Endometriosis and organochlorinated environmental pollutants: a case-control study on Italian women of reproductive age.

Abstract

Endometriosis is a common gynecologic disease characterized by the ectopic growth of endometrial tissue. In industrialized countries, it affects approximately 10% of women of reproductive age. Its etiology is unclear, but a multifactorial origin is considered to be most plausible. Environmental organochlorinated persistent pollutants, in particular dioxins and polychlorinated biphenyls (PCBs), have been hypothesized to play a role in the disease etiopathogenesis. However, results of studies carried out on humans are conflicting.
We evaluated the exposure to organochlorinated persistent pollutants as a risk factor for endometriosis.
We conducted a case-control study in Rome on 158 women comprising 80 cases and 78 controls. In all women, serum concentrations of selected non-dioxin-like PCBs (NDL-PCBs) and dioxin-like PCBs (DL-PCBs), 1,1-dichloro-2,2,-bis(4-chlorophenyl)-ethene (p,p'-DDE), and hexachlorobenzene (HCB) were determined by ion-trap mass spectrometry. DR-CALUX bioassay was employed to assess the 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity equivalent (TEQ) concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and DL-PCBs.
We found an increased risk of endometriosis for DL-PCB-118 [odds ratio (OR) = 3.79; 95% confidence interval (CI), 1.61-8.91], NDL-PCB-138 (OR = 3.78; 95% CI, 1.60-8.94), NDL-PCB-153 (OR = 4.88; 95% CI, 2.01-11.0), NDL-PCB-170 (OR = 3.52; 95% CI, 1.41-8.79), and the sum of DL-PCBs and NDL-PCBs (OR = 5.63; 95% CI, 2.25-14.10). No significant associations were observed with respect to HCB or to the sum of PCDDs, PCDFs, and DL-PCBs given as total TEQs.
The results of this study show that an association exists between increased PCB and p,p'-DDE serum concentrations and the risk of endometriosis.

