Human Reproduction, Vol.25, No.4 pp.

986994, 2010
Advanced Access publication on February 10, 2010 doi:10.1093/humrep/deq015

ORIGINAL ARTICLE Reproductive biology

Association between phthalate

exposure and glutathione S-transferase
M1 polymorphism in adenomyosis,
leiomyoma and endometriosis
Po-Chin Huang 1, Eing-Mei Tsai 2,5, Wan-Fen Li1, Pao-Chi Liao 3,
Meng-Chu Chung 1, Ya-Hui Wang 1, and Shu-Li Wang 1,4,5,*
1

Downloaded from http://humrep.oxfordjournals.org/ by guest on September 14, 2016

Division of Environmental Health and Occupational Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County
350, Taiwan 2Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 3Department of
Environmental and Occupational Health, Medical College, National Cheng Kung University, Tainan, Taiwan 4Institute of Environmental
Medicine, College of Public Health, China Medical University and Hospital, Taichung, Taiwan 5Center of Excellence for Environmental
Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

*Correspondence address. Tel: 886-37-246166 ext. 36509; Fax: 886-37-587406; E-mail: slwang@nhri.org.tw

Submitted on September 4, 2009; resubmitted on December 29, 2009; accepted on January 7, 2010

background: Phthalates are known to have estrogenic effects in cell models and experimental animals. However, the evidence regard-
ing the effects of phthalates on human reproduction is still limited. We conducted a case control study to determine whether estrogen-
dependent diseases are associated with phthalate exposure and how the glutathione S-transferase M1 (GSTM1; a major detoxication
enzyme) genotype modulates the risk.
methods: We recruited subjects who underwent laparotomy and had pathologic conrmation of endometriosis (EN) (n 28), adeno-
myosis (AD) (n 16) and leiomyoma (LEI) (n 36) from the Department of Obstetrics and Gynecology at a medical center in Taiwan
between 2005 and 2007. Controls (n 29) were patients without any of the three aforementioned gynecologic conditions. Urine
samples were collected before surgery and analyzed for seven phthalate metabolites using liquid chromatographytandem mass spec-
trometry. Peripheral lymphocytes were used for GSTM1 genotype determination.
results: Patients with LEIs had signicantly higher levels of total urinary mono-ethylhexyl phthalate (SMEHP; 52.1 versus 18.9 mg/g
creatinine, P , 0.05) than the controls, whereas patients with EN had an increased level of urinary mono-n-butyl phthalate (94.1 versus
58.0 mg/g creatinine, P , 0.05). Subjects with GSTM1 null genotype had signicantly increased odds for AD relative to those with
GSTM1 wild genotype [odds ratio (OR) 5.30; 95% CI, 1.22 23.1], even after adjustment for age and phthalate exposure. Subjects
who carried the GSTM1 null genotype and had a high urinary level of SMEHP showed a signicantly increased risk for AD (OR 10.4;
95% CI, 1.26 85.0) and LEIs (OR 5.93; 95% CI, 1.10 31.9) after adjustment for age, compared with those with GSTM1 wild-type
and low urinary level of SMEHP.
conclusions: These results suggest that both GSTM1 null and phthalate exposure are associated with AD and LEI. Larger studies are
warranted to investigate potential interaction between GSTM1 null and phthalate exposure in the etiology of estrogen-dependent gyneco-
logic conditions.
Key words: adenomyosis / leiomyoma / endometriosis / phthalate monoester / glutathione S-transferase M1

muscle hyperplasia (Devlieger et al., 2003; Bulun, 2009). LEIs, also

Introduction
The etiologies of some estrogen-dependent gynecologic conditions,
such as endometriosis (EN), adenomyosis (AD) and leiomyomas
(LEIs), are still unclear. EN is dened as the presence of endometrial
tissue outside the uterine cavity, whereas AD refers to the condition
when endometrial tissues invade the myometrium with smooth
known as broids, are dened as benign tumors of smooth muscle
(Al-Hendy and Salama, 2006) in the uterus. EN, AD and LEIs are
common gynecologic disorders presented by prolonged or heavy
menstrual bleeding, pelvic pain and infertility. The prevalence of EN
has been reported to be 2 22% in women of childbearing age,
whereas AD and LEIs have a prevalence of 20 35% in the infertility

