Human Reproduction Update, Vol.13, No.3 pp. 265–273, 2007 doi:10.

1093/humupd/dml060
Advance Access publication January 9, 2007

The Anti-Mullerian hormone and ovarian cancer

Antonio La Marca 1 and Annibale Volpe

Mother – Infant Department, Institute of Obstetrics and Gynecology, University of Modena and Reggio Emilia, Modena, Italy
1
To whom correspondence should be addressed at: Mother – Infant Department, Institute of Obstetrics and Gynecology, University of
Modena and Reggio Emilia, Policlinico di Modena, Largo del Pozzo, 41100 Modena, Italy. Tel: 39 0 594 224 379; Fax: 39 0 594 224 394;

Downloaded from https://academic.oup.com/humupd/article/13/3/265/2457878 by guest on 14 August 2021

E-mail: antlamarca@libero.it

The Anti-Mullerian hormone (AMH), which is produced by fetal Sertoli cells, is responsible for regression of Mullerian
ducts, the anlagen for uterus and Fallopian tubes, during male sex differentiation. Ovarian granulosa cells also secrete
AMH from late in fetal life. The patterns of expression of AMH and its type II receptor in the post-natal ovary indicate
that AMH may play an important role in ovarian folliculogenesis. Recent advances in the physiological role of AMH
has stimulated interest in the significance of AMH as a diagnostic marker and therapeutic agent for ovarian cancer.
Currently, AMH has been shown to be a circulating marker specifically for granulosa cell tumour (GCT). Its diagnostic
performance seems to be very good, with a sensitivity ranging between 76 and 93%. In patients treated for GCT, AMH
may be used post-operatively as marker for the efficacy of surgery and for disease recurrence. Based on the physiologi-
cal inhibitory role of AMH in the Mullerian ducts, it has been proposed that AMH may inhibit epithelial ovarian cancer
cell both in vitro and in vivo. These observations will be the basis for future research aiming to investigate the possible
clinical role of AMH as neo-adjuvant, or most probably adjuvant, therapy for ovarian cancer.

Key words: AMH/cancer therapy/diagnostic markers/epithelial ovarian cancer/granulosa cell tumour

Introduction
Ovarian cancer comprises a heterogeneous group of tumours of
three major types: epithelial, germ cell and sex cord stromal.
Epithelial tumours arise from the coelomic mesothelium, which
is capable of differentiating into both benign and malignant
tumours. Epithelial malignancies represent about 82% of all
ovarian malignancies. The predominant cell types are: serous,
mucinous and endometrioid.
The malignant germ cell tumours are believed to arise from
primitive germ cells in the ovary. These tumours represent about
5% of all ovarian malignancies. They include dysgerminoma,
embryonal carcinoma, endodermal sinus tumour, choriocarcinoma
and malignant teratomas.
The sex cord stromal neoplasms are derived from mesenchymal
stem cells in the ovarian cortex and represent about 10% of all
ovarian tumours. They include granulosa – theca cell tumours,
granulosa cell tumours (GCTs) and Sertoli –Leydig cell tumours.
GCT constitutes 3% of all ovarian tumours. Two distinct types
of GCTs have been described on the basis of clinical presentation
and histological characteristics: the juvenile and the adult form.
The more common adult form generally presents in pre- or post-
menopausal women with a median age of 50 years during presen-
tation (Pankratz et al., 1978; Bjorkholm and Silfversward, 1981).
A majority (two-thirds) of these GCT patients have endocrine
manifestations as a direct consequence of hormone secretion by
the tumour (Bjorkholm and Silfversward, 1981). In the majority
of cases the effects are estrogenic in nature, though rarely andro-
genic effects have been documented (Nakashima et al., 1984).
In the prepubertal female, these effects may manifest as preco-
cious isosexual development, whereas in premenopausal patients,
menstrual irregularities are seen (Biscotti and Hart, 1989). In older
women, the most frequent symptom is post-menopausal bleeding.
As a consequence of this hormonal secretion, an association
between GCT with both endometrial hyperplasia and adenocarci-
noma has been well documented (Salerno, 1962).
In GCT the routes of dissemination are not completely clear.
These tumours grow locally and then spread to the adjacent intra-
peritoneal organs and when systemic disease occurs it does so late
in the course of the disease. The majority of GCT present as stage I
and the 5-year survival rate is 90%.
The initial form of therapy for most patients should be a surgical
procedure so that a histological diagnosis and appropriate staging
may be obtained. In patients with disease localized to one ovary
and those wishing to preserve fertility, conservative therapy seems
to be indicated. In those with more advanced disease or in post-
menopausal women, Total abdominal hysterectomy and bilateral
salpingo-oophorectomy (TAH/BSO) would be an appropriate
initial treatment. Chemotherapy should be considered in those
patients with advanced, recurrent or metastatic disease. Although
response rates to chemotherapy are high, the impact on both disease-
free states and overall long-term survival is currently unknown.

# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For
Permissions, please email: journals.permissions@oxfordjournals.org 265
A. La Marca and A. Volpe
The GCTs have the potential to secrete estrogen and inhibin.
Hence, these products are used as markers for GCT.
In several studies, elevated serum levels of estradiol have been
found in some patients with known disease. The levels generally
fluctuate with response to or failure of treatment or with the
bulk of the disease. Unfortunately, the cases that have been
described are few and the marker itself lacks sensitivity. Although
it has been shown that the granulosa cells produce estradiol, they
do so only if testosterone is presented to them. Testosterone is
produced by the adjacent theca cells, which are present in some
ovarian tumours but not in extraovarian tumours (Lappohn
et al., 1989).
A second marker, inhibin, has also been reported in patients
with GCT. Inhibin is a dimeric glycoprotein secreted by the
gonads to regulate pituitary FSH. It exists as a dimer of two
subunits (a and bA or bB) to form inhibin A (abA) or B (abB)
(Bilezijkian et al., 2004).
Studies investigating the specific assays for inhibin A and B
have shown that almost all GCT have elevated inhibin B levels
and the majority also have elevated inhibin A levels (Robertson
et al., 1999a). Concentrations of immunoreactive inhibin and the
specific dimeric inhibin in GCT are by far the highest seen in
any form of ovarian malignancy or in other disease.
However, as a diagnostic test, neither inhibin A nor B are as
effective as a total a subunit as measured by the inhibin radio-
immunoassay (RIA) (Robertson et al., 1999a). Unfortunately,
the inhibin RIA is limited as a diagnostic test due to its lack of
practicability (5-day assay). A two-site immunofluorometric
assay (aC IFMA) was subsequently developed (Robertson et al.,
1999b). However, the aC IFMA is not appropriate for wide-spread
diagnostic use as it has proved difficult to maintain the assay
reliability between different batches of antisera. In conclusion
there is controversy surrounding the use of different antisera and
methodologies, which have resulted in conflicting results.
More than 10 years ago, an alteration in anti-Mullerian hormone
(AMH) [also called mullerian inhibiting substance) secretion was
described in a patient affected by GCT (Gustafson et al., 1992).
After this first observation, several studies investigating the
dynamics of serum AMH in patients with ovarian malignancies
have been published.
In the present article, the main findings and possible clinical
implications of these studies are explored.
AMH and AMH receptors
AMH is a member of the transforming growth factor (TGF) super-
family. AMH is strongly expressed in Sertoli cells from testicular
differentiation up to puberty and to a much lesser degree in granu-
losa cells from birth up to menopause (Munsterberg and
Lovell-Badge, 1991; Lee and Donahoe, 1993; Josso et al., 1998;
Durlinger et al., 1999; Durlinger et al., 2001; Ikeda et al., 2002).
AMH seems to act only in the reproductive organs (Lee and
Donahoe, 1993). The most striking effect of AMH is its capacity
to induce regression of the Müllerian ducts, the anlage of the
female internal reproductive organs. In the absence of AMH,
Müllerian ducts of both sexes develop into the uterus, Fallopian
tubes and the upper part of the vagina (Munsterberg and
Lovell-Badge, 1991). During fetal life, only the male testis
expresses the hormone (Munsterberg and Lovell-Badge, 1991).
266
In the female, AMH expression begins post-natally and acts in
modulating follicular growth (Durlinger et al., 2001; Ikeda
et al., 2002) and preventing recruitment of non-dominant follicles
(Durlinger et al., 1999; Josso et al., 1998).
Granulosa cells of primary follicles show homogeneous AMH
expression, whereas in larger follicles, AMH is mainly produced
in cells near the oocyte and in a few cells surrounding the
antrum. AMH continues to be expressed in the growing follicles
in the ovary until they have reached the size and differentiation
state at which they are to be selected for dominance by the
action of pituitary FSH.
In mouse this occurs at the early antral stage in small growing
follicles (Durlinger et al., 2002), whereas in human it is in antral
Downloaded from https://academic.oup.com/humupd/article/13/3/265/2457878 by guest on 14 August 2021
follicles of size 4– 6 mm (Weenen et al., 2004). Thus, AMH is
expressed in follicles that have undergone recruitment from the
primordial follicle pool and have not been selected for dominance.
AMH is not expressed in atretic follicles and theca cells (Rey
et al., 2000) (Figure 1).
AMH employs a heteromeric receptor system consisting of a
single membrane spanning serine threonine kinase receptors of
types I and II, respectively. The type II receptor imparts ligand-
binding specificity and the type I receptor mediates downstream
signalling when activated by the type II receptor (Figure 2). The
AMH type II receptor (AMH-RII) is localized to the mesenchyme
around the Müllerian duct in the urogenital ridge of both the male
and female rat and mouse. It is interesting to note that loss of func-
tion mutations in the AMH-RII as well as the AMH ligand itself, are
causes of persistent Müllerian Duct Syndrome in humans (Imbeaud
et al., 1994). In the ovary of rats, the AMH-RII is expressed both in
granulosa and theca cells (Ingraham et al., 2000).
Role of AMH in ovarian physiology
AMH is considered as a negative regulator of the early stages of follicu-
lar development (Themmen 2005). Earlier studies have shown that
AMH inhibits the first meiotic division of diplotene oocytes in imma-
ture rats (Ueno et al., 1989), but this has not been confirmed by others
(Tsafriri et al., 1988). In addition, AMH blocks the proliferation of
human granulosa–luteal cells in vitro (Kim et al., 1992) and the con-
centration of AMH in follicular fluid is inversely proportional to the
mitotic indices of granulosa cells in vivo (Seifer et al., 1993). Thus,
AMH appears to have an autocrine role in maturation of normal fol-
licles (Figure 3).
Based on this observation it has been hypothesized that AMH
could be one of the factors involved in determining the responsive-
ness of ovarian follicles to FSH during cyclic recruitment
(Skinner, 2005). It has been recently shown that oocytes
up-regulate AMH expression in granulosa cells in a fashion that
is dependent upon the developmental stage of the oocyte
(Salmon et al., 2004). This observation leads to the hypothesis
that oocytes in the pool of growing follicles could control the
pool of primordial follicles by modulating the expression of the
inhibiting factor AMH.
Circulating AMH in women
In males, AMH levels after birth are low, but rapidly rise to peak
values by late infancy, then slowly decrease to the adult range at
puberty (Josso et al., 1990; Lee et al., 1996). In women, AMH
 AMH and ovarian cancer

Downloaded from https://academic.oup.com/humupd/article/13/3/265/2457878 by guest on 14 August 2021

