßA, ßB, ßC, follistatin, activin receptors (ACTRIA, ACTRIB, ACTRIIA, ACTRIIB) and ß-glycan genes. There was no significant difference in the relative expression of these genes between the groups. Villous content of inhibin A, activin A and follistatin were also not different between the two groups. Maternal serum levels of inhibin A were significantly lower in the miscarriage group compared to the controls. The decreased maternal levels of inhibin A in miscarriage patients could be due to a decrease in placental mass prior to embryonic demise. This finding also confirms that the trophoblast is the major source of inhibin A after the luteo-placental shift in early pregnancy. Key words: activin/activin receptors/ß-glycan/follistatin/inhibin
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Introduction |
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It is well-established that high concentrations of inhibin A, activin A and follistatin are present in the maternal circulation throughout pregnancy (Muttukrishna et al., 1995
, 1996; Fowler et al., 1998). In early pregnancy, inhibin A is produced by the feto-placental unit (Muttukrishna et al., 1997a) and corpus luteum until the luteo-placental shift occurs (Treetampinich et al., 2000). Placenta is a source for inhibin/activin subunits, dimeric proteins, follistatin and activin receptors throughout pregnancy (Petraglia et al., 1991; Petraglia, 1997; Peng et al., 1999; Debieve et al., 2000; Manuelpillai et al., 2001). Placental explants at term (Keelan et al., 1998; Riley et al., 2000), first trimester (Manuelpillai et al., 2003) and trophoblasts at term in culture (Mohan et al., 2001) have also been found to secrete inhibin A and activin A.
Inhibins (–ß dimers) and activins (ß–ß dimers) are members of the transforming growth factor ß (TGFß) family. Follistatin is a single chain glycoprotein with a high affinity to activin. There are two types of activin receptors, ACTRI (A and B) and ACTRII (A and B). ACTRIIA and ACTRIIB are membrane receptors to which activin binds and promotes the recruitment and phosphorylation of type I receptor serine kinase which then regulates gene expression by activating Smad proteins (Carcamo et al., 1994; Attisano et al., 1996). Binding of activin to Type II receptor stabilizes the receptor complexes and activates signal transduction. Inhibin can also bind to Type II activin receptors but it does not activate the receptor complex. Recently, type III TGFß receptor, ß-glycan was shown to function as an inhibin co-receptor with ACTRII (Lewis et al., 2000). ß-Glycan binds inhibin with high affinity and enhances binding in cells co-expressing ACTRII and ß-glycan. ß-Glycan also confers inhibin sensitivity to cell lines that otherwise respond poorly to this hormone (Lewis et al., 2000).
Although the proteins and receptors are expressed in the placenta, the exact role of these proteins in pregnancy is yet unclear. However, maternal circulating concentrations of inhibins and activins are increased in pregnancy complications such as pre-eclampsia (Petraglia et al., 1995; Muttukrishna et al., 1997b, 2000) and fetal growth restriction (Bobrow et al., 2002). Inhibin and ß subunit gene and protein expression is also altered in placental tissue from pre-eclamptic pregnancies (Silver et al., 2002; Casagrandi et al., 2003).
The most common pregnancy complication is miscarriage, affecting 
20% of pregnancies (Alberman, 1992). Serum concentrations of inhibin A are significantly lower than gestation-matched control pregnancy serum in patients with miscarriages (Phipps et al., 2000; Muttukrishna et al., 2002; Luisi et al., 2003). Since the placenta is a major source of inhibin and related proteins in pregnancy, the objectives of this study were: (i) to investigate the villous expression of inhibin/activin subunits, follistatin, activin receptors and ß-glycan genes; (ii) to investigate the villous protein content of inhibin A, activin A and follistatin in miscarriages.
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Materials and methods |
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Chorionic villous samples (n=12) were obtained from women presenting with a sporadic missed miscarriage at the time of surgical uterine evacuation between 8 and 12 gestational weeks. In nine women, maternal serum samples were also taken just before the surgical procedure.
Villous samples (n=12) from women with normal pregnancy, undergoing termination of pregnancy at 8–12 weeks gestation for social reasons, were taken at the time of surgical termination and used as controls. In 10 women, maternal serum samples were also taken just before the surgical procedure.