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volume 117 | number 7 | July 2009 • Environmental Health Perspectives
Research
The possible role of the exposure to envi-
ronmental chemicals as a co-causal factor
in the etiology of endometriosis has been
the object of scientific debate in the last 20
years. Endometriosis is a common gyneco-
logic disease characterized by the ectopic
growth of endometrial tissue and is often
associated with pelvic pain and/or infertil-
ity. It affects approximately 10% of women
of reproductive age in Italy as well as in
other industrialized countries (Eskenazi and
Warner 1997; Gruppo Italiano per lo Studio
dell’Endometriosi 1994), and its prevalence
and severity are reported to be increasing in
developing countries (Donnez et al. 2002).
Its etiology is unclear, although a multifac-
torial origin, resulting from the contribution
of immunologic, genetic, and environmental
factors, is considered to be most plausible.
The hypothesis that exposure to immunotoxic
endocrine-disrupting environmental pollut-
ants could play a role in the disease etiology
first arose from the study of Rier and cowork-
ers (Rier et al. 1993). These authors observed
a dose-dependent increase of incidence and
severity of spontaneous endometriosis in a
colony of monkeys chronically exposed to
dioxin [2,3,7,8-tetrachloro dibenzo-p-dioxin
(2,3,7,8-TCDD)], the most toxic member
of the family of polychlorinated dibenzo-p-
dioxins (PCDDs) and polychlorinated diben-
zofurans (PCDFs), 210 different molecules
or congeners generally referred to as dioxins.
Although criticized by some scientists (Guo
2004; Hitchin 1994), this study opened the
way to a number of studies on the relation-
ship between the disease and environmental
pollutants. Major criticism involved the inci-
dental nature of the observation—endometri-
osis was not a prospectively defined end point
of the experiment, and its presence in mon-
keys was observed many years after the end of
the treatment—and appropriateness of statis-
tical analysis of the results. The small sample
size (24 animals assigned to three groups of
8 animals each), the limited values of dioxin
exposure levels (a low-dose and a high-dose
group), and the use of linear regression and
t-test based on normality assumption were
the study elements deemed to be more critical
in interpreting study results. Together with
dioxins, polychlorinated biphenyls (PCBs),
a family of persistent and bioaccumulative
industrial compounds widely used until the
1980s, have been the pollutants principally
investigated as to their possible role in the
disease onset or progression. In the general
population, diet accounts for over 90% of
total exposure to dioxins and PCBs.
PCBs comprise 209 different congeners
which, according to the structure, are grouped
into 12 dioxin-like PCB (DL-PCB) conge-
ners, with no or only one chlorine in the ortho
position, and non-dioxin-like PCBs (NDL-
PCBs), characterized by the presence of two
or more chlorines in the ortho positions. The
latter are normally much more abundant
than DL-PCBs in environmental, food, and
human specimens. The most abundant con-
geners in human tissues include NDL-PCBs
28, 52, 101, 138, 153, and 180—referred
to as ‘‘indicators’’ (Appel 2003) because
conventionally they are used to estimate the
overall PCB content in specimens of biologi-
cal origin (EFSA 2005)—along with a few
others such as DL-PCB-105, DL-PCB-118,
DL-PCB-156, and DL-PCB-167, and
NDL-PCB-170, PCB-138, PCB-153, and
PCB-180, are prevalent in all human tissues
and account for 50–80% of total PCB con-
tent in serum (Glynn et al. 2000).
DL-PCBs have been the first PCB con-
geners to be considered in association with
endometriosis because, as dioxins, they bind
to the aryl hydrocarbon receptor (AhR) and
elicit the same spectrum of toxic activities
Address correspondence to E. De Felip, Unit of
Toxicological Chemistry, Department of the
Environment and Primary Prevention, National
Institute for Health, Viale Regina Elena, 299 00161
Rome, Italy. Telephone: 39 06 4990 2904. Fax: 39
06 4990 2836. E-mail: defelip@iss.it
*These authors contributed equally to this work.
The authors thank P. Cerenzia and D. Pallante,
Department of Gynaecology and Obstetrics,
University Sapienza, Rome, Italy, for their contribu-
tion in data collection.
This study was supported by a grant from the
Italian National Institute for Health and the Italian
Ministry of Health (Nr. 0R/2C).
DR-CALUX method has been used under license
agreement Nr. RLA-0306 ISS.
The authors declare they have no competing
financial interests.
Received 10 October 2008; accepted 31 March 2009.
Endometriosis and Organochlorinated Environmental Pollutants:
A Case–Control Study on Italian Women of Reproductive Age
Maria Grazia Porpora,1,* Emanuela Medda,2,* Annalisa Abballe,3 Simone Bolli,2 Isabella De Angelis,3
Alessandro di Domenico,3 Annamaria Ferro,1 Anna Maria Ingelido,3 Antonella Maggi,4
Pierluigi Benedetti Panici,1 and Elena De Felip3
1Department of Gynaecology and Obstetrics, Sapienza University of Rome, Rome, Italy; 2National Centre for Epidemiology, Surveillance
and Health Promotion, National Institute for Health, Rome, Italy; 3Department of the Environment and Primary Prevention, National
Institute for Health, Rome, Italy; 4Service for Biotechnology and Animal Welfare, National Institute for Health, Rome, Italy
Background: Endometriosis is a common gynecologic disease characterized by the ectopic growth
of endometrial tissue. In industrialized countries, it affects approximately 10% of women of repro-
ductive age. Its etiology is unclear, but a multifactorial origin is considered to be most plausible.
Environmental organochlorinated persistent pollutants, in particular dioxins and polychlorinated
biphenyls (PCBs), have been hypothesized to play a role in the disease etiopathogenesis. However,
results of studies carried out on humans are conflicting.
oBjective: We evaluated the exposure to organochlorinated persistent pollutants as a risk factor for
endometriosis.
Methods: We conducted a case–control study in Rome on 158 women comprising 80 cases and
78 controls. In all women, serum concentrations of selected non-dioxin-like PCBs (NDL-PCBs)
and dioxin-like PCBs (DL-PCBs), 1,1-dichloro-2,2,-bis(4-chlorophenyl)-ethene (p,p´-DDE), and
hexachlorobenzene (HCB) were determined by ion-trap mass spectrometry. DR-CALUX bioassay
was employed to assess the 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity equivalent (TEQ) concen-
trations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs),
and DL-PCBs.
results: We found an increased risk of endometriosis for DL-PCB-118 [odds ratio (OR) = 3.79;
95% confidence interval (CI), 1.61–8.91], NDL-PCB-138 (OR = 3.78; 95% CI, 1.60–8.94), NDL-
PCB-153 (OR = 4.88; 95% CI, 2.01–11.0), NDL-PCB-170 (OR = 3.52; 95% CI, 1.41–8.79), and
the sum of DL-PCBs and NDL-PCBs (OR = 5.63; 95% CI, 2.25–14.10). No significant associa-
tions were observed with respect to HCB or to the sum of PCDDs, PCDFs, and DL-PCBs given as
total TEQs.
conclusions: The results of this study show that an association exists between increased PCB and
p,p´-DDE serum concentrations and the risk of endometriosis.
key words: biomonitoring, case–control study, dioxins, endometriosis, PCBs. Environ Health
Perspect 117:1070–1075 (2009). doi:10.1289/ehp.0800273 available via http://dx.doi.org/ [Online
31 March 2009]

