Activin and follistatin in female reproduction

Molecular and Cellular Endocrinology 225 (2004) 45–56

Activin and follistatin in female reproduction Shanthi Muttukrishna a,∗ , Dionne Tannetta b , Nigel Groome c , Ian Sargent b a

Department of Obstetrics and Gynaecology, Royal Free University College Medical School, 86–96 Chenies Mews, London WC1E 6HX, UK b Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, UK c School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, UK

Abstract Activin and follistatin were initially identified in the follicular fluid based on their effects on pituitary FSH secretion in the mid-1980s. It is now evident that activin, follistatin and activin receptors are widely expressed in many tissues where they function as autocrine/paracrine regulators of a variety of physiological processes including reproduction. The major function of follistatin is to bind to activin with high affinity and block activin binding to its receptors. Total activin A and follistatin are also found in the maternal circulation throughout pregnancy. Activin A levels are increased in abnormal pregnancies such as pre-eclampsia, fetal growth restriction and gestational hypertension. The placenta, vascular endothelial cells and activated peripheral mononuclear cells (PBMC) may all contribute to the raised levels of activin A in pre-eclampsia with unaltered follistatin in pre-eclamptic placenta, PBMCs or vascular endothelial cells suggesting the availability of ‘free’ activin A that could be biologically active in these cells. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Activin; Follistatin; Peripheral mononuclear cells

1. Isolation and characterisation 1.1. Activin Purification of inhibin from follicular fluid led to the discovery of two other molecules that modulated pituitary FSH release. The molecule that stimulated pituitary FSH release and FSH ␤ subunit mRNA expression was termed activin (Ling et al., 1986; Vale et al., 1986). Activin was characterised as a dimer of inhibin beta subunits linked with disulphide bonds with a molecular weight of ∼25 kDa. Activin exists as homodimers or heterodimers of the beta subunits forming activin A (␤A␤A), activin B (␤B␤B) and activin AB (␤A␤B). Recent cloning experiments have provided evidence for the possible existence of three further ␤ subunits. Activin ␤c was cloned from human liver cDNA library and possessed 53 and 51% homology to the amino acid sequence of the mature ␤A and ␤B subunits (Hötten et al., 1995). An activin ␤D subunit cDNA has been cloned from Xenopus laevis cDNA library and showed 63% identity to the ␤C protein (Oda et al., 1995), and more recently an activin ␤E-subunit has been cloned from a mouse ∗

Corresponding author.

liver cDNA library. The predicted mature region of this subunit showed >60% homology to the amino acid sequences of activin ␤C and ␤D subunits, and approximately 45% homology to activin ␤A and ␤B subunits (Fang et al., 1996). Loveland et al. (1996) detected the expression of ␤C subunit mRNA in human ovary, placenta and testis samples and also in rat testis and spleen. Todate, only purified recombinant activin C and activin E have been produced (Kron et al., 1998) and the biological activity of these proteins are yet to be reported. However, activin ␤D subunit has been shown to possess activin-like bioactivity in Xenopus embryos (Oda et al., 1995). The existence of these recently cloned ␤ subunits raises the issue of the possible existence of five activin homodimers and potentially ten heterodimers. It remains to be established which, if any, of these homo- and heterodimers are actually synthesised naturally and their potential biological role remain obscure. 1.2. Follistatin The second molecule purified from follicular fluid was found to suppress pituitary FSH release from cultured anterior pituitary cells and was termed follistatin (Ying, 1988; Ueno et al., 1987; Robertson et al., 1987). Follistatin is a cysteine rich monomeric polypeptide structurally unrelated to

0303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2004.02.012

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inhibin or activin. It is encoded by a single gene and exists in several size variants of (Mr 32–39 kDa). It was subsequently discovered that follistatin binds to activin with high affinity (Nakamura et al., 1990). There are two major splice variants; FS315 and FS288. Although both forms appear to have a similar binding affinity for activin (Sugino et al., 1993), FS288 also has a high affinity for heparin (Sugino et al., 1993; Sumitomo et al., 1995). Subsequent experiments have shown that complexes of activin and FS288 become associated with cell surface proteoglycans through follistatin’s heparin binding site, and are thereafter endocytosed and broken down by lysosomal enzymes (Hashimoto et al., 1997). Therefore, FS288 may provide a mechanism by which activin is targeted for degradation. Although FS315 has the same affinity for activin as FS288, it has a poor affinity for cell surface proteoglycans. There is evidence to suggest that FS315 is the predominant form of follistatin in human circulation (Schneyer et al., 1996). Its possible functions could be as a store of follistatin in circulation, a carrier of activin to the target tissues or cells and/or a mechanism by which activin escapes the degradative pathway via binding to FS288. However, the actual functions of FS315 are yet to be elucidated and follistatin physiology is crucial to our understanding of these proteins and how they might regulate activin bioavailability.