& The Author 2010. 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
Phthalate and GSTM1 polymorphism in estrogen-dependent diseases
clinic and 2025% in premenopausal women, respectively, and varies
by ethnic groups (Devlieger et al., 2003; Guo, 2005; Al-Hendy and
Salama, 2006). Susceptibility to these estrogen-dependent gynecologic
conditions depends on complex interactions between immunologic,
hormonal, environmental and genetic factors (Giudice and Kao, 2004).
Previous studies have revealed that some extensively used plasticizers,
known as phthalates, are possibly associated with EN (Cobellis et al., 2003;
Reddy et al., 2006a, b). Phthalates are added to plastics to make them soft
and exible, to cosmetics as a vehicle for fragrances and to many other
daily products, such as building materials, childrens toys and medical
devices (Schettler, 2006). Recent reports have shown that phthalates
are widely added in considerable amounts (up to 5%) to cosmetics and
personal care products, and that they raise the levels of exposure to
urinary phthalate monoesters rapidly when these products are used
daily (Houlihan et al., 2002; Duty et al., 2005), particularly in women.
Phthalates are estrogenic and anti-androgenic endocrine disruptors
that may prolong menstrual cycles and increase the proportion of pre-
mature menopause in animal models (Moore, 2000; Ma et al., 2006).
Toxicological evidence has shown that some phthalates, such as
butyl benzyl phthalate (BBzP), di-n-butyl phthalate (DnBP) and
di-(2-ethylhexyl) phthalate (DEHP), may alter or mimic estradiol (E2)
in vivo and in vitro (Harris et al., 1997; Okubo et al., 2003; Jin et al.,
2008). However, whether phthalate exposure results in adverse
effects on the human reproductive system is largely unknown.
Several studies have examined a relationship between estrogen-
dependent gynecologic conditions and genetic polymorphisms of
human detoxication enzymes, including N-acetyltransferase 2, gluta-
thione S-transferases (GST) M1 and T1 (Baranova et al., 1997,
1999) and cytochrome P450 (CYP) 1A1 (Juo et al., 2006). Previous
studies have suggested that the GSTM1 null mutation is associated
with EN in French (Baranova et al., 1997, 1999), Greek (Arvanitis
et al., 2003) and Indian (Babu et al., 2005) populations. However,
other studies have concluded that neither GSTM1 mutations nor
CYP 1A1 is linked to EN in Korean (Hur et al., 2005), Taiwanese
(Juo et al., 2006) and English (Hadeld et al., 2001) populations. The
inconsistent results could be due to small sample size or different
genetic backgrounds between ethnic groups, and therefore whether
genetic variability acts as a critical factor in EN is still under debate
(Guo, 2005). Furthermore, for complex gynecologic conditions, such
as EN and AD, it is likely that gene environmental interactions are
more important and relevant than genetics alone.
The present study was aimed to explore the possible interaction
between phthalates and GSTM1 on estrogen-dependent gynecologic
conditions, including AD, EN and LEIs. We rst examined the relation-
ship between gynecologic conditions and phthalate exposure using
urinary phthalate monoesters as the exposure biomarkers, and deter-
mined whether GSTM1 polymorphisms can modify individual risk for
such conditions. The results of this study will also help us further
understand the possible adverse effects of phthalates and provide
hints on the role of GST enzymes in phthalate detoxication.
Materials and Methods
Subject recruitment
We recruited patients who had undergone laparotomy in the Department
of Obstetrics and Gynecology of Kaohsiung Medical University Hospital
987
(KMUH) in Taiwan between 2005 and 2007, and had pathologic conr-
mation of EN, AD and LEIs. The diagnoses of EN and AD were based
on the pathologic results of the presence of endometrial tissue outside
the uterine cavity and within the myometrium with smooth muscle hyper-
plasia, respectively. The diagnosis of LEIs was based on the pathologic
nding of a benign tumor within the uterine smooth muscle. In this
study, 28 cases of EN, 16 cases of AD and 36 cases of LEIs were included.
Women who underwent laparotomy for other clinical reasons and did
not have EN, AD or LEIs, as conrmed by pathology, served as the control
group. The laparotomy was performed to exclude subjects with pelvic
masses, such as uterine and ovarian tumors. Women who had been pre-
viously diagnosed with these estrogen-dependent gynecologic conditions
were also excluded. All cases and controls were of Chinese descent.
This protocol was approved by the Institutional Review Board of KMUH
and informed consent was obtained from the participants before the
study.
Demographic characteristics
Downloaded from http://humrep.oxfordjournals.org/ by guest on September 14, 2016
The demographic data of each subject were obtained from an interviewed
questionnaire during the recruitment process. The recorded character-
istics included age, body mass index (BMI), education, age of menarche,
duration of menstrual cycle, history of abnormal uterine bleeding, parity,
hormone therapy, intrauterine device (IUD) use, oral contraceptive use,
caffeinated drink consumption, cigarette smoking, alcohol consumption,
a family history of gynecologic diseases and dietary habits.
Blood and urine collection
Blood samples were collected in tubes with EDTA and stored at 48C for
20 30 min. Blood samples were centrifuged at 1620 g for 15 min and
lymphocytes obtained from the blood sample (8 ml) were collected and
analyzed for the GSTM1 genotype. A 20 30 ml urine sample was col-
lected in a 250 ml glass vessel and immediately transferred into a 12 ml
amber glass bottle for phthalate monoesters and creatinine analysis. To
prevent possible contamination of the urine samples, all the glassware
had been washed in methanol, acetonitrile and acetone, and then was
sealed with aluminum foil before sample collection.
Genotype determination
The GSTM1 genotype was determined by polymerase chain reaction
(PCR) based on a method described previously (Shinka et al., 1998).
PCR, containing 100 ng of genomic DNA, was incubated at 948C for
5 min and subjected to 35 cycles at 948C for 30 s, 588C for 60 s, 728C
for 60 s and a nal extension at 728C for 10 min. The presence of the
GSTM1 allele was shown as a fragment of 215 bp on 2% agarose gel.
The primers used were 50 -GAACTCCCTGAAAAGCTAAAGC-30 and
50 -GTTGGGCTCAAATATACGGTGG-30 .
Urinary phthalate monoesters analysis
Standards of phthalate metabolites, including mono-methyl phthalate
(MMP), mono-ethyl phthalate (MEP), mono-n-butyl phthalate (MnBP),
mono-benzyl phthalate (MBzP), mono-(2-ethylhexyl) phthalate (MEHP),
mono-(2-ethyl-5-oxo-hexyl) phthalate (5oxo-MEHP) and mono-(2-ethyl-
5-hydroxyhexyl) phthalate (5OH-MEHP), and their corresponding
13
C4-labeled compounds were purchased from Cambridge Isotope Lab-
oratories (Andover, MA, USA). Formic acid (FA), acetic acid and
b-glucuronidase (Helix pomatia) were purchased from Sigma-Aldrich
(St Louis, MO, USA). Methanol (HPLC grade) was purchased from
Merck (Darmstadt, Germany). Deionized water was acquired from a Milli-
pore system (Milford, MA, USA). Seven urinary phthalate monoesters
were analyzed using on-line solid-phase extraction (SPE) coupled with
988