Figure 1. AMH immunostaining in normal human adult ovary. (A) Primordial follicles are negative (arrowheads), whereas a weak positive reaction can already be
observed in the cytoplasm of a few cells in a primary follicle (arrow). (B) All granulosa cells of pre-antral follicles are AMH-positive. (C) AMH-positive and AMH-
negative cells are intermingled in the cumulus oophorus of a pre-ovulatory follicle. (D) Only a few granulosa cells surrounding the antrum (ANT) are AMH-positive.
Thecal cells (TC) are not immunoreactive (original magnifications—A and B: 200; C: 100; and D: 300) (Reprinted from “Anti-Müllerian hormone is a specific
marker of sertoli- and granulosa-cell origin in conadal tuitors” by Rey R. et al. (2000) Human Pathology, 31:1202-1208, with permission from Elsevier).

serum levels are almost undetectable at birth (Rajpert-De Meyts time in young normovulatory women (de Vet et al., 2002), and
et al., 1999), with a subtle increase noted after puberty (Hudson to correlate with age, FSH and the number of antral follicles. In
et al., 1990). a recent study, a group of women was studied twice and the inter-
Serum AMH levels have been measured at different time-points val between the two visits ranged from 1.1 to 7.3 years. A
during the menstrual cycle, suggesting minimal fluctuation reduction in mean AMH levels of about 38% was observed,
(La Marca et al., 2006a). Minimal fluctuations in serum AMH whereas the number of antral follicles and the levels of FSH and
levels may be consistent with continuous non-cyclic growth of inhibin B did not change (de Vet et al., 2002). The decrease in
small follicles. AMH with advancing age may occur before changes in the
Hence, AMH is relatively convenient to determine, especially
as it seems to exhibit a relatively stable expression during the men-
strual cycle, making it an attractive determinant of ovarian activity
(Gruijters et al., 2003).
Recent studies indicate the AMH as a novel measure of ovarian
reserve. Serum AMH levels have been shown to decrease over

Figure 3. Role of AMH in human folliculogenesis. In women, AMH

Figure 2. AMH receptor binding. Upon ligand binding to the type II receptor,
the type I receptor is recruited into the receptor complex and becomes phos-
phorylated by the type II receptor. Activation of the type I receptor results in
the phosphorylation of the downstream Smad proteins. Phosphorylated
receptor-specific Smads translocate to the nucleus where they regulate gene
expression.
expression can first be observed in granulosa cells of primary follicles, and
expression is strongest in pre-antral and small antral follicles (4 mm). AMH
expression disappears in follicles of increasing size and is almost lost in follicles
larger than 8–10 mm, where only very weak staining remains, restricted to the
granulosa cells of the cumulus. This expression pattern suggests that AMH may
play a role in initial recruitment and in the selection of the dominant follicle:
AMH seems to be a negative regulator of the early stages of follicular develop-
ment. AMH prevents recruitment of non-dominant follicles and reduces the
responsiveness of ovarian follicles to FSH during cyclic recruitment by
Stephen et al. (2002) Highly purified Müllerian inhibiting substance inhibits
human ovarian cancer in vitro. Clin Cancer Res. 8, 2640-2646.

267
A. La Marca and A. Volpe
currently known ageing-related variables and this indicates that
serum AMH levels may be the best marker for ovarian ageing
and menopausal transition.
AMH and AMH receptor in ovarian cancer
Granulosa cell tumour
In the first description of increased AMH in serum from a patient
with GCT (Gustafson et al., 1992), the AMH gene expression in
neoplasm specimens was also investigated. RNA was extracted
and analysed by northern blotting showing that a 2.0 kb AMH
RNA transcript was present.
In a small series of ovarian tumours, AMH expression was
detected in all of the GC tumours but not in the seven epithelial
carcinomas (Matias-Guiu et al., 1998). Using paraffin-fixed sec-
tions and antibody directed to AMH, authors demonstrated that
AMH immunoreactivity was always present in the GC tumours
but was not as strong or diffuse as alpha-inhibin. The finding of
AMH expression in both juvenile- and adult-type GCT of either
ovarian or metastatic localization has been recently confirmed
(Rey et al., 2000) (Figure 4).
Dutertre et al. (2001) developed a murine transgenic model of
GCT of the ovary. They showed that driving the expression of
the simian virus 40 (SV40) oncogene by the AMH promoter
Figure 4. AMH-immunostaining of primary GCT. (A) Juvenile-type GCT of
ovarian localization. (B) Adult-type GCT of ovarian localization. In all
cases, staining is in the cytoplasm of positive cells (original magnifications—
A: 300; B: 500) (for permission see fig. 1).
268
induces gonadal tumourigenesis in transgenic mice (Peschon
et al., 1992; Dutertre et al., 1997). They also showed that granu-
losa cell tumours of less than 100 mg (10-fold the size of
the normal ovary) were positive for both AMH and AMH-RII
mRNA and proteins. Moreover, expression was not found in
larger tumours (Dutertre et al., 2001). They also showed that
AMHR-II was expressed on the cell surface and was able to
bind to its ligand AMH. They concluded that GCT of transgenic
mice produce AMH and secrete it into the blood when the
tumour is relatively small. In advanced stages, the GCT invades
organs and develops metastases and loses both AMH and
AMH-RII expression. Considering the inhibitory effect of AMH
on GCT growth (Kim et al., 1992), this phenomenon could indi-
Downloaded from https://academic.oup.com/humupd/article/13/3/265/2457878 by guest on 14 August 2021
cate a counter-selection to favour further tumour progression.
Epithelial ovarian cancer
More recently, the AMH-RII expression has been found in human
epithelial ovarian cancer (Masiakos et al., 1999). Human epithelial
ovarian cancer cells and ascites cells isolated from patients with
ovarian papillary serous cystoadenocancer were processed and
analysed by RT – PCR for the detection of AMH-RII mRNA.
AMH-RII transcripts were detected in cell lines from 10 of the
15 patients. In 89% of the cases, the presence of AMH-RII
mRNA was predictive of the binding to AMH. In the solid
tumours from four patients AMH-RII immunostaining was also
demonstrated.
AMH as circulating tumour marker for GCT
Gustafson et al. (1992) have demonstrated abnormalities of AMH
secretion in relation to ovarian malignancy in stored sera from 18
patients. Elevated serum concentrations of AMH was present in
four women with GCT. Serum AMH levels were normal in six
women who had undergone resection of GCT 2– 45 months
before and who were disease-free. AMH was undetectable in
serum from women with metastatic epithelial ovarian cancer and
in three women with previously resected ovarian carcinoma. Simi-
larly, AMH was undetectable in serum from one woman with
Fallopian tube carcinoma and from a woman with mesonephric
adenocancer. Two women with bilateral gonadoblastoma or
bilateral dysgerminoma AMH did not exhibit increased serum
AMH levels.
Hence, based on these findings it seems that AMH is increased
only in the serum from women with GCT.
One patient with GCT was followed in the clinical course for
about 6 years and the results of hormonal evaluation demonstrated
that serum AMH levels decreased after each of the subsequent
major tumour resections. Furthermore, elevations of AMH pre-
ceded or accompanied recurrences of disease.
A successive study confirmed that AMH is a serum marker
limited to GCT. Rey et al., 1996 showed normal AMH levels in
93.3% of patients with non-gynaecologic cancer or gynaecologic
cancer other than GCT. In eight of nine patients with progressive
GCT, AMH levels were above the normal range, and in one patient
with clinical remission of the disease, AMH was below the detec-
tion limit.
In their longitudinal study of 11 patients with recurrent GCT
(Rey et al., 1996), AMH showed a good correlation with evolution