2. Hypothalamo-pituitary-ovarian axis 2.1. Is there an endocrine role for circulating activin on pituitary FSH? Total activin A has been measured in the circulation of women throughout the menstrual cycle (Muttukrishna et al., 1996). Activin A varies in a biphasic manner with higher levels around the mid cycle and luteo-follicular transition period. Serum activin A levels are maximum during the luteo-follicular transition period when inhibin A and inhibin B levels are a minimum and the rise of serum FSH suggests that activin A might have an endocrine role to play in the rise of FSH in the early follicular phase. We have observed a rise in luteal phase activin A concentration in the circulation in the raised FSH group (FSH ≥ 10 mIU/ml) compared to the normal FSH group (FSH < 10 mIU/ml) further supporting our proposition that activin A might be involved in the early follicular phase FSH rise (Muttukrishna et al., 2000a). A recent study looking at the levels of activin A in ageing men and women has reported a steep increase in both genders (Baccarelli et al., 2001) when FSH levels rise as they get older supporting our observation. Circulating follistatin levels have been measured during the menstrual cycle by several groups. With the exception of one report which showed lower levels of follistatin during luteal phase (Gilfillan and Robertson, 1994) all other studies irrespective of the assays used, reported no changes in follistatin levels throughout the menstrual cycle (Khoury et al., 1995; Kettel et al., 1996;

McConnell et al., 1998; Evans et al., 1998). The absence of ‘activin free’ follistatin during the menstrual cycle as measured in the activin free follistatin assay format (McConnell et al., 1998) suggests that most of the circulating follistatin exists in an activin-bound state and has led investigators to question an endocrine role for follistatin. Activin has been shown to directly stimulate FSH secretion in rat (Vale et al., 1986; Ling et al., 1986) and sheep pituitary cells (Muttukrishna and Knight, 1991). Follistatin antagonises the effects of activin in these models. Since activin is also synthesised in the pituitary, the inhibitory effect of follistatin on the pituitary may be due to the neutralisation of the endogenous activin rather than a direct effect of follistatin on pituitary FSH release. Recent studies using human fetal pituitary cells from mid gestation terminations have shown that activin A has a stimulatory effect and follistatin has an inhibitory effect on pituitary FSH and LH release (Blumenfeld and Ritter, 2001).

3. Are activin and follistatin autocrine/paracrine regulators in the hypothalamo-pituitary axis? In the rat brain, activin beta A subunit immunoreactivity has been identified in the preoptic and septal areas of the hypothalamus, regions in which GnRH perikarya are located (Knight, 1996a). Activin has been shown to stimulate a GnRH-secreting neuronal cell line (Gonzalez-Manchon et al., 1991) raising the possibility of the involvement of brain activin containing neurons playing a role in the regulation of GnRH release. As mentioned above, activin was initially identified in the follicular fluid and was thought to have an endocrine effect on pituitary FSH production. However, in the early 1990s there were several reports on the existence of inhibin/activin subunits and follistatin in the pituitary cells of various species (Mather et al., 1992; Bilezikjian et al., 1993a, 1993b; Farnworth et al., 1995) suggesting the production of dimeric activin and follistatin by the pituitary gonadotrophs themselves. Activin B has been reported to be the major form of activin produced by rat anterior pituitary cells (Bilezikjian et al., 1994). Therefore, these molecules have an autocrine and/or paracrine effect on pituitary gonadotrophin secretion. Corrigan et al. (1991) provided the first evidence that antibodies to activin B suppress FSH secretion from cultured rat pituitary cells. Further studies with sheep pituitary cells confirmed the presence of intra-pituitary activin (Padmanabhan et al., 1995a,b). Studies by Liu et al. (1996a,b) showed the secretion of immunoreactive activin A and follistatin by rat anterior pituitary cells. Recent studies have shown that folliculostellate cells are a major source of follistatin in both rat (Bilezikjian et al., 2003) and primates (Kawakami et al., 2002). The evidence so far suggests that activin A is the major form of activin in circulation and activin B is produced in the pituitary. Therefore, in the pituitary ovarian axis, we