Villous tissue pieces were taken and rinsed several times in sterile phosphate-buffered saline to remove any blood and snap-frozen in liquid nitrogen. Tissue samples were stored at –80°C until RNA or protein extraction. Control and miscarriage placental tissues were from normal karyotypes. Serum samples were stored at –20°C for assays.
Gestational age was determined from the first day of the last menstrual period and confirmed by ultrasound measurement of the fetal crown–rump length in ongoing pregnancies. Written consent was obtained from each woman after receiving complete information on the procedure. Approval for this study was obtained from the joint UCL/UCLH ethics committees on the ethics of human research.
RNA extraction and cDNA synthesis
The Trizol reagent (Life Technologies Inc., USA) was used to isolate total RNA according to the manufacturer's protocol. Briefly, 100–200 mg of placental tissue was placed in a clean 1.5 ml Eppendorf tube. A total of 500 µl of Trizol was added and the tissue was homogenized; 200 µl of chloroform was added to the tube and the solution was incubated at room temperature for 5 min. It was then centrifuged at 1089 g for 15 min at 4°C. The upper aqueous phase was transferred to a fresh Eppendorf tube, and 500 µl isopropanol was added, incubated at room temperature for 5 min and centrifuged at 1089 g for 10 min. The RNA pellet was washed in 1 ml of 75% ethanol and centrifuged again at 1089 g for 5 min. The resulting pellet was dried and resuspended in 80% ethanol made up in diethylpyrocarbonate (DEPC)-treated water (Sigma–Aldrich, UK) and stored at –80°C. The concentration of RNA was analysed on a spectrophotometer (Jenway Ltd, UK). The ratio between the absorbance at 260 and 280 nm was >1.6.
Two micrograms of total RNA was reverse-transcribed into cDNA for each RNA sample extracted from placental tissue using TaqMan reverse transcription kit (Applied Biosystems Ltd, UK) according to the manufacturer's protocol.
Each cDNA sample obtained was checked by TaqMan (Applied Biosystems Ltd) PCR reaction, using 18S primers. PCR was carried out using the following cycle: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. PCR products were run on a 2% agarose gel.
Real-time PCR
Real-tme PCR was used for relative gene expression using an ABI Prism 7700 Sequence Detection System thermal cycler (Applied Biosystems, USA). The sequences for each gene are as previously published (Casagrandi et al., 2003).
A stock placental cDNA pool was prepared as a standard stock and used for all real-time assays. PCR reaction mixes for each standard and samples were prepared in separate tubes, using TaqMan Universal PCR master mix, primers, probe and cDNA.
All samples were assayed in triplicate and an aliquot of 25 µl reaction mix was transferred to each well of a MicroAmp optical 96-well reaction plate (Applied Biosystems, USA). The thermocycler parameters were: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
Control gene expression
Due to the nature of the miscarriage tissue the collection of samples and the storage conditions were critical. Several housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), human ß-actin, human large ribosomal protein (RPLPO) and r18S RNA were tested to choose the best housekeeping gene. 18S expression was not significantly different between the miscarriage and normal villous tissue. Therefore, an 18S pre-designed assay from Applied Biosystems was used to detect the expression of the housekeeping gene 18S in each sample using the same standard preparation. Target gene expression was normalized with 18S gene expression in each sample and the ratio between the target and 18S was expressed in all samples.
Placental homogenates
Extracts of first trimester placental tissue were prepared by homogenizing frozen samples in 4 volumes of Tris-buffered saline containing phenylmethylsulphonyl fluoride (PMSF; 1 mmol) and aprotinin (20 IU/ml). After centrifugation for 15 min at 6000 g, the supernatants were collected and stored at –40°C.
Hormone assays
Inhibin A was measured using a two-site enzyme-linked immunosorbent assay (ELISA) that has been previously validated for human serum by Muttukrishna et al. (1994). The minimum detection limit of this assay for human recombinant inhibin A (National Institute for Biological Standards, Potters Bar, Herts, UK) was 2 pg/ml. Intra- and inter-assay variation were 4.5 and 5.1% respectively.
Activin A was measured using a two-site ELISA specific for ‘total’ activin A (follistatin bound + unbound activin A) as described by Muttukrishna et al. (1996). The detection limit of this assay for human recombinant activin A (Genentech, USA) was 50 pg/ml. Intra- and inter-assay variations were 8.5 and 9.8% respectively.