liquid chromatography/electrospray ionization tandem mass spectrometry
(LC/ESI-MS/MS), which was adapted from a previous study (Lin et al.,
2004; Huang et al., 2007). Briey, 1 ml aliquots of the sample containing
750 ml urine, 50 ml of 2000 ppb 13C4-labeled phthalate monoesters as
internal standards, 200 ml of 100 mM ammonium acetate buffer (pH
6.5) and 10 ml of b-glucuronidase were incubated at 378C for 90 min
for deconjugation. The deconjugation reaction was stopped by the
addition of 20% acetic acid/can (50 ml). The mixture was passed
through a 0.2 mm PVDF membrane lter (MSF-3; Advantec MFS, Inc.,
Pleasanton, CA, USA) and stored at 48C prior to loading onto the analyti-
cal system. Then, the urine mixture was loaded onto the on-line SPE car-
tridge (C18 trap cartridge, 2.0  55 mm, 3 mm; Merck) and washed with
2% FA/H2O at a ow rate of 600 ml/min for 10 min before the switching
valves (6-ports; Valco Europe, Schenkon, Switzerland) were triggered to
start the LC gradient and initiate chromatography on the Chromolith
column Flash RP-18e column (4.6  50 mm; Merck). The gradient
program was started with a mobile phase from 0.1% FA/H2O at a ow
rate of 600 ml/min to 100% MeOH in 10 min. The LC eluent was split
into a 1:20 ratio before entering the mass spectrometer (API365 triple
quadrupole; PE Sciex, Throhill, Ontario, Canada). After gradient analysis
was completed, the Chromolith column was washed with MeOH for
5 min and re-equilibrated with 2% FA/H2O for 1 min before the next
injection. The precursor to product ion transitions of seven phthalate
monoesters and their corresponding 13C4-labeled compounds were the
same as reported in a previous study (Kato et al., 2005).
The dynamic concentration ranges of the calibration curves for MMP,
MEP, MBzP, 5OH-MEHP and 5oxo-MEHP were 0.67 1300 ng/ml,
whereas the dynamic concentration ranges of the calibration curves for
MnBP and MEHP were 0.67 670 ng/ml. The intra-day variations of
these phthalate monoesters in urine at three different concentrations
(25%, 50% and 75%) within the dynamic range of the individual substance
were all ,10% with intra-day recoveries at 100 + 20%. The detection
limits of MMP, MEP, MnBP, MBzP, MEHP, 5OH-MEHP and 5oxo-MEHP
were 3.4, 2.2, 1.6, 0.99, 0.55, 0.23 and 0.26 ng/ml, respectively. The accu-
racy of the analytical approach was tested against two reference urine
samples with different known phthalate monoester concentrations. The
samples were received from the laboratory inter-comparison program
(www.g-equas.de) in 2006. The relative errors of the ve urinary monoe-
sters (MnBP, MBzP, MEHP, 5OH-MEHP and 5oxo-MEHP) were ,16%,
whereas MMP and MEP were not included in the reference sample.
Statistical analysis
The Kruskal Wallis and the Wilcoxon rank sum tests were used to evalu-
ate the difference in demographic data among all groups and between
groups, respectively. We also used the Wilcoxon rank sum test to
assess the difference in phthalate exposure between cases and controls.
All phthalate metabolite measurements were log transformed to approxi-
mate normal distribution. As 5OH-MEHP and 5oxo-MEHP are both the
further metabolites of MEHP, we calculated the sum of MEHP (SMEHP)
by combining the levels of 5oxo-MEHP, 5OH-MEHP and MEHP measure-
ments to evaluate the total exposure of DEHP.
Deviation from the Hardy Weinberg equilibrium (HWE) was examined
using a x2 test. Genotypic effects were evaluated for three estrogen-
dependent gynecologic conditions. We also categorized the subjects
into two groups by the median levels of phthalate metabolites to evaluate
the joint effects of GSTM1 polymorphism. Then, we evaluated the odds
ratio (OR) of individual case groups carrying the GSTM1 null genotype
using logistic regression. We used logistic regression to assess whether
there was a signicant risk increase between the case group with higher
phthalate exposure and the control group. Potential covariance was
adjusted in these two simple models. Further, stepwise backward logistic
Huang et al.
regression analysis was carried out to adjust for signicant covariates,
including age and BMI. We used the control group as a reference in the
logistic model and a cut-off point of P , 0.2 was applied as the criterion
for backward variable selection. In addition, we put only the most sensitive
exposure variable (urinary phthalate metabolites) into the nal models to
avoid confounding co-linearity.
In order to assess the interaction of phthalate exposure and GSTM1
polymorphism, four groups are categorized as follows: those with
GSTM1 and below-median SMEHP exposure, those with GSTM1 and
above-median SMEHP exposure, those with GSTM1 null type and below-
median SMEHP exposure, and those with GSTM1 null type and above-
median SMEHP exposure. We used two dummy variables for evaluation
of interaction. Dummy variable 1 (DV1): the presence and absence of
GSTM1 genotype in our subjects is coded as 0 and 1, respectively.
Dummy variable 2 (DV2): the exposure level of sum (MEHP) lower and
higher than the median in our subjects is coded as 0 and 1, respectively.
Then, we used DV1  DV2 as an interaction term in the model. The level
of statistical signicance was set at 0.05 for all analyses. All analyses were
performed using SAS 9.0 software.
Downloaded from http://humrep.oxfordjournals.org/ by guest on September 14, 2016
Results
Demographic characteristics of the study
participants
The demographic characteristics of the controls and patients with EN,
AD or LEIs are shown (Table I). There was a signicant difference in
age, using the Wilcoxon rank sum test, between the AD and the LEI
groups and the control group, but not the EN group. However, the
EN group had a signicantly lower BMI (21.5 + 2.9 kg/m2) than the
controls (23.9 + 4.8 kg/m2). No signicant differences in menstrual
and medical histories, such as age of menarche, abnormal uterine
bleeding, menstrual cycle, hormone therapy, IUD use and oral contra-
ceptive use, and lifestyles, such as caffeinated drink consumption, ciga-
rette smoking and alcohol consumption, were found among the
control and disease groups.
Levels of phthalate monoesters
The distribution of seven urinary phthalate monoesters (creatinine-
adjusted and non-adjusted) among the disease and control groups is
shown in Table II. Among the three disease groups, the most obvious
difference in urinary phthalate monoesters when compared with con-
trols was for the LEI group. The creatinine-adjusted (mg/g-c) levels of
MMP (65.8 mg/g-c), MEHP (6.0 mg/g-c) and SMEHP (52.1 mg/g-c) in
the LEI patients were signicantly higher than controls (P , 0.05). For
the EN group, we found increased median levels of all phthalate metab-
olites, except for MEP, but signicant differences was found in
MnBP (94.1 mg/g-c) and 5oxo-MEHP (19.0 mg/g-c), and SMEHP
(42.4 mg/g-c) reached a marginal signicance (P , 0.1). The difference
in urinary phthalate monoester levels was least evident for the AD
group.
ORs for estrogen-dependent gynecologic
conditions with different GSTM1 genotypes
The frequencies and crude- and adjusted OR of the GSTM1 poly-
morphisms among all groups are presented in Table III. Genotype of
GSTM1 was in HWE in both cases and controls. The frequencies of
GSTM1 null type in control, EN, AD and LEIs were 34.5%, 42.9%,
Phthalate and GSTM1 polymorphism in estrogen-dependent diseases 989

