European Journal of Obstetrics & Gynecology and Reproductive Biology 167 (2013) 194198

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European Journal of Obstetrics & Gynecology and

Reproductive Biology
journal homepage: www.elsevier.com/locate/ejogrb

Anti-Mullerian hormone as a marker of ovarian reserve following chemotherapy

in patients with gestational trophoblastic neoplasia
Akira Iwase a,b,*, Atsuko Sugita a,b, Wakana Hirokawa a, Maki Goto a,b, Eiko Yamamoto a,
Sachiko Takikawa a, Tatsuo Nakahara a, Tomoko Nakamura a, Mika Kondo a,b, Fumitaka Kikkawa a
a
b

Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
Department of Maternal and Perinatal Medicine, Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 2 August 2012
Received in revised form 5 November 2012
Accepted 30 November 2012

Objective: The loss of primordial follicles from gonadal damage caused by chemotherapy results in
decreased ovarian reserve. To assess the impact of chemotherapy for patients with gestational
trophoblastic neoplasia (GTN) on the ovarian reserve, we evaluated the post-chemotherapy serum antiMullerian hormone (AMH) levels.
Study design: In 22 patients with GTN receiving chemotherapy, serum AMH levels were measured after
the administration of chemotherapy and compared with serum AMH levels measured in patients with
hydatidiform mole who did not receive chemotherapy, as a control. We also analyzed differences in the
serum AMH levels following the administration of different anti-cancer agents.
Results: The serum AMH levels measured in the GTN group after chemotherapy was administered
(median 1.18, range 0.323.94 ng/mL) signicantly decreased in comparison to those measured in the
control group (median 4.22, range 0.776.53 ng/mL, P = 0.002). Serum AMH levels were signicantly
lower in the patients who had received a regimen including etoposide than in the patients who had not
received treatment with etoposide (0.71 vs. 1.30 ng/mL, P = 0.027).
Conclusion: Our results suggest that chemotherapy administered to treat GTN does indeed affect the
ovarian reserve, especially in patients who receive a medication regimen that includes etoposide.
Measuring their serum AMH levels might therefore be helpful for counseling GTN patients regarding
their ovarian reserve.
2012 Elsevier Ireland Ltd. All rights reserved.

Keywords:
Anti-Mullerian hormone
Chemotherapy
Gestational trophoblastic neoplasia
Ovarian reserve

1. Introduction
Improvements in the survival rates over the last decades have
made maintaining the reproductive potential after treatment for
malignant diseases an important quality of life issue [1]. The
depletion of oocytes as a result of chemotherapy is not reversible
and is considered to be the most critical side effect of
chemotherapy related to fertility preservation. Recently, the
effects of chemotherapy on reproductive capacity, and especially
on ovarian function, have become more apparent because the
number of patients surviving hematologic malignancies and breast
cancer has increased [2].
Gestational trophoblastic neoplasia (GTN), which originates in
the products of conception, may threaten the life and health of the
patient if not properly treated. The majority of patients with GTN

* Corresponding author at: Department of Obstetrics and Gynecology, Nagoya

University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 4668550, Japan. Tel.: +81 52 744 2261; fax: +81 52 744 2268.
E-mail address: [email protected] (A. Iwase).
0301-2115/$ see front matter 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ejogrb.2012.11.021

are successfully treated with chemotherapy using a single anticancer drug. Therefore, in patients with low-risk GTN, reproductive
capacity is not considered to be signicantly affected by
chemotherapy. On the other hand, patients with high-risk GTN,
who are at greater risk of rapid disease development, require
treatment with multi-agent chemotherapy, which might affect the
post-treatment ovarian function [3,4].
Anti-Mullerian hormone (AMH), a member of the transforming
growth factor-b superfamily, is produced by the granulosa cells of
preantral and early antral follicles [5]. The serum AMH concentration has been established as a novel and reliable marker of the
ovarian reserve [6], and it also closely correlates with both the antral
follicle count obtained by ultrasonography and the number of
oocytes retrieved during in vitro fertilization treatment. Moreover,
recent studies have suggested that AMH could be a valuable marker
of follicle depletion in childhood cancer survivors and women who
have undergone treatment for breast cancer [79].
In the current study, the serum AMH concentrations were
measured in order to assess the ovarian reserve levels in patients
with GTN previously treated with chemotherapy. These levels
were compared with those measured in patients who underwent

A. Iwase et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 167 (2013) 194198

curettage to treat hydatidiform moles and who did not receive

chemotherapy.