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Organochlorinated pollutants and endometriosis
Environmental Health Perspectives • volume 117 | number 7 | July 2009
1071
through the same mechanism of action.
Indeed, DL-PCBs, rather than dioxins, were
suggested to be associated with an endometri-
otic effect (Rier et al. 2001) when blood sam-
ples from exposed monkeys were analyzed
years after the experiment was completed and
significant concentrations of these compounds
were detected, possibly originating from con-
taminated food.
On the epidemiologic side, several stud-
ies have been conducted to investigate the
potential relationship between endometrio-
sis and dioxins and/or PCBs, including the
non-dioxin-like congeners, but their results
are conflicting. Major differences in study
design, analytical methods, and the number
and kinds of congeners measured make com-
parability (if any) between different studies
limited, as recently discussed by some authors
(Anger and Foster 2008; Heilier et al. 2008).
In the last decade, the hypothesis of a correla-
tion between the disease and environmentally
persistent organohalogenated compounds has
been extended to include other organoha-
logenated pollutants present in human tis-
sues such as polybrominated biphenyls
(PBBs) (Hoffman et al. 2007), hexachloro-
benzene (HCB), and 1,1-dichloro-2,2-bis(4-
chlorophenyl)-ethene (p,p´-DDE) (Lebel et al.
1998; Tsukino et al. 2005), all characterized
by endocrine-disrupting and immunotoxic
activity (Agency for Toxic Substances and
Disease Registry 2002; Blanck et al. 2000;
Davis et al. 2005; Foster et al. 1992, 1995;
Halloway et al. 2005; Reed et al. 2007;
Windham et al. 2005). No significant associa-
tion for these pollutants has been evidenced
in the studies performed to date.
In previous studies carried out to inves-
tigate a possible association between organo-
chlorinated compounds and endometriosis,
we found significantly higher concentrations
of some of the most abundant PCBs and
higher concentrations of p,p´-DDE, in women
affected by the disease (Porpora et al. 2006;
Quaranta et al. 2006). We also observed that
such increased concentrations were associ-
ated with altered natural killer (NK) immune
responses (Quaranta et al. 2006). On the con-
trary, no increase in blood concentrations of
dioxin-like chemicals (PCDDs, PCDFs, and
the sum of the 12 DL-PCBs) was observed
in women affected by the disease at different
degrees (De Felip et al. 2004).
The present study was funded by Italy’s
Ministry of Health and National Institute
for Health in the context of research activities
aimed to characterize the risk for women’s
reproductive health from exposure to persis-
tent organic pollutants of environmental ori-
gin. It is the largest study carried out in Italy
on the possible association between endo-
metriosis and persistent organochlorinated
pollutants of high toxicologic relevance.
Methods
Patients. Of the 312 women who under-
went laparoscopy between January 2002
and December 2005 at the Department of
Gynaecology and Obstetrics, Policlinico
Umberto I, University of Rome Sapienza, for
endometriosis or other benign gynecologic
conditions, 158 patients were enrolled in the
study. The vast majority of women participat-
ing in the study underwent surgery between
March 2004 and October 2005. The protocol
of this study was approved by the Sapienza
University, School of Medicine, Institutional
Review Board. All patients met the inclu-
sion criteria: 18–45 years of age, residence in
Rome in the last 5 years, no breast-feeding
history, absence of immunologic, hormonal
disorders, or chronic diseases, and no occupa-
tional exposure to PCBs or pesticides.
All enrolled women signed an informed
consent form. A physician unaware of the
indications to laparoscopy administered a
questionnaire before surgery which docu-
mented age, education, job, medical, gyneco-
logic and obstetric history, height and weight,
and smoking and dietary habits. The ques-
tionnaire was designed to obtain information
on potential confounders, including metabolic
diseases, gravidity, parity, and weight changes
in the last years. A detailed medical and gyne-