Follistatin was measured using a two-site ELISA developed by Evans et al. (1998). The sensitivity of this assay was 20 pg/ml. Intra- and inter-assay variations were 6.8 and 9.15% respectively.
Statistical analysis
All individual values were log-transformed and Student's t-tests (Graph pad prism, USA) were carried out to test the difference between the cases and controls.
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Control gene expression
Threshold cycle values were compared for all miscarriage and normal placental RNA preparations. r18S RNA was the least affected in miscarriage samples and the variability between the cases and controls was not significant (Figure 1). Therefore, all gene expression data were normalized against 18S expression.
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Inhibin/activin subunits and follistatin gene expression
The mean ratio of inhibin
subunit:18S gene expression in miscarriage placentae (0.97±0.25) was similar to that of the controls (1.67±0.66, Figure 2a). The mean ratio of inhibin/activin ßA subunit:18S in miscarriage placental tissue (2.73±0.77) was not significantly different from the control placental tissue (2.45±0.75, Figure 2b). The ratios inhibin/activin ßB:18S and ßC subunit:18S in miscarriage placental tissue (10.3±3.35, 2±0.85 respectively) were not significantly different from the controls (5.87±2.6, 3.48±2.86 respectively; Figure 2c and d). The mean ratio of follistatin:18S gene expression in miscarriage placentas (3.2±2.23) was not altered compared to the controls (1.26±0.82, Figure 2e).
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Expression of activin receptors and ß-glycan genes
The mean ratio of ACTRIA:18S and ACTRIB:18S in the miscarriage tissue (2.1±0.7, 3.13±1.29 respectively) was not significantly altered from that of the controls (2±1.3, 3.97±2.84 respectively; Figure 3a and b). ACTRIIA:18S and ACTRIIB:18S expression in the miscarriage placental tissue (0.74±0.28, 1.4±0.33 respectively) was also not altered from the control tissue (0.94±0.53, 1.41±0.84 respectively; Figure 3c and d).
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ß-Glycan:18S ratio was not significantly altered in miscarriage placental tissue (4.47±1.93) compared to the controls (2±0.66, Figure 3e).
Inhibin A, activin A and follistatin contents in placental homogenates
Inhibin A concentration in miscarriage placental tissue (462±273 pg/mg protein) was not altered from that of the control placental tissue (405±112.7 pg/mg protein). Total activin A and follistatin concentrations in miscarriage placental tissue (1410±324, 347±86 pg/mg tissue respectively) were also not different from that of the control placental tissue (1455±588, 439±88 pg/mg protein respectively).
Inhibin A, activin A and follistatin in maternal serum
Maternal serum levels of inhibin A were significantly lower (P<0.01) in miscarriage patients compared to the controls. Serum activin A levels tended to be low in the miscarriage patients compared to the controls. However, the difference did not reach statistical significance (P=0.08). Follistatin levels were not significantly altered between the two groups (Table I).
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Discussion |
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It has been previously shown that maternal serum levels of inhibin A are significantly lower in miscarriages compared to normal ongoing pregnancies matched for gestational age (Phipps et al., 2000
; Muttukrishna et al., 2002; Luisi et al., 2003; Wallace et al., 2004). Inhibin A has been also reported to be an early marker of subsequent miscarriage in IVF pregnancies prior to the appearance of clinical symptoms (Lockwood et al., 1997).
Our data show that inhibin and ßA subunit gene expression are not significantly altered in miscarriage villous tissue compared to villous tissue from normal pregnancies matched for gestational age. Previous studies have shown that expression of hCG and ßA subunit gene is decreased in the villous tissue of miscarriage patients compared to controls (Henderson et al., 1992), suggesting down-regulation of hCG genes in miscarriages. Recently it was shown that, in pregnancies in which the fetus had died long before the clinical symptoms of miscarriage, the placental hCG was lower than in the age-matched normal pregnancies (Greenwold et al., 2003). Conversely, in those pregnancies in which the fetus appears intact, having died recently, the placenta contained higher than normal hCG. However, overall immunohistochemical staining for ß subunit hCG in the trophoblast tissue obtained from missed miscarriages showed no significant difference in the comparison with tissues obtained from normal pregnancies, indicating the ability of the placenta to stabilize in utero after fetal death (Greenwold et al., 2003). In the present study, the samples were taken 
7 days after the fetal demise and therefore even if there was a comparable change in inhibin subunit expression, it would not have been apparent.