195

After the completion of the chemotherapy, all patients received

follow-up visits at certain intervals.
2.3. Hormonal measurements

2. Materials and methods

Patients who underwent chemotherapy to treat GTN at Nagoya

University Hospital from January 2007 to December 2011 were
recruited for this study. The inclusion criteria were as follows: (1)
patients 45 years of age and younger and (2) patients with no
evidence of any other endocrine disorders, including thyroid
dysfunction, hyperprolactinemia, or Cushings syndrome. The
exclusion criteria were as follows: patients currently receiving
hormonal treatment, including oral contraceptives, and patients
having undergone previous ovarian surgery. Patients who previously underwent dilatation and curettage to treat hydatidiform
mole were included as age-matched controls. This study was
approved by the ethics committee of Nagoya University Graduate
School of Medicine, and informed consent was obtained from all
patients.

Blood assays for bhCG were routinely performed during each

outpatient visit. Blood samples for assaying AMH and folliclestimulating hormone (FSH) were obtained at one point on 2nd7th
day of the menstrual cycle during the follow-up from 1 to 42
months after chemotherapy. For each sample, the serum was
separated from the whole blood, transferred to a sterile
polypropylene tube, and stored at 80 8C until assayed. The
serum AMH concentrations were measured using an enzyme
immunoassay kit according to the manufacturers instructions (EIA
AMH/MIS, IMMUNOTECH, Marseille, France). For the AMH assay,
the intra-assay and inter-assay coefcients of variation were below
12.3% and 14.2%, respectively. The serum FSH concentrations were
measured using an ELISA kit according to the manufacturers
instructions (Elegance FSH ELISA Kit; Bioclone, Marrickville,
Australia). For the FSH assay, the intra-assay and inter-assay
coefcients of variation were 3.4% and 4.2% at 6.920.3 IU/L, and
6.7% and 6.6% at 7.636.7 IU/L, respectively.

2.2. Diagnosis and chemotherapy

2.4. Statistical analysis

A detailed physical examination, transvaginal ultrasonography,

magnetic resonance imaging, radiologic investigation, including
chest X-ray and computed tomography, and measurement of
serum human chorionic gonadotropin b (bhCG) concentration
were performed on each patient to conrm the diagnosis of GTN.
All of the patients were scored according to the International
Federation of Gynecology and Obstetrics (FIGO) staging system
and classied into low-risk and high-risk GTN groups [10]. The
chemotherapy regimens used in this study were determined in
accordance with the FIGO staging. Methotrexate (MTX) and/or
actinomycin-D (ACD) were administered to the patients in the lowrisk GTN group. A regimen that included etoposide (ETP) was used
to treat both the patients in the high-risk GTN group and patients
in the low-risk group who showed resistance to MTX and ACD.

Data were analyzed using the SigmaPlot 11 software program

(Systat Software Inc., San Jose, CA, USA). We used either the Mann
Whitney U-test (between 2 groups) or a KruskalWallis one-way
analysis of variance on ranks (among 3 groups) to compare the
patient characteristics, variables, serum FSH levels, and serum
AMH levels, since the variables did not pass the normality test. A Pvalue of <0.05 was considered to be statistically signicant.

2.1. Patients

3. Results
Twenty-two women of mean age 35.1 (range 2042) years were
recruited. Table 1 presents the patients clinical characteristics,
including the time from last treatment and serum AMH level for

Table 1
The clinical and hormonal characteristics of the patients with gestational trophoblastic neoplasia after the administration of chemotherapy.
Patients

Age at
evaluation (y)

Time from
last treatment (m)

Stage:
score

Low/
high-risk

Regimen

MTX
(mg)

ACD
(mg)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

31
20
37
20
44
34
42
39
40
29
39
34
37
42
39
32
26
39
40
39
33
37

5
8
12
12
15
24
24
30
30
30
30
36
36
36
36
36
42
1
1
6
24
36

I: 2
I: 2
III: 3
I: 2
I: 5
III: 3
I: 3
III: 4
I: 4
III: 2
III: 4
III: 3
I: 2
III: 2
III: 3
III: 3
I: 1
I: 1
III: 3
IV: 16
I
IV: 9

Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
High
Low
High

MA  6, ACD  1
MTX  2, ACD  3, EA  6
MA  3, ACD  6
MA  6
MA  6, EA  8
MA  4
MA  7
MA  5, ACD  3
MA  4
MA  3, ACD  3
MA  4, ACD  2
MA  3, ACD  3
MA  2, ACD  1, EA  2
MA  3, ACD  3
MA  3, ACD  3
ACD  6, MA  2
ACD  3, MA  2
MA  3
MA  2
MEA  16
MEA  3
EMA/CO  9

80
160
240
480
480
320
560
400
320
240
320
240
160
240
240
160
240
240
160
7200
1350
1350

12
18
18
12
28
8
14
16
14
12
12
12
10
12
12
16
12
6
4
32
4.8
9

ETP
(mg)

Others

Pregnancy

2400
Yes
3040
Yes
Yes
Yes
Yes

Yes
800

5120
960
2700

AMH
(ng/mL)
0.32
0.89
2.41
1.98
0.52
1.24
1.79
1.77
0.40
1.18
1.12
1.79
0.32
1.36
0.62
3.51
3.94
0.60
1.24
0.32
1.16
1.18

CPA 8100 mg
VCR 13.5 mg
Note: MTX: methotrexate; ACD: actinomycin D; ETP: etoposide; CPA: cyclophosphamide; VCR: vincristine; MA: MTX + ACD; EA: ETP + ACD; MEA: MTX + ETP + ACD; EMA/CO:
ETP + MTX + ACD/CPA + VCR.

