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 dis-
covery 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 sub-
units 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 experi-
ments 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 re-
cently an activin ␤E-subunit has been cloned from a mouse
∗ Corresponding author.
liver cDNA library. The predicted mature region of this sub-
unit showed >60% homology to the amino acid sequences
of activin ␤C and ␤D subunits, and approximately 45% ho-
mology 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 re-
mains 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 ante-
rior pituitary cells and was termed follistatin (Ying, 1988;
Ueno et al., 1987; Robertson et al., 1987). Follistatin is a cys-
teine 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
46 S. Muttukrishna et al. / Molecular and Cellular Endocrinology 225 (2004) 45–56
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 vari-
ants; 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 asso-
ciated with cell surface proteoglycans through follistatin’s
heparin binding site, and are thereafter endocytosed and bro-
ken down by lysosomal enzymes (Hashimoto et al., 1997).
Therefore, FS288 may provide a mechanism by which ac-
tivin 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 cir-
culation (Schneyer et al., 1996). Its possible functions could
be as a store of follistatin in circulation, a carrier of ac-
tivin 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 un-
derstanding 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 re-
cent 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 lev-
els have been measured during the menstrual cycle by sev-
eral 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 mea-
sured 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 secre-
tion 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 ac-
tivin is also synthesised in the pituitary, the inhibitory effect
of follistatin on the pituitary may be due to the neutralisa-
tion 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 stim-
ulatory effect and follistatin has an inhibitory effect on pi-
tuitary 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 immunoreactiv-
ity has been identified in the preoptic and septal areas of
the hypothalamus, regions in which GnRH perikarya are lo-
cated (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 regu-
lation 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 in-
hibin/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 anti-
bodies to activin B suppress FSH secretion from cultured rat
pituitary cells. Further studies with sheep pituitary cells con-
firmed 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 fol-
liculostellate 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 ma-
jor form of activin in circulation and activin B is produced
in the pituitary. Therefore, in the pituitary ovarian axis, we
 S. Muttukrishna et al. / Molecular and Cellular Endocrinology 225 (2004) 45–56
HYPOTHALAMUS
GnRH
+
Activin B PITUITARY
+ +
Activin A
- Fig. 2. Schematic diagram showing follicular growth related changes in
- FSH
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 se-
cretion (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 fac-
tors. Follicular granulosa cells and granulosa-lutein cells of
the corpus luteum are the principal site of expression of in-
hibin/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 treat-
ment for in vitro fertilisation (IVF) does not significantly al-
ter circulating activin A in women (Lockwood et al., 1996)
suggesting a largely extragonadal source for activin A. How-
ever, 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 exces-
sive production of activin, causes uncontrolled prolifera-
tion of granulosa cells and ovarian tumour development
47
granulosa cell production of activin and follistatin.
(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 undif-
ferentiated 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 go-
nadotrophin dependent stage of development. Undifferen-
tiated rat granulosa cells express relatively little follistatin
in comparison to cells from more advanced follicles (Shi-
masaki et al., 1989; Nakatani et al., 1991) suggesting the
presence of a relative excess of ‘free’ activin A that is biolog-
ically active. Collectively, studies from different species (re-
viewed 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 gran-
ulosa cells revealed that activin has an anti-steroidogenic ac-
tion, inhibiting basal and gonadotrophin stimulated P450cc
expression, progesterone secretion, P450 aromatase activ-
ity 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 re-
duces LH-induced androgen production and opposes the
48 S. Muttukrishna et al. / Molecular and Cellular Endocrinology 225 (2004) 45–56
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 al-
pha, ␤A, ␤B and follistatin mRNA and subunit proteins
(Roberts et al., 1993; Sidis et al., 1998) and oocytes ex-
press 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 sug-
gesting a role for activin in the maturation of oocytes.
Activin accelerates in vitro maturation of oocytes in mon-
keys (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 recom-
binant activin A increased their developmental compe-
tence 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 preimplan-
tation 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.
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 biologi-
cally 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 availabil-
ity 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 sug-
gesting a tightly regulated mechanism within the ovarian
follicle.
5. Activin and follistatin in the endometrium
and decidua
Human decidual expression of inhibin ␣ and in-
hibin/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 de-
cidualisation, ␣, ␤A and ␤B subunits were expressed by
the decidualised stroma. Expression of ␤A and ␤B sub-
units 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 bind-
ing 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 preg-
nancy occurs or breaks down every 28 days if it does not.
Activin A is known to promote proliferation and differenti-
ation 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
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 progres-
sively 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 syn-
thesis 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 follis-
tatin (Petraglia et al., 1990, 1992). Several in vivo studies
 S. Muttukrishna et al. / Molecular and Cellular Endocrinology 225 (2004) 45–56 49