Our recent study (Muttukrishna et al., 2004) also showed that placental extracts collected early in pregnancy contain large quantities of inhibin A, confirming our previous observation (Muttukrishna et al., 1997a). However, activin A and follistatin do have several sources such as the placenta, peripheral mononuclear cells (Tannetta et al., 2003), vascular endothelial cells (Phillips et al., 2001) and bone marrow (Mather et al., 1997). After 6–7 weeks gestation, the trophoblast is considered to be the principal source of estrogens and progesterone (Csapo and Pulkkinen, 1978). Thus pregnancy failure after this time can be mainly linked to placental dysfunction either secondary to a fetal abnormality or as a result of primary trophoblast dysfunction.
Using real-time PCR, we have shown for the first time the expression of inhibin/activin subunits, follistatin, activin receptors and ß-glycan genes in normal and abnormal early pregnancies. The villous tissue content of inhibin A, activin A and follistatin proteins was also not significantly altered in miscarriages compared to the controls. However, in this study serum concentrations of inhibin A were 70% lower in women with clinical symptoms of sporadic miscarriages compared to controls matched for gestational age, consistent with previous studies (Phipps et al., 2000; Muttukrishna et al., 2002; Luisi et al., 2003; Wallace et al., 2004). These observations suggest that circulating levels of inhibin A in miscarriages may be decreased because of reduced placental mass and/or decreased secretion by the trophoblasts into the circulation and/or reduced luteal function rather than any changes in gene expression or protein production in the placenta. Since maternal serum activin A and follistatin levels are not significantly lower in miscarriages compared to controls, the secretory mechanism may not be affected because the same cell types express inhibin/activin subunits and follistatin subunits (Petraglia et al., 1991).
Human placentation is characterized by the progressive infiltration of the uterine endometrium and superficial myometrium and transformation of the utero-placental circulation (Brosens et al., 2002). Up to 10 weeks of gestation, the trophoblast also forms a shell with plugs at the tip of the utero-placental arteries limiting the entry of maternal blood inside the placenta (Jauniaux et al., 2003a). In early pregnancy failure, independently of the cause, there is poor trophoblastic invasion, with limited transformation and plugging of the utero-placental arteries (Hustin et al., 1990; Greenwold et al., 2003; Hempstock et al., 2003; Jauniaux et al., 2003b). This results in an early and diffuse entry of maternal blood inside the placenta with rapid degeneration of the trophoblast and dislocation of the villous structure (Jauniaux et al., 2003b). In pre-eclampsia, there is a partial transformation of the utero-placental vessels (Brosens et al., 2002) with the untransformed vessels retaining their vasoreactive capacity (Hung et al., 2004). This results in an ischaemia–reperfusion phenomenon with chronic oxidative stress (Hung et al., 2001, 2004).
In pre-eclampsia, the expression of several protein and growth factor genes is increased (Tsoi et al., 2001; Sagawa et al., 2002). In pre-eclampsia in the third trimester, inhibin and ßA subunit gene expression is also increased in the placenta, reflected in higher serum levels of inhibin A and activin A, and this may be due to oxidative stress. In miscarriages, there is a temporary attempt to stabilize after the initial oxidative insult which is associated with an increased hCG expression (Greenwold et al., 2003). After 5–7 days there is irreversible damage with increased apoptosis and a decrease in numbers of mitotic cells (Hempstock et al., 2003).
We speculate that inhibin A levels in the serum are lower than the controls in miscarriages because of a decrease in total placental mass. Although the placenta also produces activin A, other cells also secrete activin A in pregnancy (Mather et al., 1997; Phillips et al., 2001; Tannetta et al., 2003). Therefore, placental contribution of activin A and follistatin may not be entirely reflected in circulating concentrations. Clearly, future studies should investigate miscarriages in detail based on the time of fetal demise and also the relationship between placental mass and maternal circulating levels of inhibin A, activin A and follistatin.
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Acknowledgements |
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This project was funded by a Wellcome Trust project grant (No.059743). We thank the Department of Haematology, UCL for allowing us to use the real-time PCR machine (ABI 7700).
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Submitted on August 19, 2004; accepted on August 20, 2004.
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