cologic history was taken, and all patients
underwent clinical and ultrasound examina-
tions. For each woman, the body mass index
(BMI) was calculated. Before laparoscopy, a
blood specimen of approximately 30 mL was
collected from the cubital vein in Vacutainer
tubes and centrifuged. Serum specimens were
stored at –20°C until subjected to analysis.
A 10-mm laparoscopy was performed under
general anesthesia. The presence of endo-
metriosis was confirmed by histologic analy-
sis of lesions, and the disease was staged in
80 women according to the revised American
Society of Reproductive Medicine (ASRM)
classification. The control group consisted of
women without complaints of infertility or
pelvic pain who were undergoing laparoscopy
for benign gynecologic conditions and had
no visual evidence and histologic features of
endometriosis in random peritoneal biopsies.
The questionnaire administered to docu-
ment dietary habits included 16 questions on
the frequency of consumption (times/month)
of various milk, meat, and fish products.
Analysis of serum samples: polychloro-
biphenyls and pesticides. Serum samples were
added with a mixture of 13C-labeled internal
standards (Cambridge Isotope Laboratories
Inc., Andover, MA, USA) comprising PCB-
28, PCB-52, PCB-101, PCB-118, PCB-138,
PCB-153, PCB-156, and PCB-180, HCB,
and p,p´-DDE and kept overnight at 4°C.
Before extraction, we thawed the spiked sam-
ples at room temperature, added a 4:1 (vol/vol)
mixture of formic acid and iso-propanol (J.T.
Baker, Phillipsburg, NJ, USA), and sonicated
them. Extraction was performed with five
6-mL aliquots of n-hexane (Merck KGaA,
Darmstadt, Germany) by manual shaking, fol-
lowed by centrifugation at 3,500 revolutions
per minute (rpm) for 5 min. We removed the
n-hexane aliquots collected from centrifuga-
tion, pooled them in a centrifuge tube, and
carefully concentrated them. Concentrated
sulfuric acid (H2SO4, Carlo Erba, Milan,
Italy) was added to the n-hexane extracts; the
two phases were vigorously shaken and then
separated by centrifugation (3,500 rpm for 20
min). We reduced the volume of the purified
extracts and transferred them into 1-mL vials
to undergo instrumental analysis.
Instrumental analysis was carried out by
using ion trap mass spectrometry (IT-MS)
(Polaris Q; Thermofisher Scientific Inc.,
Waltham, MA, USA) coupled with high-
resolution gas chromatography (GC). A
RTX-5MS 60-m length, 0.25-mm i.d. cap-
illary column (Restek Corporation, State
College, PA, USA) coated with a 0.25-µm
film was employed to separate the extracted
compounds. The initial oven temperature
of 70°C was increased to 190°C at a rate of
30°C/min, subsequently to 280°C at 5°C/min
and to 330°C at 20°C/min, and maintained
at 330°C for 4 min (total GC run time was
28 min). The injector was operated in the
splitless mode with a 1.5-min splitless time.
The initial injector temperature of 70°C was
increased to 280°C at a rate of 14.5°C/sec.
The transfer line and the ion source tem-
peratures were set at 290 and 250°C, respec-
tively. The IT-MS detector was operated in
the electron ionization mode (70 eV) and
MS/MS mode. Selected daughter ions used
for quantification were (analyte, daughter
ion): PCB-28, PCB-186; PCB-52, PCB-257;
PCB-101, PCB-291; PCB-105, PCB-256;
PCB-118, PCB-256; PCB-138 and PCB-153,
PCB-325; PCB-156 and PCB-167, PCB-290;
PCB-170 and PCB-180, PCB-361; HCB-
249; p,p´-DDE-248. Data were processed
using the XCALIBUR software (Thermofisher
Scientific Inc.). Based on the 13C-labeled
compounds employed, recovery ranges were
within 75–110% for all compounds. Analytic
reliability was warranted by the use of an in-
house validated method (Ingelido et al. 2008).
The laboratory has a consolidated experience
in the analysis of halogenated organic micro-
contaminants and periodically participates
in interlaboratory exercises concerning the
analysis of PCDDs, PCDFs, PCBs, organo-
chlorinated pesticides, and some brominated
flame retardants in dietary, biological, and
environ mental matrices.
Analysis of serum samples: compounds
with dioxin-like activity. We conducted the
analysis of compounds eliciting dioxin-like