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A. Iwase et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 167 (2013) 194198

(with chemotherapy) were 5.76 (1.9210.58) and 5.24 (1.339.45)

IU/L, respectively, and no signicant differences were noted
(P = 0.539; Fig. 1A). In contrast, the serum AMH levels in the
GTN group measured after the administration of chemotherapy
(median 1.18, range 0.323.94 ng/mL) were found to be signicantly decreased in comparison to those measured in the control
(median 4.22, range 0.776.53 ng/mL, P = 0.002; Fig. 1B).
We then analyzed whether the type of chemotherapy regimen
administered affected the post-chemotherapy AMH levels differently. There was a signicant difference in the serum AMH levels
among the control, ETP and ETP+ groups (P = 0.001). We also
found a signicant decrease in the serum AMH levels in the ETP
group, compared with the control group (1.30 vs. 4.22 ng/mL,
P = 0.006). The serum AMH levels in the patients who were
administered ETP signicantly decreased compared to those in
patients who were not administered ETP (0.71 vs. 1.30 ng/mL,
P = 0.027), but no signicant differences in age, time from last
treatment, dose of MTX, or dose of ACD were observed (Table 2).
We also analyzed the correlation between the serum AMH
levels and cumulative doses of MTX or ACD. The post-chemotherapy serum AMH levels in the GTN patients did not show a
signicant correlation with the cumulative doses of MTX
(r = 0.243, P = 0.275; Fig. 2A), or ACD (r = 0.083, P = 0.712;
Fig. 2B).
4. Comments

Fig. 1. The serum levels of (A) FSH and (B) AMH in the patients with hydatidiform
mole who did not receive chemotherapy (w/o CT) and those with GTN who did
receive chemotherapy (CT). The data are represented by box-and-whisker plots. The
solid lines and the dotted lines inside the boxes represent the median and the mean,
respectively. The upper and the lower limits of the boxes and the whiskers indicate
the 75th and 25th, and 90th and 10th percentiles, respectively. The closed circles
indicate the outliers.

each patient. The chemotherapy regimens and doses used in this

study are also detailed in Table 1. The regimens used were: MTX
alone, ACD alone, MA (MTX and ACD), EA (ETP and ACD), MEA
(MTX, ETP and ACD), and EMA/CO (ETP, MTX, ACD, cyclophosphamide and vincristine). Chemotherapy with MTX and/or ACD alone
was administered to sixteen patients, while six patients received
chemotherapy that included ETP. Six patients became pregnant
following the administration of chemotherapy.
We then compared the serum AMH and FSH levels in these
patients measured after the administration of the chemotherapy
with those of the nine age-matched control patients (ranging from
24 to 43 years old) who received treatment for hydatidiform mole
without chemotherapy. The median (range) levels of serum FSH in
the control group (without chemotherapy) and the GTN group

Chemotherapeutics may affect both a patients future ovarian

function and her subsequent fertility. A complete depletion of
oocytes caused by chemotherapy is not reversible even with
todays treatments. Therefore, fertility preservation is considered
important for eligible patients. The current options for fertility
preservation primarily consist of embryo, oocyte, and ovarian
tissue cryopreservation [1]. Moreover, accurate predictive markers
could have clinical applicability in choosing between chemotherapeutic agents and deciding to use gonadotropin releasing
hormone agonists to protect a patients ovaries [11].
In the current study, we analyzed 22 patients who underwent
chemotherapy to treat GTN. In comparison with those measured in
the control patients with hydatidiform mole who did not receive
chemotherapy, the serum AMH levels, but not the basal FSH levels,
were found to be signicantly lower in the patients with GTN who
underwent chemotherapy. To the best of our knowledge, this is the
rst report to use serum AMH levels to demonstrate that ovarian
reserve is decreased following chemotherapy to treat GTN. The
serum AMH level is thus suggested to be a useful predictive marker
to evaluate the effect of GTN treatment. Given the fact that none of
the patients in our study developed permanent ovarian failure,
cryopreservation of embryos, oocytes or ovarian tissue does not
seem necessary for GTN patients. Intervention, however, using
infertility treatments including assisted reproductive technologies
might be needed, especially for relatively older GTN patients,

Table 2
Comparison between patients with and without etoposide administration.