Table I. Sensitivity of AMH measurement as marker of GCT
Reference N Type of AMH assay
Gustafson (1992) 4 Traditional
Rey (1996) 9 Traditional
Lane (1999) 8 (JGCT) Traditional
9 (AGCT)
Long (2000) 31 New ultrasensitive
JGCT, Juvenile granulosa-cell tumor.
AGCT, Adult granulosa-cell tumor.
of the disease during follow-up in nine cases. Serum AMH fell to
undetectable values after successful treatment or rose early in
cases of recurrence of the disease. These findings indicate that
AMH can be used to evaluate the efficacy of the treatment. Inter-
estingly, comparing AMH and alpha-inhibin, it has been shown
that serum alpha-inhibin fluctuated between normal and high
values more frequently than did AMH. AMH has also been
measured intraoperatively in cystic fluid from two children with
juvenile GCT, showing elevated concentrations in comparison
with circulating ones, thus indicating the local production (Rey
et al., 1996).
Globally, AMH serum levels seem to be elevated in 76% (Lane
et al., 1999) to 93% (Long et al., 2000) of GCT patients (Table I).
The mean AMH level was 190.3 ng ml21 (range 2 – 1124 ng ml21)
in patients with GCT in one study (Lane et al., 1999). In patients in
whom serial measurements were made following initial tumour
resection, the length of time that an elevation in AMH levels
preceded clinical detection of a recurrence was 3 months (Lane
et al., 1999).
An ultrasensitive ELISA assay for AMH was developed some
years ago (Long et al., 2000). The new ultrasensitive ELISA
assay is a sandwich-type assay where sensitivity is as low as
0.1 ng ml21 (in comparison with 2 ng ml21 of the traditional
assay used in the studies published before the year 2000). Using
the new assay, Long et al. (2000) found that sensitivity, specificity,
positive predictive value (PPV) and negative predictive value
(NPV) for the presence of GCT were 93, 96, 93, 96%, respectively.
The relevance of using an ultrasensitive assay is demonstrated
by the fact that in 6 of 31 patients the new and the traditional
assays gave discordant results. Indeed in these patients, AMH
was undetectable using the traditional assay but readily detectable
with the ultrasensitive AMH ELISA (Long et al., 2000).
AMH as a chemotherapeutic agent for epithelial
ovarian cancer
About two-thirds of the new cases of epithelial ovarian cancer
diagnosed every year are at an advanced stage (Greenlee et al.,
2001). Once the tumour has spread beyond the confines of the
ovary, surgery alone is unlikely to provide adequate therapy.
After aggressive debulking surgery, primary chemotherapy with
curative intent with platinum and taxanes is the standard of care.
Carboplatin and cisplatin are equally effective, but carboplatin
has less toxicity and is the most common platinum compound
used as first line in combination with paclitaxel.
In patients with advanced epithelial ovarian disease, the overall
response to primary platinum-based therapy is 70 – 80%, with
one-half of these patients achieving complete clinical response
AMH and ovarian cancer
Sensitivity (%) Specificity (%) PPV (%) NPV (%)
100
88.8
75
78
93 96 93 96
and one-quarter reaching complete pathological response.
One-half of those patients with complete pathological response
Downloaded from https://academic.oup.com/humupd/article/13/3/265/2457878 by guest on 14 August 2021
will relapse. It is expected that 70 – 80% of all patients with
advanced ovarian cancer will need second-line therapy (Kalil
and McGuire, 2002). The object of second-line therapy is to
reduce symptoms, minimize toxicity and prolong the progression-
free interval, with palliative intent.
AMH may also be applicable as a chemotherapeutic agent for
epithelial ovarian cancer. AMH induces regression of the Muller-
ian ducts, which arise from a common embryological precursor to
the surface epithelium of the ovary, from which epithelial ovarian
tumours originate (La Marca et al., 2006b). The inhibitory effect
of the hormone is believed to be consequent to its binding to the
AMH-RII. Indeed, expression of AMH-RII has been demonstrated
at the transcriptional and translational level in a series of human
ovarian cancer cell lines (Masiakos et al., 1999).
Masiakos et al. (1999) demonstrated that the growth of five of
six epithelial ovarian carcinoma cell lines expressing AMH-RII
was inhibited by exogenous recombinant AMH. Moreover, 56%
of primary ascites cells seem to bind AMH (Masiakos et al.,
1999). Using the colony inhibition assay, it has been demonstrated
that AMH binding to its receptor is followed by inhibition of the
cell growth (Chin et al., 1991; Masiakos et al., 1999; Ha et al.,
2000; Stephen et al., 2001).
The growth inhibition correlates with a block in cell cycle
progression and is characterized by apoptosis. These events are
mediated by induction of the cyclin-dependent kinase inhibitor
p16, which results in the up-regulation of the E2F family of
transcription factors that are regulated by members of the pocket
protein family (Ha et al., 2000).
In a recent study human ovarian cancer cells were implanted in
immunosuppressed mice and treated with recombinant AMH
(Stephen et al., 2002). Two to three weeks of AMH treatment
was followed by a significant reduction in the graft size ratio
indicating that AMH delivered parenterally may cause inhibition
of human cancer cell lines in vivo (Figure 5).
Conclusions
Ovarian cancer is one of the most frequent gynaecological malig-
nancies. Three major types are recognized: epithelial, stromal and
germ cell. Epithelial ovarian tumours are the most common human
ovarian carcinomas accounting for 82% of all the human ovarian
cancers. The majority of these cancers present at III or IV stage,
hence the 5-year mortality rate for the disease is about 70% (Can-
nistra, 1993).
GCT constitutes 3% of all ovarian tumours. GCT is divided in to
the adult and the juvenile form. Of women with adult GCT, 90%
269
A. La Marca and A. Volpe