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Porpora et al.
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volume 117 | number 7 | July 2009 • Environmental Health Perspectives
activity by the dioxin receptor (DR or AhR)-
driven chemically activated luciferase expres-
sion bioassay (DR-CALUX; BioDetection
Systems, Amsterdam, The Netherlands), a
bioanalytic tool used to detect AhR active
compounds, such as dioxins, present in dif-
ferent environmental and biological matrices
(Hoogenboom et al. 1999; Murk et al. 1997).
DR-CALUX cells were purchased from
BioDetection Systems. We cultured them
routinely in a 5% CO2 atmosphere at 37°C in
Alpha MEM supplemented with 10% heat-
inactivated fetal calf serum (GIBCO BRL,
Gaithersburg, MD, USA). The luciferase
assay system, Britelite kit, was purchased
from PerkinElmer Life and Analytical Science
(Boston, MA, USA).
Analysis involved liquid-liquid extraction
of blood serum with a 97:3 (vol/vol) mix-
ture of n-hexane and diethyl ether and lipid
removal by eluting the extract on a silica gel
column concentrated H2SO4. The purified
extract was quantitatively transferred to a vial,
evaporated, and dissolved in dimethyl sulfox-
ide (DMSO) for DR-CALUX measurement.
DR-CALUX cells were seeded in 96-multi-
well plates (Packard ViewPlate, PerkinElmer)
and incubated for 24 hr in a CO2 incubator.
We performed the cell treatments in tripli-
cate, adding to each well 100 µL of sample
extract diluted in culture medium just before
use, using DMSO as vehicle (0.5%, vol/vol
DMSO in culture medium). After 24 hr of
incubation at 37°C, cell monolayers were
checked under an inverted microscope to
exclude any cytotoxic effects of the extracts.
We removed the exposure medium, washed
the cells with phosphate-buffered saline
with calcium and magnesium (pH 7.4), and
lysed them with 100 µL Britelite solution.
Light production was immediately measured
with a MicroBeta luminescence counter
(MicroBeta Jet, 1450 LSC, PerkinElmer). We
always included a standard calibration curve
of 2,3,7,8-TCDD in the experiments and
simultaneously analyzed it with the samples
(Sanderson et al. 1996; Windal et al. 2005).
Eight 2,3,7,8-TCDD concentrations (0.3–300
pM/well), dissolved in DMSO and processed
as described for sample extracts, were tested.
Results expressed as relative light unit values
were transformed into 2,3,7,8-TCDD toxicity
equivalents (TEQs) using the BioDetection
Excel file. Results were expressed in picograms
of DR-CALUX-TEQs (C-TEQs) per gram
of serum fat (pgC-TEQs/g fat). The limit of
detection was calculated as the signal measured
from the DMSO solvent control on each well
plate plus three times its standard deviation.
Lipid analysis. Concentrations of total
cholesterol, phospholipids, and triglycer-
ides were determined by enzymatic methods
(Ingelido et al. 2008) and the use of colori-
metric kits (Futura System s.r.l., Rome, Italy).
Sample size. In setting the study design,
sample size estimation was performed to
determine the number of women per group
sufficient to detect a true odds ratio (OR)
between 2.5 and 3.0. With a power of 80%,
type 1 error of 5%, and 0.20 probability of
exposure in controls, we calculated that 64–94
subjects (ORs = 3.0 and 2.5, respectively) were
required per group. A retrospective power
analysis based on 80 cases and 78 controls
enrolled in the study and a significance level
of α set to 5% provided a power estimation
of 72.6% to detect a risk of 2.5 and 88% to
detect a risk of 3.0.
Statistical analysis. Serum concentrations
of all the analytes determined were subjected
to statistical analysis. Differences in analyte
levels between groups were investigated; geo-
metric rather than arithmetic means were
employed. The statistical significance of differ-
ences between cases and controls was assessed
by Student’s t-test.
The concentration distribution of each ana-
lyte was divided by tertiles, and women were
classified at low, medium, and high exposure.
Using unconditional logistic regression anal-
ysis to adjust for potential confounders, the
adjusted ORs and 95% confidence intervals
(CIs) were estimated for the second and third
tertile of PCBs, C-TEQs, HCB, and p,p´-DDE.
Variables included in the final model were age
(years), BMI (kilograms per square meter),
smoking habits (nonsmokers, exsmokers, smok-
ers), and evidence of relevant weight modifica-
tions in the last 5 years (> 10 kg). In addition,
with the aim to investigate whether different
histopathologic features of endometriosis were
related to different etiologic risk factor pat-
terns, patients were classified according to the
presence of ovarian endometrioma, peritoneal
lesion, and deep lesions, and the groups were
compared. The relationship between the sever-
ity of disease, according to the revised American
Society of Reproductive Medicine (ASRM)
classification and the detected chemical levels
Table 1. Laparoscopic findings and stage of endo-
metriosis classified according to rASRM (1997) in
women affected by endometriosis (cases).
Laparoscopic findings
Stage of endometriosis (rASRM)
I
II
III
IV
Ovarian endometrioma
Yes
No
Peritoneal lesions
Yes
No
Type of peritoneal lesion
Typical
Subtle
Both
Deep endometriosis
Yes
No
Cases [no. (%)]
8 (10.0)
5 (6.2)
44 (55.0)
23 (28.7)
72 (90.0)
8 (10.0)
45 (56.2)
35 (43.7)
23 (53.5)
6 (13.9)
14 (32.6)
6 (7.5)
74 (92.5)
Table 2. Sociodemographic characteristics of cases (n = 80) and controls (n = 78).
Characteristics
Age [years (mean ± SD)]
Age (years)
≤ 25
26–35
≥ 36
No. of childrenb
0
1
2
BMI (mean ± SD)
Relevant weight modifications in the
last 5 years (> 10 kg)b
Yes
No
Food consumption [times/month (mean ± SD)]
Milk and dairy products
Meat
Fish
Age at menarche [years (mean ± SD)]
Breast-fed as childrenb
Yes
No
Unknown
Smoking status
Nonsmokers
Ex-smokers
Smokers (cigarettes/day)
1–9
10 –19
≥ 20
Controls
29.5 ± 6.1
Cases
31.6 ± 6.0
p-Valuea
0.03
18 (23.1)
46 (59.0)
14 (17.9)
12 (15.0)
46 (57.5)
22 (27.5) 0.23
74 (94.9)
4 (5.1)
0 (—)
22.4 ± 4.6
74 (96.1)
2 (2.6)
1 (1.3)
21.1 ± 2.8
0.99
0.03
9 (11.5)
69 (88.5)
8 (10.1)
71 (89.9) 0.98
33 ± 16
25 ± 10
7 ± 5
12.1 ± 1.4
41 ± 25
25 ± 12
8 ± 6
12.2 ± 1.5
0.02
0.93
0.49
0.84
55 (70.5)
13 (16.7)
10 (12.8)
56 (70.9)
15 (19.0)
8 (10.1) 0.83
41 (53.9)
7 (9.2)
44 (55.0)
13 (16.2)
8 (10.5)
13 (17.1)
7 (9.3)
7 (8.8)
8 (10.0)
8 (10.0) 0.54
Values shown are mean ± SD or no. (%)
aStudent’s t- test, chi-square test, Fisher test. bNumbers do not add up due to missing values.