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HYPOTHALAMUS

GnRH

+ Activin B

PITUITARY

+ -

+ Activin A

FSH

-

Fig. 2. Schematic diagram showing follicular growth related changes in granulosa cell production of activin and follistatin.

follistatin

follistatin

OVARY

Fig. 1. Summary of autocrine/paracrine and endocrine actions of activins and follistatin in the pituitary-ovarian axis.

propose that activin A has an endocrine and activin B has an autocrine/paracrine role on regulating pituitary FSH secretion (Fig. 1).

4. What are the functions of activin and follistatin in the ovary? The control of folliculogenesis lies with the interactions between the pituitary gonadotrophins and intra-ovarian factors. Follicular granulosa cells and granulosa-lutein cells of the corpus luteum are the principal site of expression of inhibin/activin ␤A, ␤B subunits and follistatin. An in vivo study has shown that pituitary down regulation with GnRH agonist for three weeks followed by recombinant FSH treatment for in vitro fertilisation (IVF) does not significantly alter circulating activin A in women (Lockwood et al., 1996) suggesting a largely extragonadal source for activin A. However, human granulosa-luteal cell culture studies show that activin A is stimulated by FSH, LH and hCG (Muttukrishna et al., 1997a) suggesting that granulosa/luteal cell secretion of activin A is gonadotrophin dependent. Evidence from in vitro follicle cultures, granulosa cells, theca cells and oocytes suggest that activins and follistatin synthesised by granulosa cells exert local autocrine–paracrine actions. 4.1. Granulosa cells Activin promotes cell proliferation in rat granulosa cells (Li et al., 1995; Miro and Hillier, 1996) and human granulosa-lutein cells (Rabinovici et al., 1990). Targeted gene deletion of inhibin alpha subunit, resulting in excessive production of activin, causes uncontrolled proliferation of granulosa cells and ovarian tumour development

(Matzuk et al., 1992). In contrast, in activin receptor type IIB ‘knockout’ mice, follicle development was arrested at an early antral stage confirming a key role for activin in granulosa cell proliferation and differentiation (Nishimori and Matzuk, 1996). Activin can promote FSH receptor expression on undifferentiated rat granulosa cells (Hasegawa et al., 1988; Xiao et al., 1992). This is particularly significant since it could explain how a follicle at the late preantral to early antral stage progresses from a gonadotrophin independent to a gonadotrophin dependent stage of development. Undifferentiated rat granulosa cells express relatively little follistatin in comparison to cells from more advanced follicles (Shimasaki et al., 1989; Nakatani et al., 1991) suggesting the presence of a relative excess of ‘free’ activin A that is biologically active. Collectively, studies from different species (reviewed by Knight and Glister, 2001) and human (Schneyer et al., 2000) have shown that activin secretion is decreased whilst, inhibin and follistatin production is increased, as a follicle develops to become a dominant follicle (Fig. 2). A recent study in PCOS patients showed a high follistatin and low activin A in the circulation (days 3–5) of these patients (Eldar-Geva et al., 2001) suggesting that low activin levels could contribute to the follicular arrest in PCOS. Studies involving granulosa cells from both immature and mature marmoset follicles showed that activin can enhance basal and gonadotrophin stimulated P450 aromatase activity (Hillier and Miro, 1993). Studies on luteinised human granulosa cells revealed that activin has an anti-steroidogenic action, inhibiting basal and gonadotrophin stimulated P450cc expression, progesterone secretion, P450 aromatase activity and oestradiol production (Cataldo et al., 1994; Eramaa et al., 1995). Follistatin reversed the effect of activin on progesterone secretion and had no effect in the absence of activin in luteinised human granulosa cells (Cataldo et al., 1994) suggesting that follistatin on its own may not have an effect on granulosa cell function other than modulating activin availability. 4.2. Theca cells Treatment of human and rat theca cells with activin reduces LH-induced androgen production and opposes the