Age at evaluation (y)

Time from last treatment (m)
MTX (mg)
ACD (mg)
ETP (mg)
AMH (ng/mL)

Control (n = 9)

ETP

31.0 (29.5, 38.5)

NA
NA
NA
NA
4.22 (1.92, 5.86)

38.0 (31.3, 39.8)

30.0 (12.0, 36.0)
240 (240, 320)
12 (12, 14)
NA
1.30 (0.75, 1.93)

Note: Median (25th, 75th percentile).

a
KruskalWallis one-way analysis of variance on ranks among 3 groups.
b
MannWhitney U-test between ETP and ETP+.

(n = 16)

ETP+ (n = 6)

37.0 (29.8, 40.3)

19.5 (8.0, 36.0)
915 (160, 2813)
17 (7.95, 29)
2550 (960, 3040)
0.71 (0.32, 1.16)

0.668a
0.736b
0.175b
0.650b
NA
0.001a

A. Iwase et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 167 (2013) 194198

197

patients who subsequently have normal pregnancies is limited to

54% [18]. ETP prevents re-ligation of DNA strands, and thereby
inhibits DNA synthesis. Therefore, the gonadotoxicity of etoposide is
not negligible, similar to the effects of alkylating agents that cause
DNA crosslinks, although the fact that such patients have often
undergone a hysterectomy might be another reason for the limited
number of pregnancies in the high-risk GTN patients.
Although treatment with MTX or ACD did not show any
correlation between doses and post-chemotherapy AMH levels in
this study (data not shown), our results indicate that patients
receiving regimens with MTX and/or ACD have lower AMH levels
than control patients with hydatidiform mole who do not receive
chemotherapy. Recently, Oriol et al. reported that single-dose MTX
treatment for ectopic pregnancy does not compromise ovarian
reserve in terms of serum AMH levels [19]. The dose of MTX used in
that study, however, was much smaller (1 mg/kg of body weight).
In our study, the median dose of MTX in the ETP-negative regimens
was 240 mg/body. Our results suggest that multiple administration of MTX might affect ovarian reserve.
In conclusion, this is the rst study in which AMH was used to
assess the ovarian reserve after chemotherapy for GTN. Our results
suggest that treatment with ETP causes the ovarian reserve to
decrease, and even treatment with MTX and/or ACD affects ovarian
reserve in patients with low-risk GTN. Our results may be helpful
for counseling of GTN patients who are concerned about their
ovarian reserve. Further studies to evaluate pre- and postchemotherapy serum AMH levels in GTN patients would provide
detailed information regarding ovarian reserve.
Fig. 2. Correlation of the post-chemotherapy serum AMH levels with the cumulative
doses of MTX (A) and ACD (B). r is the Pearson correlation coefcient.

considering the signicant decrease in the ovarian reserve after

chemotherapy, especially by regimens including ETP.
A patients basal FSH level has been widely used as an ovarian
reserve marker. Increased FSH levels are, however, present at the
very end of the reproductive life span [12]. Moreover, de Vet et al.
reported that the FSH levels were stable in normo-ovulatory
women, whereas the serum AMH levels decreased during a twoyear follow-up [6]. We recently reported that serum AMH levels
signicantly decrease after cystectomy to treat ovarian endometrioma, while basal FSH levels do not change after this procedure
[13,14]. In the current study, we observed similar results. The basal
FSH levels did not signicantly increase in the women treated with
chemotherapy who showed approximately 70% lower serum AMH
levels compared with the control women without chemotherapy.
Partridge et al. reported that serum AMH levels, but not basal FSH
levels, signicantly decrease in premenopausal women following
the administration of chemotherapy to treat early-stage breast
cancer [9]. Therefore, a patients FSH level is not sufciently
informative to assess the ovarian reserve and damage to ovarian
function caused by invasive treatment.
The type of chemotherapy agent (multi-agent or single-agent),
length of exposure time, and amount of cumulative doses may
inuence the decrease of the ovarian reserve. Alkylating agents, such
as cyclophosphamide and procarbazine, are known to be highly
gonadotoxic. Rosendahl et al. reported signicantly higher AMH
values in patients who did not receive alkylating agents [15]. In the
current study, we found a signicant difference in serum AMH levels
between patients receiving etoposide-negative regimens and those
receiving etoposide-positive regimens. ETP has been considered to
be a key agent for high-risk GTN [3]. Newlands et al. stated that EMA/
CO is an effective and well tolerated regimen for patients with highrisk GTN [16], but some patients fail to recover ovarian function
following EMA/CO treatment [17]. MEA and EA regimens are
additional options for high-risk and/or persistent GTN, but the rate of

Acknowledgement
We appreciate the technical assistance by Yoshinari Nagatomo.
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