Downloaded from https://academic.oup.com/humupd/article/13/3/265/2457878 by guest on 14 August 2021

Figure 5. Effects of AMH on ovarian cancer. OVCAR 8 is a human ovarian cancer cell line, which expresses the AMH-RII. OVCAR 8 tumours were implanted
under the renal capsules of nude mice after 3 weeks. (A) Control tumour shows neovascularization and minimal necrosis or inflammation. (magnification: 40). (B)
AMH-treated tumour (10-mg day213 weeks delivered i.p.) is about one-third the size of the control tumour and has minimal necrosis or inflammation
(magnification:  40) (Reprinted from “Highly purified Müllerian inhibiting substance inhibits human ovarian cancer in vivo” by Stephen et al. Clinical Cancer
Research 2002, 8: 2640– 2646, with permission from American Association for Cancer Research).

present with stage I and the survival rate at 5 years remains very
high (Bjorkholm and Silfverswaard, 1981). The recurrence for
adult GCT is late occurring: 4 – 6 years after the first treatment.
In contrast, a large subset experience recurrence of juvenile
GCT occurs within one year, with a mean of 6 months (Calaminus
et al., 1997).
In recent years, the advances in the physiological role of AMH
has stimulated interest in the significance of AMH as diagnostic
marker and therapeutic agent for ovarian cancer. At this
moment, AMH has only been shown to be a circulating marker
for GCT. Its diagnostic performance seems to be very good,
with a sensitivity ranging between 76 and 93%. AMH seems to
be superior to alpha-inhibin and estradiol in the early diagnosis
and in the follow-up of GCT.
It is largely recognized that AMH and alpha-inhibin
exhibit a higher degree of sensitivity than estradiol in pro-
gressive GCT. Indeed, estradiol production by GCT is
widely variable (Lappohn et al., 1989) while AMH seems to
be a marker of comparable value to alpha-inhibin. However,
AMH is elevated only in sex cord stromal tumours, while