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Organochlorinated pollutants and endometriosis
Environmental Health Perspectives • volume 117 | number 7 | July 2009
1073
was assessed by analysis of variance and chi-
square test. Multiple linear regression analysis
was used to test the association between log-
transformed PCB concentrations in serum and
dietary habits (milk, meat, fish) after checking
for potential confounders.
Results
A total of 158 women were enrolled in the
study, 80 cases and 78 controls. Among
cases, there were eight patients with stage I
endometriosis, 5 with stage II, 44 with stage
III, and 23 with stage IV. In particular, deep
endometriotic nodules were present in 6
women (7.5%), ovarian endometriomas were
observed in 72 patients (90.0%), and perito-
neal implants in 45 cases (56.2%) (Table 1).
The control group consisted of 78 women with
other benign gynecologic conditions, including
eight uterine myomas and 70 adnexal masses,
with no laparoscopic evidence of endometriosis
and random peritoneal biopsies negative for
the disease at histologic analysis.
The main characteristics of the studied pop-
ulation, as derived from the administered ques-
tionnaires, are shown in Table 2. Mean age at
interview was slightly higher in cases (31.6 ± 6.0
years) compared with controls (29.5 ± 6.1 years).
Moreover, cases had a lower BMI (21.1 ± 2.8)
than controls (22.4 ± 4.6). Both these differences
were taken into account using a multivariate
analysis. Age at menarche, having been breast-
fed, smoking habits, and alcohol consumption
did not significantly differ between the groups.
Dietary habits, described by the analysis of the
dietary intakes of the main categories of food-
stuffs, were not significantly different in the case
of meat and fish. Average monthly consumption
of milk and dairy products was slightly differ-
ent among cases and controls (41 vs. 33 times/
month), but the associated incremental risk of
endometriosis could be considered negligible
(OR = 1.02; 95% CI, 1.00–1.04).
Geometric means and pertinent CIs of
the analytes assessed in cases and controls are
shown in Table 3. The concentrations of all
the analyzed PCBs were higher in cases than
in controls. Significant differences (p < 0.05)
were found for PCB-101, PCB-156, and
PCB-170, and highly significant differences
(p < 0.01) were found for PCB-52, PCB-118,
PCB-138, PCB-153, and PCB-180. p,p´-DDE
and total PCB serum concentrations, as the
sum of all the 11 congeners analyzed, were also
significantly higher (relative increase: 44.9 and
48.4%, respectively) in cases than in controls.
As for the AhR ligands that elicit a
response of the DR-CALUX system [dioxins,
DL-PCBs, and possibly other (pseudo) planar
aromatic pollutants present in serum], no sig-
nificant difference was detected between cases
and controls, despite the observed increase of
two DL-PCBs (PCB-118, p = 0.0002, and
PCB-156, p = 0.01) in women with endo-
metriosis. However, as noted above, C-TEQs
account for all compounds with dioxin-like
activity, with PCBs 118 and 156 likely provid-
ing a minor contribution.
Table 4 shows the frequency distributions
of all the analytes divided by tertiles, as well as
the results of the multivariate analysis adjusted
for age at interview, smoking habits, BMI, and
weight loss. Concentrations of PCB congeners
118, 138, 153, and 170 are associated with a
significant increased risk of endometriosis for
the second and third tertile when compared
with the lowest tertile. Risk of endometriosis
appears to be significant (OR = 3.05; 95% CI,
1.25–7.42) also for the highest serum concen-
trations of PCB-180 (≥ 60.5 ng/g fat).
As to the sum of all PCB congeners,
patients in the mid and upper tertiles have a 4-
to 5-fold increased risk of having the disease.
Covariate-adjusted ORs showed that no
increased risk of endometriosis was associated
with serum levels of HCB, p,p´-DDE, and
total C-TEQs, the latter determined by the
DR-CALUX bioassay.
To explore a possible correlation between
analyte serum concentrations and the disease
type and localization, we stratified cases by
peritoneal, deep, or ovarian endometriosis
and compared levels of total PCBs, C-TEQs,
HCB, and p,p´-DDE among groups. No dif-
ferences in mean analyte levels could be found
Table 3. Serum concentrations [geometric mean (95% CI); ng/g fat] of PCBs, C-TEQs, HCB, and p,p´-DDE in
cases and controls.