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action of inhibin (Hsueh et al., 1987; Hillier, 1991). The effects were reversed by follistatin consistent with its role as an activin binding protein. 4.3. Oocyte cumulus complex and embryo Cumulus granulosa cells express inhibin/activin alpha, ␤A, ␤B and follistatin mRNA and subunit proteins (Roberts et al., 1993; Sidis et al., 1998) and oocytes express activin receptors (Cameron et al., 1994; Sidis et al., 1998). Human oocyte cumulus complexes retrieved at IVF also secrete activin A in culture and significantly higher levels of activin A were secreted by good quality oocytes (Lau et al., 1999) supporting the studies suggesting a role for activin in the maturation of oocytes. Activin accelerates in vitro maturation of oocytes in monkeys (Alak et al., 1996) and human (Alak et al., 1998), an effect that was inhibited by follistatin. Exposure of denuded or cumulus-enclosed bovine oocytes to recombinant activin A increased their developmental competence to form blastocysts (Silva and Knight, 1998), an effect that was reversed by follistatin. There are reports on the expression of activin ␤A subunit, follistatin and activin receptors in rat and mouse embryos (Manova et al., 1992; Roberts and Barth, 1994), pig embryos and uterus (Van de Pavert et al., 2001) and bovine oocytes and embryos (Yoshioka et al., 1998). Human preimplantation embryos express the inhibin/activin beta A subunit gene at the blastocyst stage. However, activin receptor genes (types I and II) were detectable from four-cell to the blastocyst stage of human embryos (He et al., 1999) suggesting a role for activin in early human embryo development.

5. Activin and follistatin in the endometrium and decidua Human decidual expression of inhibin ␣ and inhibin/activin ␤A and ␤B subunits was first reported by Petraglia et al. (1990). Human endometrial expression of inhibin ␣, ␤A and ␤B subunit genes and proteins have been studied across the menstrual cycle and in early pregnancy. The glandular epithelium expresses all three subunits in the early phase of the cycle. Following the onset of decidualisation, ␣, ␤A and ␤B subunits were expressed by the decidualised stroma. Expression of ␤A and ␤B subunits is maintained in the glandular epithelium whilst ␣ subunit is down regulated (Jones et al., 2000). There is an up regulation of activin in decidualisation (Popovici et al., 2000). Activin receptors and follistatin are also expressed by human endometrial stromal cells (Jones et al., 2002). Co-expression of activin subunits, receptors and the binding protein follistatin suggests there is local regulation of activin action in the endometrium. Human endometrium is dynamic and undergoes proliferation, differentiation or decidualisation and regulates trophoblast invasion if pregnancy occurs or breaks down every 28 days if it does not. Activin A is known to promote proliferation and differentiation in various cell types and the presence of its receptors and subunits in the endometrial tissue supports a role for activin in decidualisation and implantation. However, the production of the dimeric proteins by endometrial cells is yet to be confirmed and the factors controlling activin and follistatin production by different endometrial cell types warrants further investigation.

6. Activin and follistatin in human pregnancy 4.4. Corpus luteum Activin promotes the proliferation of cultured human granulosa-lutein cells decreases basal and hCG induced progesterone secretion by granulosa-luteal cells (Rabinovici et al., 1990; Di Simone et al., 1994) whilst, follistatin reverses the effect of activin on progesterone secretion (Cataldo et al., 1994). Follistatin expression is increased by hCG in luteinised granulosa cells (Tuuri et al., 1994) suggesting that follistatin could play an important role in gonadotrophin-dependent luteal support mechanisms by controlling the availability of ‘free’ activin that is biologically active in the luteal cells. Almost all in vitro studies in the ovary have been carried out with activin A and the effects of other molecular forms of activin remains to be investigated. The limited availability of these molecular forms has hindered the research in this field. Within the ovary, activin affects cell proliferation, differentiation, oocyte maturation and hormone secretion. Almost all effects could be reversed by follistatin suggesting a tightly regulated mechanism within the ovarian follicle.