have indicated a feto-placental source for these proteins in

Activin A (pg/ml/mg DNA

2000 **
early pregnancy (Birdsall et al., 1997; Muttukrishna et al.,
1997b; Lockwood et al., 1997, 1998). Activin receptor pro-
teins were localised to the syncytium in the first and second
trimester and to vascular endothelial cells of villous blood 1000
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 0
Control TNF
trimester of pregnancy, Riley et al. (1998) reported barely
100ng/ml
detectable levels of activin A in coelomic fluid and unde-
tectable levels in the amniotic fluid whilst, follistatin levels Fig. 4. Human umbilical vein endothelial cell (HUVEC) secretion of
were high in the coelomic fluid and low in the amniotic fluid. activin A in culture (24 h) in the presence of TNF-␣ (100 ng/ml). Student’s
t-text: ∗∗ P < 0.01.
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
7. Abnormal pregnancies
fluid had measurable levels of activin A (Muttukrishna
et al., 2002a). In term amnion, decidual and placental cell
Effective prediction of an abnormal pregnancy by non
and explant cultures pro inflammatory cytokines IL-1␤ and
invasive means could be very useful in the obstetric care
TNF-␣ preferentially stimulated activin A production with
of patients. Although pregnancy is a physiological condi-
limited effects on follistatin (Keelan et al., 1998, 2002).
tion, there are complications in pregnancy that can cause
Term placental trophoblasts secrete activin A in culture
maternal and fetal morbidity and mortality. There are inva-
and cytokines such as EGF, IL-1␤ and TNF-␣ have a
sive methods such as amniocentesis and chorionic villous
stimulatory effect on its secretion (Mohan et al., 2001).
sampling that are accurate in predicting fetal chromosomal
However, follistatin secretion was not detectable in these
abnormality but in the recent years, obstetricians have been
cultures.
increasingly using ultrasound measurements to monitor
Peripheral mononuclear cells (PBMC) are known to
fetal outcome. However, these measurements are not pre-
secrete activin A in response to pro-inflammatory stim-
cise in very early pregnancy and are somewhat subjective.
uli (Eramaa et al., 1995; Shao et al., 1992, 1998; Abe
Hence, serum markers of placental and fetal abnormal-
et al., 2001). Several types of endothelial cells, rele-
ity that could increase the predictive rate of ultrasound
vant to pregnancy, such as umbilical vein endothelial
measurements in early pregnancy would be helpful in mon-
cells (Kozian et al., 1997; McCarthy and Bicknell, 1993)
itoring most women with pregnancy complications. The
and vascular endothelial cells of myometrial blood ves-
ability to predict these could benefit research in treating
sels (Schneider-Kolsky et al., 2001) and placental vessels
these patients before the onset of some of these com-
(Schneider-Kolsky et al., 2002) express ␤A subunit mRNA.
plications such as pre-eclampsia, fetal growth restriction
We have shown that activin A is not secreted from PBMCs
and pre-term labour and could lead to therapeutic inter-
obtained from pregnant women in the resting state but
ventions.
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 7.1. Early pregnancy loss
secretion by PBMCs or HUVECs was not detectable in the
medium. 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
500 in sporadic and unexplained recurrent (greater then three
**
Activin A (pg/ml)

consecutive miscarriages for no known reason) miscarriage

400 **
patients (Muttukrishna et al., 2002b). However, circulating
300 levels of activin A, follistatin and follistatin:activin A ratio
200 were found to be largely unaltered (Muttukrishna et al.,
100 2002b). This discrepancy between inhibin A and activin A
levels suggest that although the feto-placental unit is known
0
control LPS TNF
to be a source of activin A in early pregnancy (Muttukrishna
1 ng/ml 10ng/ml et al., 1997b; Birdsall et al., 1997), there must be extra
placental sources as well. Recently Luisi et al. (2003) have
Fig. 3. Normal pregnant peripheral blood mononuclear cell (PBMC,
reported significantly lower levels of activin A in miscar-
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 riages, but only when the miscarriage was complete, which
t-test: ∗∗ P < 001. may explain the differences in these findings.
50 S. Muttukrishna et al. / Molecular and Cellular Endocrinology 225 (2004) 45–56
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 con-
centration 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 ac-
tivin 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 syn-
drome 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 dis-
ease in the second trimester, it could lead to more effective
management of the condition. Early studies looking at cir-
culating activin A and inhibin A in pre-eclampsia patients
showed a marked increase in patients with the disease com-
pared 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 hy-
pertension alone, activin A levels were higher than the con-
Activin A (ng/ml)
1000 Control ( n=20)
PE<34 ( n=10)
100 PE34-36 ( n=10)
PE>37 ( n=10)
GH(n=10)
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. Follis-
tatin 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 pla-
centa. 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 ac-
tivin 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) be-
cause of underlying placental dysfunction (FGR). The fetus
 S. Muttukrishna et al. / Molecular and Cellular Endocrinology 225 (2004) 45–56
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 con-
dition. This observation was confirmed by a recent study,
which reported a rise in serum activin A in FGR pregnan-
cies 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 pa-
tients and in pregnant women in labour at term compared
to pregnant women not in labour (Petraglia et al., 1995). In-
creased 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 ac-
tivin 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), sug-
gesting 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 sev-
eral sources in pregnancy. Various studies have evaluated the
changes in activin A and follistatin in abnormal pregnan-
cies (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 evi-
dent that activin A could be useful in predicting some gesta-
tional 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.
51
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 ac-
tivin 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 produc-
tion. 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 appreci-
ation 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 circula-
tion 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 de-
velopment in various species. In mice, activin A increases the
rate of morula formation and the velocity of embryo cleav-
age (Orimo et al., 1996). Activin is a mesoderm-inducing
factor in the amphibians and has a potent effect on em-
bryo 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
52 S. Muttukrishna et al. / Molecular and Cellular Endocrinology 225 (2004) 45–56
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.
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 avail-
ability 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
in this field. Various activin forms and antibodies for sub-
units, receptors and follistatin are now commercially avail-
able leaving no doubt that substantial further progress will
be made in the next few years.
Acknowledgements
Shanthi Muttukrishna acknowledges the research funding
from the Wellcome trust, UK.
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