270

inhibin may be elevated in different types of cancer (Healy
et al., 1993).
Doubts remain on the possible role of AMH in the pathogenesis
of GCT. Indeed, AMH is considered a powerful inhibitor of
granulosa cell proliferation. Hence, the high serum AMH levels
found in patients with GCT, are in contrast with the progressive
characteristic of the disease. One possible explanation is that the
binding of AMH-RII by its ligand in tumour cells is not followed
by the inhibitory signal. Otherwise from animal studies it has been
hypothesized that the inhibitory signal is functional when the
tumours are limited (Dutertre et al., 2001). Successively, when
the tumours become progressive and reach a large dimension,
they lose AMH and AMH-RII expression. This could be a
counter selection to favour further tumour progression (Dutertre
et al., 2001).
In patients treated for GCT, AMH may be followed post-
operatively as a marker for efficacy of surgery and disease recur-
rence. Following surgery, AMH decreases to normal values within
days to weeks. The AMH half-life is estimated to be 48 h (Vigier
et al., 1983). Hence, reduced levels of AMH should be detected
72 h after successful surgery. Indeed, bilateral ovariectomy per-
formed for benign disease is followed by undetectable AMH
levels 3 – 5 days after surgery (La Marca et al., 2005).
In the past, anti-AMH antibodies were not commercially avail-
able. Only few laboratories in the world performed the assay and
this has precluded the diffusion of AMH as a marker for GCT. The
recent availability of ultrasensitive commercial ELISA will permit
its clinical application (Rey et al., 1999; Long et al., 2000). This
ELISA, which is valid both for males and females, is a sandwich-
type assay where sensitivity is as low as 0.1 ng ml21 (in compari-
son with 2 ng ml21 for the traditional assay used in the studies
published before 2000). More recently, a new double-antibody
ELISA for AMH has been developed, with a detection limit of
,0.078 ng ml21 (Al-Qahtani et al., 2005).
The specificity of the commercially available ultrasensitive
assay was tested by assaying samples containing known concen-
trations of most of the AMH-related members of a large family
of growth and differentiation factors: TGF-b, inhibin A and B,
inhibin pro-aC subunit and activin A. No cross reactivity was
observed in any of the cases (Long et al., 2000).
Based on the published studies it is possible to elaborate the fol-
lowing recommendations for the use of AMH as a marker of GCT:
(i) High serum AMH levels are found in 76 – 93% of patients
with GCT.
(ii) Serum AMH levels should normalize within few days fol-
lowing surgery.
(iii) Persistent AMH levels are indicative of residual disease.
(iv) When bilateral ovariectomy is performed AMH must be
undetectable in serum. Even very low levels may be sug-
gestive of residual disease.
(v) A post-operative rise in serum AMH levels may precede
the clinical recurrence. Hence, the clinical examination
and imaging should be anticipated.
(vi) AMH levels should be obtained every 6 months for at least
5 years after the surgery. However, it should not be
ignored that a late recurrence (up to 30 years after initial
treatment) has been reported in up to 33% of patients
(Stenwig et al., 1979).
AMH and ovarian cancer
(vii) Recurrences in juvenile GCTs occur within a year of
initial diagnosis at a mean of 5 – 6 months. Hence, in
juvenile GCT, AMH serum levels should be determined
every month following the surgery.
At this moment, data on the role of AMH measurement in moni-
toring the response to chemotherapy is lacking.
These recommendations are based on a limited number of
studies. Hence, they should be validated in clinical trials.
Several authors have also hypothesized a role for recombinant
AMH in the treatment of epithelial ovarian cancer (Stephen
et al., 2001, 2002). Based on the physiological inhibiting role of
AMH on the mullerian ducts, researchers have demonstrated
that AMH inhibits epithelial ovarian cancer cell in vitro (Fuller
Downloaded from https://academic.oup.com/humupd/article/13/3/265/2457878 by guest on 14 August 2021
et al., 1982, 1985; Stephen et al., 2002; Masiakos et al., 1999).
In a small series, it has been shown that 56% of ovarian cancer
ascite cells express the receptor for AMH, and when tested in
solid tumours from patients with epithelial ovarian cancer, in
each case AMH-RII was expressed (Masiakos et al., 1999). The
administration of recombinant AMH in immunosuppressed mice
with human epithelial ovarian cancer implants has been followed
by reduced dimensions of the graft (Stephen et al., 2002).
This of course will be the basis for future research aiming to
investigate the possible clinical role of AMH as neo-adjuvant or
most probably adjuvant therapy for ovarian cancer.
References
Al-Qahtani A, Muttukrishna S, Appasamy M, Johns J, Cranfield M, Visser JA,
Themmen AP and Groome NP (2005) Development of a sensitive enzyme
immunoassay for anti-Müllerian hormone and the evaluation of potential
clinical applications in males and females. Clin Endocrinol 63,267–273.
Bilezikjian LM, Blount AL, Leal AM, Donaldson CJ, Fischer WH and Vale
WW (2004) Autocrine/paracrine regulation of pituitary function by
activin, inhibin and follistatin. Mol Cell Endocrinol. 225,29–36.
Biscotti CV and Hart WR (1989) Juvenile granulosa cell tumors of the ovary.
Arch Pathol Lab Med 113,40– 46.
Bjorkholm E and Silfversward C (1981) Prognostic factors in granulosa-cell
tumors. Gynecol Oncol 11,261–274.
Calaminus G, Wessalowski R, Harms D and Gobel U (1997) Juvenile granulosa
cell tumors of the ovary in children and adolescents: results from 33 patients
registered in a prospective cooperative study. Gynecol Oncol 65,447–452.
Cannistra SA (1993) Cancer of the ovary. N Engl J Med 329,1550– 1559.
Chin TW, Parry RL and Donahoe PK (1991) Human mullerian inhibiting
substance inhibits tumor growth in vitro and in vivo. Cancer Res
51,2101–2106.
de Vet A, Loven JS, de Jong FH, Themmen AP and Fauser BC (2002)
Anti-Mullerian hormone serum levels: a putative marker for ovarian
aging. Fertil Steril 77,357–362.
Donahoe PK, Fuller AF Jr, Scully RE, Guy SR and Budzik GP (1981)
Mullerian inhibiting substance inhibits growth of a human ovarian
cancer in nude mice. Ann Surg,194,472– 480.
Durlinger AL, Kramer P, Karels B, de Jong FH, Uilenbroek JT, Grootegoed JA
and Themmen AP (1999) Control of primordial follicle recruitment by
anti-Mullerian hormone in the mouse ovary. Endocrinology 140,5789–
5796.
Durlinger AL, Gruijters MJ, Kramer P, Karels B, Kumar TR, Matzuk MM,
Rose UM, de Jong FH, Uilenbroek JT and Grootegoed JA et al. (2001)
Anti-Mullerian hormone attenuates the effects of FSH on follicle
development in the mouse ovary. Endocrinology, 142,4891–4899.
Durlinger AL, Visser JA and Themmen AP (2002) Regulation of ovarian
function: the role of anti-Mullerian hormone. Reproduction 124,601–609.
Dutertre M, Rey R, Porteu A, Josso N and Picard JY (1997) A mouse Sertoli
cell line expressing anti-Mullerian hormone and its type II receptor.
Mol Cell Endocrinol 136,57–65.
Dutertre M, Gouedard L, Xavier F, Long WQ, di Clemente N, Picard JY
and Rey R (2001) Ovarian granulosa cell tumors express a functional
membrane receptor for anti-Mullerian hormone in transgenic mice.
Endocrinology 142,4040– 4046.
271
A. La Marca and A. Volpe
Fuller AF, Guy S, Budzik GP and Donahoe PK (1982) Mullerian inhibiting
substance inhibits colony growth of a human ovarian carcinoma cell
line. J Clin Endocrinol Metab 54,1051 –1055.
Fuller AF Jr, Krane IM, Budzik GP and Donahoe PK (1985) Mullerian
inhibiting substance reduction of colony growth of human gynecologic
cancers in a stem cell assay. Gynecol Oncol. 22,135– 148.
Greenlee RT, Hill-Harmon MB, Murray T and Thun M (2001) Cancer
statistics. Cancer J Clin 51,144–154.
Gruijters MJ, Visser JA, Durlinger AL and Themmen AP (2003)
Anti-Mullerian hormone and its role in ovarian function. Mol Cell
Endocrinol 211,85–90.
Gustafson ML, Lee MM, Scully RE, Moncure AC, Hirakawa T, Goodman A,
Muntz HG, Donahoe PK, MacLaughlin DT and Fuller AF (1992)