Table I Demographic factors of subjects in controls and patients of EN, AD or LEI.

Parameters Control (n 5 29) EN (n 5 28) AD (n 5 16) LEI (n 5 36) P-valuea

.............................................................................................................................................................................................
Age (years) 36.2 + 9.0 34.3 + 7.5 43.2 + 6.5b,* 41.1 + 6.8b,* ,0.001*
2 b,
BMI (kg/m ) 23.9 + 4.8 21.5 + 2.8 * 24.5 + 4.0 23.5 + 3.7 0.039*
Education
Senior high school 22 (75.9) 11 (39.3) 7 (46.7) 24 (66.7)
Technology college 4 (13.8) 10 (35.7) 3 (20.0) 8 (22.2) 0.069#
University 3 (10.3) 7 (25.0) 5 (33.3) 4 (11.1)
Age of menarche (years) 13.4 + 1.3 13.3 + 1.5 12.8 + 0.8 13.2 + 1.8 0.336
Menstrual cycle (days) 28.6 + 3.1 27.6 + 2.5 26.5 + 4.0 30.6 + 11.1 0.255
Abnormal uterine bleedingc 8 (27.6) 10 (35.7) 9 (56.3) 10 (27.8) 0.153
Hormone therapy 6 monthsc 8 (27.6) 8 (28.6) 4 (25.0) 7 (19.4) 0.828
Intrauterine device usec 8 (27.6) 5 (17.9) 6 (37.5) 10 (27.8) 0.550
c
Oral contraceptive use 8 (27.6) 7 (25.0) 4 (25.0) 5 (13.9) 0.545

Downloaded from http://humrep.oxfordjournals.org/ by guest on September 14, 2016

Coffeec 7 (24.1) 15 (53.6) 5 (31.3) 17 (47.2) 0.091#
c
Tea 15 (51.7) 17 (60.7) 5 (31.3) 18 (50.0) 0.313
Cigarette smokingc 3 (10.3) 4 (14.3) 2 (12.5) 7 (19.4) 0.778
Second-hand smokingc 15 (51.7) 13 (46.4) 6 (37.5) 13 (36.1) 0.858
Alcohol consumptionc 3 (10.3) 3 (10.7) 1 (6.3) 5 (13.9) 0.950
a
Kruskall Wallis test; *P , 0.05; #P , 0.10; as case number ,5, Fishers exact test was applied.
b
Wilcoxon rank sum test; *P , 0.05; all comparisons were relative to controls.
c
We only showed the number and percentage of subjects who reported Yes for each categorical variable.

Table II Urinary levels of phthalate metabolitesa in controls and patients of EN, AD or LEI.

Phthalate monoesters Control (n 5 29) EN (n 5 28) AD (n 5 16) LEI (n 5 36)

.............................................................................................................................................................................................
Creatinine-unadjusted (ng/ml)
MMP 28.1 (1.7 657.2) 37.8 (1.797.7) 25.8 (1.765.5) 30.8 (1.7 113.4)
MEP 37.2 (10.6 396.2) 31.6 (13.4 712.9) 33.8 (9.796.8) 28.5 (6.7 705.9)
MnBP 35.4 (5.2 247.2) 60.2 (8.2315.2) 30.2 (6.585.7) 36.2 (5.0 323.7)
MBzP 5.9 (2.1 26.2) 5.6 (2.646.0) 6.0 (1.715.2) 5.7 (1.5 27.0)
5oxo-MEHP 9.2 (0.1 38.6) 12.1 (0.165.4) 10.2 (0.1803.4) 6.2 (0.1 621.4)
5OH-MEHP 5.7 (0.1 463.4) 13.6 (0.1269.9) 9.0 (0.1511.1) 8.8 (0.1 249.3)
MEHP 0.8 (0.8 11.3) 2.7# (0.8 23.4) 3.2 (0.8145.3) 2.1 (0.8 89.3)
SMEHPb 28.5 (1.1 502.9) 28.7 (1.1305.6) 28.9 (1.11460) 28.6 (1.1 941.3)
Creatinine-adjusted (mg/g creatinine)
MMP 32.1 (6.9 2213) 52.4 (8.3357.6) 42.9 (2.2182.4) 65.8* (14.9 442.0)
MEP 71.4 (5.6 373.3) 58.0 (13.4 422.3) 53.4 (13.4 147.7) 103.7 (11.2 519.0)
MnBP 58.0 (9.8 479.0) 94.1* (8.9669.8) 36.3 (5.7201.1) 75.4# (11.8 2346)
MBzP 8.9 (2.1 38.7) 12.2 (3.094.7) 10.4 (3.140.7) 14.5 (2.8 112.7)
#
5oxo-MEHP 7.8 (0.1 98.5) 19.0* (0.1138.5) 18.5 (0.4 860.2) 11.5 (0.2 2496)
5OH-MEHP 9.9 (0.1 898.1) 16.7 (0.1885.1) 7.2 (0.1621.5) 10.5 (0.1 1001)
MEHP 3.4 (0.3 29.4) 4.2 (0.776.4) 3.4 (0.5155.6) 6.0* (0.7 1263)
SMEHPb 18.9 (2.1 974.6) 42.4# (1.2 1002) 37.9 (3.71563) 52.1* (4.5 3780)
a
Median (range); Wilcoxon rank sum test, *P , 0.05; #P , 0.10; all comparisons are relative to controls.
b
SMEHP MEHP 5oxo-MEHP 5OH-MEHP, where 5oxo-MEHP and 5OH-MEHP are the metabolites of MEHP.

68.7% and 47.2%, respectively. Subjects with the GSTM1 null-type had increased risk for subjects with the GSTM1 null type in the AD
higher crude OR for AD relative to those with wild-type (OR 4.89; group (OR 5.37; 95% CI, 1.25 23.0). The OR for the GSTM1
95% CI, 1.31 18.3). After adjustment for age, we found a small null type in the AD group was signicantly higher than in the control
990 Huang et al.

group. However, we did not nd any signicant ORs for GSTM1

..........................................................................................................................................................................................................................................................
genotypes in the EN and LEI groups, even after adjustment for BMI

P-valuea
and age, respectively.

0.188
0.133
ORs for estrogen-dependent gynecologic
conditions with different phthalates exposure

(0.72 5.53)
(0.78 6.84)
The crude and adjusted ORs of case status by above-median phthalate

95% CI
exposure are shown in Table IV. After adjustment for the GSTM1 gen-
otype and age, OR was 2.87 (P , 0.05) for LEIs in those with MEHP
LEI (n 5 36)

above the median. For those with MnBP and MEHP above the median,
19 (52.8)
17 (47.2)

the adjusted ORs for EN were 2.77 (0.68 11.3) and 2.03 (0.62
6.69), respectively, indicating that patients with higher exposure to
1.99
2.30
OR

MnBP or MEHP tended to have increased risk of EN, though

without statistical signicance.
P-valuea

Gene environmental interaction on

0.018
0.024

estrogen-dependent gynecologic conditions

Downloaded from http://humrep.oxfordjournals.org/ by guest on September 14, 2016

The ORs for GSTM1 polymorphisms and urinary phthalate monoe-
sters in the disease group using stepwise backward logistic regression
(1.31 18.3)
(1.25 23.0)

are shown in Table V. In the EN group, the OR for BMI was 0.88 (95%
Demographic factors were adjusted in the logistic regression models. BMI was adjusted for EN, whereas age was adjusted for AD and LEI, respectively.