PCB-28
PCB-52
PCB-101
PCB-105
PCB-118
PCB-138
PCB-153
PCB-156
PCB-167
PCB-170
PCB-180
Total PCBs
(pgC-TEQs/g fat)
HCB
p,p´-DDE
Controls
3.4 (2.5–4.5)
1.6 (1.3–1.9)
1.6 (1.3–1.9)
5.7 (4.1–8.1)
15.2 (13.1–17.6)
33.8 (29.0–39.3)
61.8 (51.8–73.8)
5.7 (4.7–6.9)
2.5 (2.0–3.1)
6.1 (4.8–7.7)
34.4 (29.0–40.8)
203.0 (179.4–229.8)
20.9 (17.3–25.2)
40.9 (32.6–51.4)
303.1 (247.0–371.9)
Cases
4.3 (3.1–6.0)
2.2 (1.9–2.6)
2.1 (1.7–2.5)
6.8 (4.9–9.4)
23.4 (19.7–27.9)
50.7 (43.4–59.1)
99.8 (87.5–113.8)
8.3 (6.7–10.4)
3.2 (2.6–3.9)
8.8 (7.1–10.8)
48.6 (42.0–56.2)
301.3 (271.1–334.9)
18.6 (14.5–23.9)
35.8 (28.6–44.8)
439.1 (360.4–534.9)
p-Valuea
0.3
< 0.01
0.04
0.47
< 0.01
< 0.01
< 0.01
0.01
0.08
0.02
< 0.01
< 0.01
0.47
0.41
0.01
at-Test on log-transformed values.
Table 4. Frequency distribution of serum levels of
PCB, C-TEQs, HCB, and p,p´-DDE and adjusted ORs
for endometriosis by tertiles.
Concentration Controls
(ng/g fat)
PCB-28
≤ 2.3
2.4–6.5
≥ 6.9
PCB-52
≤ 1.4
1.5–2.4
≥ 2.5
PCB-101
≤ 1.35
1.36–2.4
≥ 2.5
PCB-105
≤ 3.7
3.8–11.2
≥ 11.3
PCB-118
≤ 13.2
13.3–24.2
≥ 24.3
PCB-138
≤ 33.6
33.7–56
≥ 57
PCB-153
≤ 62
63–104
≥ 105
PCB-156
≤ 4
5–9
≥ 10
PCB-167
≤ 1.9
2–3.9
≥ 4
PCB-170
≤ 5.37
5.38–12.4
≥ 12.5
PCB-180
≤ 33.2
33.3–60.4
≥ 60.5
Total PCBs
≤ 208
209–305
≥ 306
(pgC-TEQs/g fat)
≤ 15.6
15.7–29.5
≥ 29.6
HCB
≤ 31
32–54
≥ 55
p,p´-DDE
≤ 231
232–492
≥ 493
Cases
(%)