Mullerian inhibiting substance as a marker for ovarian sex-cord tumor.
N Engl J Med 326,466 –471.
Ha TU, Segev DL, Barbie D, Masiakos PT, Tran TT, Dombkowski D,
Glander M, Clarke TR, Lorenzo HK and Donahoe PK et al. (2000)
Mullerian inhibiting substance inhibits ovarian cell growth through an
Rb-independent mechanism. J Biol Chem 275,37101– 37109.
Healy DL, Burger HG, Mamers P, Jobling T, Bangah M Quinn M, Grant P,
Day AJ, Rome R and Campbell JJ (1993) Elevated serum inhibin
concentrations in postmenopausal women with ovarian tumors. N
Engl J Med 329,1539–1542.
Hudson PL, Dougas I, Donahoe PK, Cate RL, Epstein J, Pepinsky RB
and MacLaughlin DT (1990) An immunoassay to detect human
mullerian inhibiting substance in males and females during normal
development. J Clin Endocrinol Metab 70,16– 22.
Ikeda Y, Nagai A, Ikeda MA and Hayashi S (2002) Increased expression of
Mullerian-inhibiting substance correlates with inhibition of follicular
growth in the developing ovary of rats treated with E2 benzoate.
Endocrinology 143,304– 312.
Imbeaud S, Carre-Eusebe D, Rey R, Belville C, Josso N and Picard JY (1994)
Molecular genetics of the persistent mullerian duct syndrome: a study of
19 families. Hum Mol Genet 3,125–131.
Ingraham HA, Hirokawa Y, Roberts LM, Mellon SH, McGee E, Nachtigal MW
and Visser JA (2000) Autocrine and paracrine Mullerian inhibiting
substance hormone signaling in reproduction. Recent Prog Horm Res
55,53–67.
Josso N, Legeai L, Forest MG, Chaussain JL and Brauner R (1990) An enzyme
linked immunoassay for anti-mullerian hormone: a new tool for the
evaluation of testicular function in infants and children. J Clin
Endocrinol Metab 70,23– 27.
Josso N, Racine C, di Clemente N, Rey R and Xavier F (1998) The role of
anti-Mullerian hormone in gonadal development. Mol Cell Endocrinol
145,3–7.
Kalil NG and McGuire WP (2002) Chemotherapy for advanced epithelial
ovarian carcinoma. Best Pract Res Clin Obstet Gynaecol 16,553–571.
Kim JH, Seibel MM, MacLaughlin DT, Donahoe PK, Ransil BJ, Hametz PA
and Richards CJ (1992) The inhibitory effects of Müllerian-inhibiting
substance on epidermal growth factor induced proliferation and
progesterone production of human granulosa-luteal cells. J Clin
Endocrinol Metab 75,911–917.
LaMarca A, De Leo V, Giulini S, Orvieto R, Malmusi S, Giannella L and Volpe
A (2005) Anti-Mullerian hormone in premenopausal women and after
spontaneous or surgically induced menopause. J Soc Gynecol Invest
12,545– 548.
La Marca A and Volpe A (2006b) Anti-Mullerian hormone (AMH) in female
reproduction: is measurement of circulating AMH a useful tool? Clin
Endocrinol (Oxf) 64,603– 610.
La Marca A, Stabile G, Artenisio AC and Volpe A (2006a) Serum
anti-Mullerian hormone throughout the human menstrual cycle. Hum
Reprod 21,3103–3107.
Lane AH Jr, Lee MM, Fuller AF, Kehas DJ, Donahoe PK and MacLaughlin DT
(1999) Diagnostic utility of Mullerian inhibiting substance determination
in patients with primary and recurrent granulosa cell tumors. Gynecol
Oncol 21,3103– 3107.
Lappohn RE, Burger HG, Bouma J, Bangah M, Krans M and de Bruijn HW
(1989) Inhibin as a marker for granulosa-cell tumors. N Engl J Med
321,790–793.
Lee MM and Donahoe PK (1993) Mullerian inhibiting substance: a gonadal
hormone with multiple functions. Endocr Rev 14,152–164.
Lee MM, Donahoe PK, Hasegawa T, Silverman B, Crist GB, Best S,
Hasegawa Y, Noto RA, Schoenfeld D and MacLaughlin DT (1996)
Mullerian inhibiting substance in humans: normal levels from infancy
to adulthood. J Clin Endocrinol Metab 81,571– 576.
272
Long WQ, Ranchin V, Pautier P, Belville C, Denizot P, Cailla H, Lhomme C,
Picard JY, Bidart JM and Rey R (2000) Detection of minimal levels of
serum anti-Mullerian hormone during follow-up of patients with ovarian
granulosa cell tumor by means of a highly sensitive enzyme-linked
immunosorbent assay. J Clin Endocrinol Metab 85,540–544.
Masiakos PT, MacLaughlin DT, Maheswaran S, Teixeira J, Fuller AF Jr,
Shah PC, Kehas DJ, Kenneally MK, Dombkowski DM and Ha TU
et al. (1999) Human ovarian cancer, cell lines, and primary ascites
cells express the human Mullerian inhibiting substance (MIS) type II
receptor, bind, and are responsive to MIS. Clin Cancer Res 5,3488–
3499.
Matias-Guiu X, Pons C and Prat J (1998) Mullerian inhibiting substance,
alpha-inhibin, and CD99 expression in sex cord-stromal tumors and
endometrioid ovarian carcinomas resembling sex cord-stromal tumors.
Hum Pathol 29,840– 845.
Munsterberg A and Lovell-Badge R (1991) Expression of the mouse
Downloaded from https://academic.oup.com/humupd/article/13/3/265/2457878 by guest on 14 August 2021
anti-mullerian hormone gene suggests a role in both male and female
sexual differentiation. Development 113,613–624.
Nakashima N, Young RH and Scully RE (1984) Androgenic granulosa cell
tumors of the ovary. A clinicopathologic analysis of 17 cases and
review of the literature. Arch Pathol Lab Med 108,786–791.
Pankratz E, Boyes DA, White GW, Galliford BW, Fairey RN and Benedet JL
(1978) Granulosa cell tumors. A clinical review of 61 cases. Obstet
Gynecol 52,718–723.
Peschon JJ, Behringer RR, Cate RL, Harwood KA, Idzerda RL, Brinster RL
and Palmiter RD (1992) Directed expression of an oncogene to Sertoli
cells in transgenic mice using mullerian inhibiting substance regulatory
sequences. Mol Endocrinol 6,1403–1411.
Rajpert-De Meyts E, Jorgensen N, Graem N, Muller J, Cate RL
and Skakkebaek NE (1999) Expression of anti-Mullerian hormone
during normal and pathological gonadal development: association with
differentiation of Sertoli and granulosa cells. J Clin Endocrinol Metab
84,3836– 3844.
Rey RA, Lhomme C, Marcillac I, Lahlou N, Duvillard P, Josso N and Bidart
JM (1996) Anti-mullerian hormone as a serum marker of granulosa cell
tumorsof the ovary: comparative study with serum alpha-inhibin and
estradiol. Am J Obstet Gynecol 174,958–965.
Rey RA, Belville C, Nihoul-Fekete C, Michel-Calemard L, Forest M.G, Lahlou
N, Jaubert F, Mowszowicz I, David M and Saka N et al. (1999) Evaluation of
gonadal function in 107 intersex patients by means of serum antimüllerian
hormone measurement. J Clin Endocrinol Metab 84,627–631.
Rey R, Sabourin JC, Venara M, Long WQ, Jaubert F, Zeller WP, Duvillard P,
Chemes H and Bidart JM (2000) Anti-Mullerian hormone is a specific
marker of sertoli- and granulosa-cell origin in gonadal tumors. Hum
Pathol 31,1202 –1208.
Robertson DM, Cahir N, Burger HG, Mamers P and Groome N (1999a) Inhibin
forms in serum from postmenopausal women with ovarian cancers. Clin
Endocrinol (Oxf) 50,381–386.
Robertson DM, Cahir N, Burger HG, Mamers P, McCloud PI, Pettersson K
and McGuckin M (1999b) Combined inhibin and CA125 assays in the
detection of ovarian cancer. Clin Chem. 45,651–658.
Salerno LJ (1962) Feminizing mesenchymomas of the ovary. An analysis of 28
granulosa-theca cell tumors and their relationship to coexistent carcinoma.
Am J Obstet Gynecol 84,731–738.
Salmon NA, Handyside AH and Joyce IM (2004) Oocyte regulation of
anti-Mullerian hormone expression in granulosa cells during ovarian
follicle development in mice. Dev Biol 266,201– 208.
Seifer DB, MacLaughlin DT, Penzias AS, Behrman HR, Asmundson L,
Donahoe PK, Haning RVJ and Flynn SD (1993) Gonadotropin-releasing
hormone agonist-induced differences in granulosa cell cycle kinetics are
associated with alterations in follicular fluid müllerian-inhibiting
substance and androgen content. J Clin Endocrinol Metab 76,711–714.
Skinner MK (2005) Regulation of primordial follicle assembly and
development. Hum Reprod Update 11,461– 471.
Stenwig JT, Hazekamp JT and Beecham JB (1979) Granulosa cell tumors of the
ovary. A clinicopathological study of 118 cases with long-term follow-up.
Gynecol Oncol 7,136– 152.
Stephen AE, Masiakos PT, Segev DL, Vacanti JP, Donahoe PK
and MacLaughlin DT (2001) Tissue-engineered cells producing
complex recombinant proteins inhibit ovarian cancer in vivo. Proc Natl
Acad Sci USA 98,3214–3219.
Stephen AE, Pearsall LA, Christian BP, Donahoe PK, Vacanti JP
and MacLaughlin DT (2002) Highly purified mullerian inhibiting
substance inhibits human ovarian cancer in vivo. Clin Cancer Res.
8,2640–2646.