CI, 0.75 1.04), whereas the OR for the urinary log MnBP was 2.92
95% CI
Table III Frequencies, crude- and adjusted-OR for GSTM1 null type in women of EN, AD and LEI.

(95% CI, 0.92 9.2), which only show marginal signicant increase in
risk. In the AD group, the OR was 1.13 (95% CI, 1.02 1.25) for
AD (n 5 16)

age and 5.30 (95% CI, 1.22 23.1) for GSTM1, showing that being
11 (68.7)
5 (31.3)

elderly or carrying the GSTM1 null type represents a signicantly

4.89*
5.37*
ORa

increased risk for AD. For the LEI group, age and the urinary level
of SMEHP were associated with an increased risk as the ORs for
age and SMEHP were 1.08 (95% CI, 1.01 1.16) and 2.64 (95% CI,
0.89 7.80), respectively.
P-valuea
0.357
0.793

We found a signicant joint effect of phthalates exposure and

GSTM1 null type on AD and LEIs (Fig. 1). When compared with
those with GSTM1 wild-type and low urinary level of SMEHP, subjects
who carried the GSTM1 null type with a high urinary level of SMEHP
(0.56 4.94)
(0.27 2.71)

had a signicantly increased risk for AD (crude OR 8.67; 95% CI,

1.34 56.3, Ptrend 0.011) and LEIs (crude OR 5.57; 95% CI,
95% CI

1.13 27.5, Ptrend 0.037). After adjustment for age, the risk
EN (n 5 28)

became even more evident as the OR increased to 10.4 (95% CI,

1.26 85.0) for AD and 5.93 (95% CI, 1.10 31.9) for LEIs. For inter-
16 (57.1)
12 (42.9)

action analysis, we found no signicant interaction between GSTM1

1.67
0.86
OR

deletion and phthalate exposure for AD (OR 1.7; 95% CI, 0.09
31.2), LEI (OR 0.6; 95% CI, 0.06 5.3) or EN (OR 3.5; 95% CI,
0.31 38.5).
Control (n 5 29)

Discussion
Reference
19 (65.5)
10 (34.5)

This is the rst study to explore the gene environmental interaction

of phthalates exposure and GSTM1 polymorphisms on EN, AD and

LEI. We found that urinary SMEHP was associated with an increased

risk of AD and LEI after adjustment of GSTM1 genotype. No associ-
ation was observed between urinary phthalate metabolites and
GST M1 null (n, %)

GSTM1 polymorphisms in EN. Previous studies have reported that

x test; *P , 0.05.
GST M1 (n, %)

some phthalates, such as DnBP, BBzP and DEHP, are associated

Adjustedb

with EN (Cobellis et al., 2003; Reddy et al., 2006a, b). Although signi-
Groups

Crude

cantly higher plasma phthalates and doseresponses were reported in

OR

these studies, recent studies have indicated that urinary phthalate

a 2
b

metabolites is a better biomarker of predicting individual phthalates

Phthalate and GSTM1 polymorphism in estrogen-dependent diseases 991

Table IV Crude- and adjusted-ORa of case status by above-median phthalate exposure.

Phthalate monoestersb EN (n 5 28) AD (n 5 16) LEI (n 5 36)

............................................. ............................................ .............................................
OR 95% CI P-value OR 95% CI P-value OR 95% CI P-value
.............................................................................................................................................................................................
#
MMP 2.53 0.87 7.39 0.089 1.48 0.425.16 0.540 2.99* 1.08 8.26 0.035
MMPadj 2.23 0.73 7.15 0.155 1.11 0.264.73 0.893 2.03 0.68 6.07 0.203
MEP 0.92 0.32 2.63 0.881 0.96 0.283.27 0.944 1.93 0.72 5.22 0.193
MEPadj 0.66 0.21 2.09 0.476 1.08 0.264.57 0.916 1.32 0.44 3.96 0.621
MnBP 3.46* 1.16 10.3 0.026 0.98 0.283.46 0.977 1.83 0.68 4.95 0.235
#
MnBPadj 2.93 0.92 9.31 0.069 0.78 0.183.33 0.737 1.36 0.46 4.00 0.581
MBzP 1.23 0.43 3.49 0.696 0.74 0.212.58 0.634 1.72 0.64 4.62 0.280
MBzPadj 1.07 0.35 3.28 0.900 1.33 0.296.13 0.713 1.40 0.48 4.05 0.537
5oxo-MEHP 1.90 0.66 5.46 0.232 1.58 0.465.41 0.464 1.23 0.46 3.28 0.678
5oxo-MEHPadj 2.03 0.64 6.37 0.225 1.41 0.345.86 0.636 1.47 0.51 4.27 0.479
5OH-MEHP 1.64 0.68 4.68 0.354 0.96 0.283.27 0.945 1.23 0.46 3.28 0.678

Downloaded from http://humrep.oxfordjournals.org/ by guest on September 14, 2016

5OH-MEHPadj 1.55 0.51 4.77 0.442 1.30 0.305.63 0.724 1.39 0.48 4.00 0.542
MEHP 1.64 0.57 4.70 0.360 0.98 0.283.46 0.977 2.90* 1.05 7.97 0.040
#
MEHPadj 1.42 0.45 4.50 0.547 0.94 0.214.09 0.929 2.70 0.92 7.89 0.070
SMEHP 2.53# 0.87 7.39 0.089 1.90 0.556.59 0.312 2.66# 0.97 7.32 0.058
SMEHPadj 2.23 0.71 6.95 0.168 1.91 0.448.25 0.384 2.64# 0.89 7.80 0.080
a
We adjusted GSTM1 polymorphism and BMI in EN, whereas age and GSTM1 genotype were adjusted for AD and LEI; control group was used as a reference; *P , 0.05, #P , 0.10.
b
We categorized our subjects into two groups according to the median levels of each urinary phthalate metabolite in all subjects.

Table V Crude- and adjusted-OR of case status by urinary phthalate monoesters using stepwise backward logistic
regressiona.