Circulating levels of ‘total’ activin A (Knight et al., 1996; Muttukrishna et al., 1996; Fowler et al., 1998; O’Connor et al., 1999) and follistatin (Fowler et al., 1998; O’Connor et al., 1999) have been reported by several groups. Levels of activin A did not vary significantly during the first and second trimesters although they were higher than menstrual cycle levels. After 24 weeks, activin A levels rise and there is a marked increase at term. Follistatin levels rise progressively throughout pregnancy reaching peak concentrations at term. A progressive rise in these hormones throughout pregnancy suggest feto-placental production. 6.1. Probable sources in pregnancy Human trophoblast contains the machinery for the synthesis of inhibin/activin subunits and follistatin (Petraglia et al., 1991, 1994; Minami et al., 1992; McCluggage et al., 1998) throughout pregnancy. The maternal decidua and foetal membranes (amnion and chorion) express specific mRNAs and subunit proteins for beta subunits and follistatin (Petraglia et al., 1990, 1992). Several in vivo studies

Activin A (pg/ml)

have indicated a feto-placental source for these proteins in early pregnancy (Birdsall et al., 1997; Muttukrishna et al., 1997b; Lockwood et al., 1997, 1998). Activin receptor proteins were localised to the syncytium in the first and second trimester and to vascular endothelial cells of villous blood vessels at term. Amniotic epithelial cells, mesenchyme and chorionic trophoblasts were also found to express activin receptor proteins (Schneider-Kolsky et al., 2002). In the first trimester of pregnancy, Riley et al. (1998) reported barely detectable levels of activin A in coelomic fluid and undetectable levels in the amniotic fluid whilst, follistatin levels were high in the coelomic fluid and low in the amniotic fluid. However, our recent study in samples collected from 8 to 12 weeks showed that activin A was undetectable in amniotic fluid samples collected <10 weeks gestation and coelomic fluid had measurable levels of activin A (Muttukrishna et al., 2002a). In term amnion, decidual and placental cell and explant cultures pro inflammatory cytokines IL-1␤ and TNF-␣ preferentially stimulated activin A production with limited effects on follistatin (Keelan et al., 1998, 2002). Term placental trophoblasts secrete activin A in culture and cytokines such as EGF, IL-1␤ and TNF-␣ have a stimulatory effect on its secretion (Mohan et al., 2001). However, follistatin secretion was not detectable in these cultures. Peripheral mononuclear cells (PBMC) are known to secrete activin A in response to pro-inflammatory stimuli (Eramaa et al., 1995; Shao et al., 1992, 1998; Abe et al., 2001). Several types of endothelial cells, relevant to pregnancy, such as umbilical vein endothelial cells (Kozian et al., 1997; McCarthy and Bicknell, 1993) and vascular endothelial cells of myometrial blood vessels (Schneider-Kolsky et al., 2001) and placental vessels (Schneider-Kolsky et al., 2002) express ␤A subunit mRNA. We have shown that activin A is not secreted from PBMCs obtained from pregnant women in the resting state but can be stimulated by proinflammatory cytokines (Fig. 3). HUVEC cells secrete activin A spontaneously and TNF-␣ stimulates activin A secretion in culture (Fig. 4). Follistatin secretion by PBMCs or HUVECs was not detectable in the medium.

500

**

400

**

300 200 100 0 control

LPS 1 ng/ml

TNF 10ng/ml

Fig. 3. Normal pregnant peripheral blood mononuclear cell (PBMC, n = 10 women) secretion of activin A in culture (24 h) in the presence of lipo polysaccharide (LPS, 1 ng/ml) and TNF alpha (10 ng/ml). Student’s t-test: ∗∗ P < 001.

Activin A (pg/ml/mg DNA

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49

**

2000

1000

0 Control

TNF 100ng/ml

Fig. 4. Human umbilical vein endothelial cell (HUVEC) secretion of activin A in culture (24 h) in the presence of TNF-␣ (100 ng/ml). Student’s t-text: ∗∗ P < 0.01.