Themmen AP (2005) Anti-Mullerian hormone: its role in follicular growth
initiation and survival and as an ovarian reserve marker. J Natl Cancer
Inst Monogr 34,18–21.
Tsafriri A, Picard JY and Josso N (1988) Immunopurified anti-Müllerian
hormone does not inhibit spontaneous resumption of meiosis in vitro of
rat oocytes. Biol Reprod 38,481– 485.
Ueno S, Kuroda T, Maclaughlin DT, Ragin RC, Manganaro TF and
Donahoe PK (1989) Mullerian inhibiting substance in the adult rat
ovary during various stages of the estrous cycle. Endocrinology
125,1060– 1066.
AMH and ovarian cancer
Vigier B, Tran D, du Mesnil du Buisson F, Heyman Y and Josso N (1983) Use
of monoclonal antibody techniques to study the ontogeny of bovine
anti-Mullerian hormone. J Reprod Fertil 69,207–214.
Weenen C, Laven JS, Von Bergh AR, Cranfield M, Groome NP, Visser JA,
Kramer P, Fauser BC and Themmen AP (2004) Anti-Mullerian
hormone expression pattern in the human ovary: potential implications
for initial and cyclic follicle recruitment. Mol Hum Reprod 10,77– 83.
Submitted on July 10, 2006; resubmitted on November 3, 2006; accepted on
November 29, 2006
Downloaded from https://academic.oup.com/humupd/article/13/3/265/2457878 by guest on 14 August 2021
273