Parameters EN AD LEI
................................................ ................................................ ................................................
OR 95% CI P-value OR 95% CI P-value OR 95% CI P-value
.............................................................................................................................................................................................
BMI 0.88 0.751.04 0.131
Age 1.13* 1.02 1.25 0.020 1.08* 1.011.16 0.023
GSTM1 5.30* 1.22 23.1 0.026 2.50 0.817.76 0.112
MnBPb 2.92# 0.929.29 0.069
b #
SMEHP 1.91 0.44 8.25 0.384 2.64 0.897.80 0.080
R-squarec 0.179 0.921 0.358 0.112 0.216 0.669
a
Stepwise backward logistic regression, , excluded from stepwise model; *P , 0.05; #P , 0.10.
b
We categorized our subjects into two groups according to the median levels of each urinary phthalate metabolite.
c
Nagelkerker R-square (R 2); Hosmer Lemeshow goodness-of-t, P-value .0.05 showed good t of individual model.

exposure due to less contamination during the process of sampling
and analysis (Silva et al., 2004; Kato et al., 2005). Determining
urinary monoesters and oxidative monoesters of phthalates in our
subjects provided the reliable exposure data. Besides, we recruited
laparotomy-conrmed controls, which reduced the selection bias
from the potential cases in the non-laparotomy-conrmed controls.
A few of our controls had gynecologic conditions such as endometrial
polyps and ovarian cysts. Although we found no evidence in the litera-
ture that phthalate exposures are associated with these diseases,
other estrogenic compounds such as bisphenol A (BPA) have been
associated with them (Vandenberg et al., 2007). It is thus plausible
that phthalates, which are also estrogenic, might be associated with
some of the gynecologic conditions in our control group, despite a
paucity of research in the literature. If this was true, the phthalate
exposures in our control group would be elevated leading toward
the null hypotheses of no association. It is possibly the reason why
we did not observe any correlation between phthalate exposure
and GSTM1 genotype in EN, which requires a large sample size to
clarify. However, the associations between phthalate exposure and
GSTM1 polymorphism in AD and LEI still exist.
In the present study, we found that BMI and age are potential risk
factors for EN and AD/LEI, respectively, which is consistent with
previous studies (Cramer and Missmer, 2002; Vercellini et al.,
2006), whereas other demographic characteristics, such as age at
992
Figure 1 Joint effects of total MEHP exposure and GSTM1 poly-
morphism on adenomyosis (AD) and leiomyoma (LEI) after adjust-
ment for age.
Low and high SMEHP groups were divided by the median concentrations of
SMEHP (38.9 mg/g c). GSTM1 ( ) represents the GSTM1 null-type,
whereas GSTM1 () represents the GSTM1 wild-type. Asterisk indicates
signicant difference (P,0.05) as compared to the group of low SMEHP/
wild-genotype group. Signicant trend was found for AD (P 0.011) and LEI
(P 0.037), respectively.
menarche and smoking, did not show statistical difference. The fre-
quency of the GSTM1 null genotype has been reported in different
ethnic groups, ranging from 21% to 77%, and conicting conclusions
have been offered in different populations. In the current study, the
frequency of the GSTM1 null genotype in our controls was 34.5%,
which is comparable to the Hans population (35 63%; Rebbeck,
1997; Lin et al., 2003). The frequency of GSTM1 null type in EN is
42.9%, which is comparable to the Indian population (39%; Babu
et al., 2005), and approximates the Hans population (51%; Ding
et al., 2004). Although our results of GSTM1 polymorphisms in EN
and controls were different from Japanese and Korean studies
(Morizane et al., 2004; Hur et al., 2005), our results are in agreement
with most previous studies reporting no association between GSTM1
and EN.
Our data reveal that the risk of SMEHP exposure in AD and LEI
with the GSTM1 null genotype was signicantly increased. DEHP, the
parent compound of MEHP, MEHHP, MEOHP and other phthalates,
has estrogenic effects in vivo and in vitro studies (Harris et al., 1997; Jin
et al., 2008), although a few studies have conicting ndings (Hong
et al., 2005). No human studies have focused on the interaction
between phthalates exposure and GSTM1 genotyping in women
with AD and LEI. The effects of other estrogenic endocrine disrup-
tors on AD may provide some clues. One study showed that
low-dose diethylstilbestrol exposure may affect the development of
AD in female mice (Huseby and Thurlow, 1982). Another study
reported that long-term exposure to BPA, which has similar estro-
genic activity to phthalates, can cause AD in female CD-1 mice
Huang et al.
(Newbold et al., 2007). Therefore, we suggest that both GSTM1
null and phthalate exposure are associated with AD and LEI, but
our study lacked power to test for interactions. The potential
effects from phthalates exposure modied by estrogen-receptor-
related genotypes or other enzymes on the estrogen-dependent
gynecologic diseases are worthy of future investigation. Larger
studies to investigate potential interaction between GSTM1 null and
phthalate exposure in the etiology of estrogen-dependent gynecologic
conditions are warranted.
Interestingly, we also found a signicant correlation between urinary
MEHP and MnBP (Pearsons correlation coefcient: r 0.31, P
0.001), which is also reported in another study (Becker et al.,
2009). Low correlation between urinary MEHP and MnBP may indi-
cate that the exposure sources of their parent compounds, DEHP
and DnBP, in our subjects could partly come from similar sources. It
is reported that high percentages of DEHP and DnBP exposure in
humans come from food intake, possibly by contamination in the
Downloaded from http://humrep.oxfordjournals.org/ by guest on September 14, 2016
process of food preparation and leaching out from plastic containers
(Schettler, 2006). In addition, DEHP and DnBP are also used in cos-
metic and personal care products (DiGangi et al., 2002; Houlihan