7. Abnormal pregnancies Effective prediction of an abnormal pregnancy by non invasive means could be very useful in the obstetric care of patients. Although pregnancy is a physiological condition, there are complications in pregnancy that can cause maternal and fetal morbidity and mortality. There are invasive methods such as amniocentesis and chorionic villous sampling that are accurate in predicting fetal chromosomal abnormality but in the recent years, obstetricians have been increasingly using ultrasound measurements to monitor fetal outcome. However, these measurements are not precise in very early pregnancy and are somewhat subjective. Hence, serum markers of placental and fetal abnormality that could increase the predictive rate of ultrasound measurements in early pregnancy would be helpful in monitoring most women with pregnancy complications. The ability to predict these could benefit research in treating these patients before the onset of some of these complications such as pre-eclampsia, fetal growth restriction and pre-term labour and could lead to therapeutic interventions. 7.1. Early pregnancy loss Inhibin A has been shown to be a marker of a viable pregnancy in early IVF pregnancies (Lockwood et al., 1998; Treetampinich et al., 2000) and is significantly lower in sporadic and unexplained recurrent (greater then three consecutive miscarriages for no known reason) miscarriage patients (Muttukrishna et al., 2002b). However, circulating levels of activin A, follistatin and follistatin:activin A ratio were found to be largely unaltered (Muttukrishna et al., 2002b). This discrepancy between inhibin A and activin A levels suggest that although the feto-placental unit is known to be a source of activin A in early pregnancy (Muttukrishna et al., 1997b; Birdsall et al., 1997), there must be extra placental sources as well. Recently Luisi et al. (2003) have reported significantly lower levels of activin A in miscarriages, but only when the miscarriage was complete, which may explain the differences in these findings.

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7.2. Down’s syndrome It is generally agreed that inhibin A levels are raised in patient’s with Down’s syndrome pregnancies but the reports on activin A levels have been conflicting. Some studies have reported a significant rise in activin A concentration compared to control pregnancies between 10 and 14 weeks (Spencer et al., 2001; Dalgliesh et al., 2001) whereas, Cuckle et al. (1998) have reported a rise in activin A levels in only 27% of the patients with Down’s syndrome pregnancy compared to controls between 13 and 16 weeks gestation. Placental mRNA expression of activin beta A subunit was not significantly altered in placental tissue obtained from Down’s syndrome pregnancies at term (Lambert-Messerlian et al., 1998; Debieve et al., 2000). However, activin A levels in the Downs syndrome placental extract were higher than control placental extracts at term (Dalgliesh et al., 2001). In the second trimester, amniotic fluid levels of activin A were slightly lower in Downs syndrome pregnancies compared to controls whilst, follistatin levels were not affected (Wallace et al., 1999).

8. Gestational hypertension and pre-eclampsia Pre-eclampsia is one of the commonest causes of fetal and maternal morbidity and mortality. Gestational hypertension is defined as sustained diastolic pressure ≥ 90 mmHg from previously lower levels whereas pre-eclampsia is defined as gestational hypertension with sustained proteinuria ≥ 0.3 mg/24 h. The pathogenesis of pre-eclampsia is believed to have placental origins and the removal of the placenta is the treatment for severe pre-eclampsia (Redman, 1991). When pre-eclampsia is clinically apparent in the third trimester, intervention with anti-hypertensive drugs often does little to slow the progression of the disease. Therefore, if a biochemical or biophysical marker predicts the disease in the second trimester, it could lead to more effective management of the condition. Early studies looking at circulating activin A and inhibin A in pre-eclampsia patients showed a marked increase in patients with the disease compared to gestation matched controls (Petraglia et al., 1995; Muttukrishna et al., 1997c). Various other studies using the same assays have confirmed these findings (Fraser et al., 1998; Laivuori et al., 1999; Silver et al., 1999; D’Antona et al., 2000). Silver et al. (1999) reported a rise in activin A levels in patients with gestational hypertension. Our own longitudinal study (Muttukrishna et al., 2000b), looking at samples obtained from 10 to 12 weeks up to term, shows that patients who go on to develop pre-eclampsia before 34 weeks gestation have higher levels of activin A at 15–18 weeks gestation compared to normal pregnant controls. As the onset of the disease is delayed, the levels of activin A diverge from the controls at a later gestation (∼21 weeks gestation; Fig. 5). In patients who developed gestational hypertension alone, activin A levels were higher than the con-

Activin A (ng/ml)

50

1000

Control ( n=20) PE<34 ( n=10) PE34-36 ( n=10) PE>37 ( n=10) GH(n=10)