et al., 2002). The co-exposure of DEHP and DnBP may occur as
women tend to use various types of these products, such as deodor-
ant, perfume, hair spray, nail polish etc. in their daily routine.
However, lacking an integrated toxic equivalence factor of phthalates
may underestimate the joint effects of total phthalates exposure and
GSTM1 polymorphism in our subjects.
The present study had some limitations. First, although the small
sample size may limit our interpretation, our results provide useful
data for further investigation. Further, studies with a large sample
size are needed. Second, other gynecologic conditions in controls
may derive from estrogenic compounds, such as BPA or phthalates,
which may lead toward the null result of no association. Third, we
did not have age-matched controls for each estrogen-dependent gyne-
cologic condition. Because the presenting age of women with EN, AD
and LEIs was quiet different, we used laparotomy-conrmed criteria
for subject recruitment. Then, we used age as a covariance in the
regression models. Fourth, because phthalates are ubiquitous in our
environment and consumer products, it is generally hypothesized
that phthalate exposure in humans is continual and the intensity is
gradually increased, especially in women (DiGangi and Norin, 2002;
DiGangi et al., 2002; Houlihan et al., 2002; Duty et al., 2005). In
addition, recent studies have also reported the prenatal, post-natal
and childrens exposure to phthalate, and its potential effects in
early gene expression during embryonic stem cell and neonates
(Wolff et al., 2007; Becker et al., 2009; Engel et al., 2009; Huang
et al., 2009; van Dartel et al., 2009). However, the missing evidence
of phthalate exposure during the critical period for development of
measured outcomes is still a limitation of this study. Fifth, because
we set 0.05 as the signicant level of each statistic testing, it may
produce a few false associations by multiple testing. Owing to different
phthalates exposure in humans from different sources, it is helpful to
identify possible exposure proles of phthalate in human subject by
multiple comparison of phthalate exposure between cases and con-
trols. In order to validate our ndings, we used stepwise backward
logistic regression model to avoid the false association. Therefore,
our ndings still provide important information for future studies on
estrogen-dependent gynecologic conditions.
Phthalate and GSTM1 polymorphism in estrogen-dependent diseases
Conclusions
We found a signicantly increased risk for AD and LEI in subjects with
high MEHP exposure and GSTM1 deletions. However, we did not
observe any signicant interaction between phthalates exposure and
GSTM1 polymorphisms in EN, AD or LEI, probably limited by the
sample size. We suggest that both GSTM1 null and phthalate exposure
are associated with AD and LEI, but our study lacked power to test for
interactions. Larger studies to investigate potential interaction
between GSTM1 null and phthalate exposure in the etiology of
estrogen-dependent gynecologic conditions are warranted.
Acknowledgements
We are greatly indebted to the gynecologists of the Department of
Obstetrics and Gynecology, Kaohsiung Medical University Hospital,
Kaohsiung, Taiwan, and our colleagues, especially for Ms Yi-Hsin
Chang, at the National Health Research Institutes, Miaoli, Taiwan,
for subject recruitment and sample collection.
Funding
We are grateful for the nancial support from the National Health
Research Institutes (grant nos: EO-095-PP-09 and EO-096-PP-09).
References
Al-Hendy A, Salama S. Gene therapy and uterine leiomyoma: a review.
Hum Reprod Update 2006;12:385 400.
Arvanitis DA, Koumantakis GE, Goumenou AG, Matalliotakis IM,
Koumantakis EE, Spandidos DA. CYP1A1, CYP19, and GSTM1
polymorphisms increase the risk of endometriosis. Fertil Steril 2003;
79(Suppl 1):702 709.
Babu KA, Reddy NG, Deendayal M, Kennedy S, Shivaji S. GSTM1, GSTT1
and CYP1A1 detoxication gene polymorphisms and their relationship
with advanced stages of endometriosis in South Indian women.
Pharmacogenet Genomics 2005;15:167 172.
Baranova H, Bothorishvilli R, Canis M, Albuisson E, Perriot S,
Glowaczower E, Bruhat MA, Baranov V, Malet P. Glutathione
S-transferase M1 gene polymorphism and susceptibility to
endometriosis in a French population. Mol Hum Reprod 1997;
3:775 780.
Baranova H, Canis M, Ivaschenko T, Albuisson E, Bothorishvilli R,
Baranov V, Malet P, Bruhat MA. Possible involvement of arylamine
N-acetyltransferase 2, glutathione S-transferases M1 and T1 genes in
the development of endometriosis. Mol Hum Reprod 1999;5:636 641.
Becker K, Goen T, Seiwert M, Conrad A, Pick-Fu H, Muller J,
Wittassek M, Schulz C, Kolossa-Gehring M. GerES IV Phthalate
metabolites and bisphenol A in urine of German children. Int J Hyg
Environ Health 2009;212:685 692.
Bulun SE. Endometriosis. New Engl J Med 2009;15:268 279.
Cobellis L, Latini G, De Felice C, Razzi S, Paris I, Ruggieri F, Mazzeo P,
Petraglia F. High plasma concentrations of di-(2-ethylhexyl)-phthalate
in women with endometriosis. Hum Reprod 2003;18:1512 1515.
Cramer DW, Missmer SA. The epidemiology of endometriosis. Ann N Y
Acad Sci 2002;955:11 22.
Devlieger R, DHooghe T, Timmerman D. Uterine adenomyosis in the
infertility clinic. Hum Reprod Update 2003;9:139 147.
DiGangi J, Norin H. Pretty NastyPhthalates in European Cosmetics
Products. USA: Healthcare Without Harm, and in association with
993
Womens Environmental Network and Swedish Society for Nature
Conservation, 2002.
DiGangi J, Schettler T, Cobbing M, Rossi M. Aggregate Exposures to
Phthalates in Humans. USA: Healthcare Without Harm, and in
association with Womens Environmental Network and Swedish
Society for Nature Conservation, 2002.
Ding Y, Chen ZF, Lin RY, Wang XF, Ding JB, Ai XZ, Wen H. Relationship
between endometriosis and glutathione S-transferase M1 T1 genes of
the Uygurs and Hans in Xinjiang. Zhonghua Fu Chan Ke Za Zhi 2004;
39:101 104.
Duty SM, Ackerman RM, Calafat AM, Hauser R. Personal care product use
predicts urinary concentrations of some phthalate monoesters. Environ
Health Perspect 2005;113:1530 1535.