100 10 1 8-12

15-18 20-24 27-30

35-38

Gestation (weeks) Fig. 5. Maternal serum concentrations of activin A in pregnant controls, patients who developed pre-eclampsia (i) <34 weeks (PE < 34); (ii) 34–36 weeks (PE 34–36) and at term (PE > 37) and patients who developed gestational hypertension. Serial samples were taken from each patient at 8–12, 15–18, 20–24, 27–30 and 35–38 weeks of gestation.

trols from 27 to 30 weeks gestation suggesting that activin A is also altered in extra placental sources (Muttukrishna et al., 2000b). Several other studies have looked at the levels of activin A at 15–18 weeks and reported higher levels in patients who subsequently developed pre-eclampsia (Cuckle et al., 1998; Aquilina et al., 1999; Silver et al., 1999; O’Connor et al., 1999; Muttukrishna et al., 2000a,b; Keelan et al., 2002; Davidson et al., 2003). However, two studies reported no early increase in activin A levels in patients who subsequently developed pre-eclampsia (Raty et al., 1999; Grobman and Wang, 2000). The discrepancy between studies could be due to the number and/or group of patients studied, as pre-eclampsia is a wide spectrum disease. Follistatin levels also rise modestly in patients with pre-eclampsia (Keelan et al., 2002). Recent studies investigating the source for the rise in serum activin A and inhibin A have reported an increased expression of inhibin alpha and inhibin/activin beta subunit genes (Silver et al., 2002; Florio et al., 2002; Casagrandi et al., 2003) and proteins (Jackson et al., 2000; Manuelpillai et al., 2001; Bersinger et al., 2002) in the placenta. We also have evidence for raised levels of activin A secretion by PBMCs (Fig. 3), endothelial cells (Fig. 4) and placental trophoblasts in the presence of pro inflammatory cytokines that are raised in pre-eclampsia (Mohan et al., 2001; Tannetta et al., 2001) suggesting that the rise in activin A in pre-eclampsia could be from all of these sources. All the above studies are based on a small number of cases and indicate that activin A and inhibin A could be useful in predicting pre-eclampsia before the onset of the clinical symptoms. However, given the nature and spectrum of the disease, it is important to carry out a much larger study to obtain the statistical power to accurately test the predictive value of these hormones both on their own and in combination with other markers that could potentially be useful in predicting pre-eclampsia.

9. Fetal growth restriction The fetus is small in fetal growth restriction (FGR) because of underlying placental dysfunction (FGR). The fetus

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could be constitutionally small with otherwise normal and normal placental function (NSGA). Serum concentrations of activin A and activin A: follistatin ratio were found to be higher in pregnancies affected with FGR compared to controls (Bobrow et al., 2002) suggesting that ‘free’ activin A that is biologically active is in circulation in this condition. This observation was confirmed by a recent study, which reported a rise in serum activin A in FGR pregnancies compared to control and NSGA pregnancies (Wallace et al., 2003).

10. Pre-term birth Pre-term birth is the second major pathological condition that contributes towards neonatal morbidity and mortality. In most cases, the reason for premature labour is unknown and the early identification of a group that is at risk would allow early intervention with corticosteroids to reduce the risk of respiratory disorders in the new born. Maternal serum levels of activin A are higher in pre-term labour patients and in pregnant women in labour at term compared to pregnant women not in labour (Petraglia et al., 1995). Increased activin A in the fetal compartment with an increase in ␤A subunit and activin receptor IIB mRNA expression has been reported in pre-term labour (Petraglia et al., 1997). However, Coleman et al. (2000) have concluded that activin A, corticotrophin releasing hormone (CRH) and CRH binding protein are not clinically useful for the prediction of pre-term labour. A recent study using an assay for ‘free’ activin A has reported that follistatin-free activin A is not associated with pre-term labour (Wang et al., 2002), suggesting there may be very little biologically active activin A free in circulation in pre-term labour. Collectively, it is apparent that activin is secreted by several sources in pregnancy. Various studies have evaluated the changes in activin A and follistatin in abnormal pregnancies (Fig. 6). It is important to note that most studies have been carried out with limited number of patients and the observations are not always consistent. Although it is evident that activin A could be useful in predicting some gestational complications such as pre-eclampsia and fetal growth restriction, it is important to carry out much larger studies

Pre eclampsia Gestational Hypertension

Activin A

Down’s syndrome Fetal Growth Restriction Pre term labour?