Engel SM, Zhu C, Berkowitz GS, Calafat AM, Silva MJ, Miodovnik A,
Wolff MS. Prenatal phthalate exposure and performance on the
Neonatal Behavioral Assessment Scale in a multiethnic birth cohort.
Neurotoxicology 2009;30:522 528.
Giudice L, Kao L. Endometriosis. Lancet 2004;364:1789 1799.
Guo SW. Glutathione S-transferases M1/T1 gene polymorphisms and
Downloaded from http://humrep.oxfordjournals.org/ by guest on September 14, 2016
endometriosis: a meta-analysis of genetic association studies. Mol
Hum Reprod 2005;11:729 743.
Hadeld RM, Manek S, Weeks DE, Mardon HJ, Barlow DH, Kennedy SH,
OXEGENE Collaborative Group. Linkage and association studies of the
relationship between endometriosis and genes encoding the
detoxication enzymes GSTM1, GSTT1 and CYP1A1. Mol Hum
Reprod 2001;7:1073 1078.
Harris CA, Henttu P, Parker MG, Sumpter JP. The estrogenic activity of
phthalate esters in vitro. Environ Health Perspect 1997;105:802 811.
Hong EJ, Ji YK, Choi KC, Manabe N, Jeung EB. Conict of estrogenic activity
by various phthalates between in vitro and in vivo models related to the
expression of Calbindin-D9k. J Reprod Dev 2005;51:253 263.
Houlihan J, Brody C, Schwan B. Not Too Pretty: Phthalates, Beauty Products
and the FDA. Environmental Working Group, Coming Clean and Health
Care Without Harm. CA, USA. 2002. Available: http://safecosmetics.
org/downloads/NotTooPretty_report.pdf [last accessed 26 January
2010].
Huang PC, Kuo PL, Guo YL, Liao PC, Lee CC. Associations between
urinary phthalate monoesters and thyroid hormones in pregnant
women. Hum Reprod 2007;22:2715 2722.
Huang PC, Kuo PL, Ghou YY, Lin SJ, Lee CC. Association between
prenatal exposure to phthalates and the health of newborns. Environ
Int 2009;35:14 20.
Hur SE, Lee JY, Moon HS, Chung HW. Polymorphisms of the genes
encoding the GSTM1 GSTT1 and GSTP1 in Korean women: no
association with endometriosis. Mol Hum Reprod 2005;11:15 19.
Huseby RA, Thurlow S. Effects of prenatal exposure of mice to low-dose
diethylstilbestrol and the development of adenomyosis associated with
evidence of hyperprolactinemia. Am J Obstet Gynecol 1982;15:939 949.
Jin Q, Sun Z, Li Y. Estrogenic activities of di-2-ethylhexyl phthalate. Front
Med China 2008;2:303 308.
Juo SH, Wang TN, Lee JN, Wu MT, Long CY, Tsai EM. CYP17, CYP1A1
and COMT polymorphisms and the risk of adenomyosis and
endometriosis in Taiwanese women. Hum Reprod 2006;21:1498 1502.
Kato K, Silva MJ, Needham LL, Calafat AM. Determination of 16 phthalate
metabolites in urine using automated sample preparation and on-line
preconcentration/ high-performance liquid chromatography/tandem
mass spectrometry. Anal Chem 2005;77:2985 2991.
Lin J, Zhang X, Qian Y, Ye Y, Shi Y, Xu K, Xu J. Glutathione S-transferase
M1 and T1 genotypes and endometriosis risk: a case-controlled study.
Chin Med J 2003;116:777 780.
Lin LC, Tyan YC, Shih TS, Chang YC, Liao PC. Development and
validation of an isotope-dilution electrospray ionization tandem mass
994
spectrometry method with an on-line sample clean-up device for the
quantitative analysis of benzene exposure biomarker SPMA in human
urine. Rapid Commun Mass Spectrom 2004;18:1310 1316.
Ma M, Kondo T, Ban S, Umemura T, Kurahashi N, Takeda M, Kishi R.
Exposure of prepubertal female rats to inhaled di(2-ethylhexyl)phthalate
affects the onset of puberty and postpubertal reproductive functions.
Toxicol Sci 2006;93:164171.
Moore NP. The oestrogenic potential of the phthalate esters. Reprod
Toxicol 2000;14:183 192.
Morizane M, Yoshida S, Nakago S, Hamana S, Maruo T, Kennedy S. No
association of endometriosis with glutathione S-transferase M1 and T1
null mutations in a Japanese population. J Soc Gynecol Invest 2004;
11:118 121.
Newbold RR, Jefferson WN, Padilla-Banks E. Long-term adverse effects of
neonatal exposure to bisphenol A on the murine female reproductive
tract. Reprod Toxicol 2007;24:253 258.
Okubo T, Suzuki T, Yokoyama Y, Kano K, Kano I. Estimation of estrogenic
and anti-estrogenic activities of some phthalate diesters and monoesters
by MCF-7 cell proliferation assay in vitro. Biol Pharm Bull 2003;
26:1219 1224.
Rebbeck TR. Molecular epidemiology of the human glutathione
S-transferase genotypes GSTM1 and GSTT1 in cancer susceptibility.
Cancer Epidemiol Biomarkers Prev 1997;6:733 743.
Reddy BS, Rozati R, Reddy BVR, Raman NVVSS. Association of phthalate
esters with endometriosis in Indian women. BJOG 2006a;113:515 520.
Reddy BS, Rozati R, Reddy S, Kodampur S, Reddy P, Reddy R. High plasma
concentrations of polychlorinated biphenyls and phthalate esters in
Huang et al.
women with endometriosis: a prospective case control study. Fertil
Steril 2006b;85:775 779.
Schettler T. Human exposure to phthalates via consumer products. Int J
Androl 2006;29:134 139.
Shinka T, Ogura H, Morita T, Nishikawa T, Fujinaga T, Ohkawa T.
Relationship between glutathione S-transferase M1 deciency and
urothelial cancer in dye workers exposed to aromatic amines. J Urol
1998;159:380 383.
Silva MJ, Barr DB, Reidy JA, Malek NA, Hodge CC, Caudill SP, Brock JW,
Needham LL Calafat AM. Urinary level of seven phthalate metabolites in
the U.S. population from the national health and nutrition examination
survey (NANES) 1999 2000. Environ Health Perspect 2004;
112:331 338.
van Dartel DA, Pennings JL, Hendriksen PJ, van Schooten FJ, Piersma AH.
Early gene expression changes during embryonic stem cell differentiation
into cardiomyocytes and their modulation by monobutyl phthalate.
Reprod Toxicol 2009;27:93 102.
Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. Human
exposure to bisphenol A (BPA). Reprod Toxicol 2007;24:139 177.
Downloaded from http://humrep.oxfordjournals.org/ by guest on September 14, 2016
Vercellini P, Vigano P, Somigliana E, Daguati R, Abbiati A, Fedele L.
Adenomyosis: epidemiological factors. Best Pract Res Clin Endocrinol
Metab 2006;20:465 477.
Wolff MS, Teitelbaum SL, Windham G, Pinney SM, Britton JA, Chelimo C,
Godbold J, Biro F, Kushi LH, Pfeiffer CM et al. Pilot study of urinary
biomarkers of phytoestrogens, phthalates, and phenols in girls. Environ
Health Perspect 2007;115:116 121.