Fig. 6. Summary of changes in the levels of maternal serum activin A in abnormal pregnancies.

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to evaluate the normal range in the control population and a ‘cut off’ value for the disease state that would enable the use of activin A as a screening test in early pregnancy.

11. What are the functions of activin and follistatin in normal and abnormal pregnancies? It is now evident that there are several sources for activin A production in pregnancy. Activin A could act in an autocrine/paracrine or endocrine manner in different cells and tissues in pregnancy. All tissues that secrete activin also secrete follistatin which provides a tight regulation of activin activity. There are no in vivo models available to study the role of activin A on placental hormone production. However, in vitro studies provide evidence for local actions of activin A in the gestational tissue. Activin A stimulated hCG and progesterone release from cultured placental trophoblasts (Petraglia et al., 1990; Mersol-Barg et al., 1990; Steele et al., 1993) and follistatin reverses this effect (Petraglia et al., 1994). Activin A also increases the release of immunoreactive oxytocin from placental trophoblasts (Petraglia et al., 1996) and amnion-derived cells (Petraglia et al., 1993). When first trimester placental chorionic villous explants were cultured in vitro, activin A stimulated the out growth of cytotrophoblasts in to the surrounding matrix (Caniggia et al., 1997), an effect that was reversed by follistatin. There is also growing appreciation for activin’s importance as a modulator of immune function. Keelan et al. (2002) have reported that activin at lower concentrations (5 ng/ml) stimulated amnion derived IL-6 production whereas higher concentration of activin A (50 ng/ml) had an inhibitory effect on this cytokine. TNF alpha production by chorio-decidual and placental explants was inhibited by 50 ng/ml activin A suggesting that activin has both pro- and anti-inflammatory effects on gestational tissue. The high concentrations of activin A in the circulation of women with pre-eclampsia could be involved in the regulation of the intense systemic inflammatory response which characterises the maternal syndrome of this disease. Activin has been reported to have effects on embryonic development in various species. In mice, activin A increases the rate of morula formation and the velocity of embryo cleavage (Orimo et al., 1996). Activin is a mesoderm-inducing factor in the amphibians and has a potent effect on embryo differentiation in the Xenopus (Smith et al., 1990; van-den-Eijnden-Van-Raaij et al., 1990). Our recent studies in early pregnancy (Muttukrishna et al., 2002a,b) has shown that there are high levels of follistatin in fetal circulation (14–16 weeks) in the presence of low levels of activin A, suggesting that follistatin might have a role in early human fetal angiogenesis, consistent with the work of Kozian et al. (1997) who proposed a role for follistatin in angiogenesis. The presence of activin, activin receptors and follistatin in various compartments of the gestational sac and expression in various fetal tissues suggests a tightly regulated biological

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Fig. 7. Schematic representation of the role of activin and follistatin in the pituitary, ovary and the placenta.

role for activin in embryo development and pregnancy, a field which warrants further research in the future.

in this field. Various activin forms and antibodies for subunits, receptors and follistatin are now commercially available leaving no doubt that substantial further progress will be made in the next few years.

12. Conclusion and future directions Since activin and follistatin were first identified in the follicular fluid in 1986, the biology of these molecules has evolved rapidly with new sources, new targets (Fig. 7), new sensitive assays to measure different molecular forms and the identification of high affinity activin receptors and the activin binding protein (follistatin). Despite considerable progress towards identifying biological roles for activin and follistatin in the female reproductive system within the last decade, several questions remain to be addressed. Do ovarian activin and/or follistatin have any endocrine role in pituitary FSH regulation? Do different molecular forms of activin have different functions? Do activins have different functions in different cell types? Does follistatin regulate activin availability depending on cell type? What are the functions of activin and follistatin in pregnancy? Does activin have any role in placental development? What is the importance of extra placental activin in pregnancy? Limited availability of these proteins until recently has slowed down the research

Acknowledgements Shanthi Muttukrishna acknowledges the research funding from the Wellcome trust, UK.

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