(%) ORadja (95% CI)
29.49
43.59
26.92
37.50
25.00
37.50
1.0
0.35 (0.15–0.84)
0.98 (0.42–2.25)
41.03
35.90
23.08

39.74
34.62
25.64

35.90
34.62
29.49

48.72
25.64
25.64

46.15
29.49
24.36

51.28
24.36
24.36

39.74
34.62
25.64

35.90
34.62
29.49

45.95
29.73
24.32

43.24
35.14
21.62

51.35
25.68
22.90

30.2
35.8
34.0

35.90
30.77
33.33

41.03
30.77
28.21
27.50
35.00
37.50

30.00
30.00
40.00

31.25
32.50
36.25

21.25
38.75
40.00

21.25
36.25
42.50

16.25
42.50
41.25

27.50
33.75
38.75

31.25
32.50
36.25

21.25
37.50
41.25

23.75
32.50
43.75

16.25
41.25
42.50

35.7
30.4
33.9

33.75
35.00
31.25

26.25
35.00
38.75
1.0
1.51 (0.68–3.35)
1.96 (0.84–4.56)

1.0
1.29 (0.57–2.90)
1.93 (0.86–4.36)

1.0
1.23 (0.55–2.75)
1.55 (0.68–3.50)

1.0
3.17 (1.36–7.37)
3.79 (1.61–8.91)

1.0
2.37 (1.02–5.48)
3.78 (1.60–8.94)

1.0
4.64 (1.93–11.1)
4.88 (2.01–11.0)

1.0
0.98 (0.43–2.24)
1.65 (0.71–3.82)

1.0
0.97 (0.43–2.17)
1.18 (0.50–2.77)

1.0
2.71 (1.13–6.51)
3.52 (1.41–8.79)

1.0
1.33 (0.57–3.11)
3.05 (1.25–7.42)

1.0
4.64 (1.93–11.16)
5.63 (2.25–14.10)

1.0
0.52 (0.18–1.48)
0.73 (0.26–2.01)

1.0
0.91 (0.40–2.08)
0.65 (0.27–1.54)

1.0
1.54 (0.66–3.58)
2.14 (0.93–4.93)
aOR adjusted for age, smoking habits, BMI, evidence of
weight modification.

Page 5

Porpora et al.
1074
volume 117 | number 7 | July 2009 • Environmental Health Perspectives
in women with endometriosis with respect to
the different kind of disease.
Furthermore, the association between
serum concentrations of the analyzed organo-
chlorinated compounds and the stage of the
disease (according to the revised ASRM classi-
fication) was investigated. No significant dif-
ferences between groups were observed: Data
analysis showed that pollutant concentrations
did not correlate with disease severity.
The relationship between different food
intakes and values of PCBs, C-TEQs, HCB,
and p,p´-DDE was estimated by a multivari-
ate regression model. The lack of significance
(p > 0.05) of slope coefficients suggests that
food intake is not associated with the con-
centrations of the chemicals analyzed in the
present study.
Discussion
The results of the present study are in agree-
ment with findings we had obtained in two
previous investigations carried out on smaller
samples of nulliparous women, in which cases
resulted to have a significant increase in blood
concentrations of PCB-118, PCB-138, PCB-
153, PCB-180, the sum of the mentioned 11
PCBs (Porpora et al. 2006; Quaranta et al.
2006), as well as of p,p´-DDE (Quaranta et al.
2006). In particular, a greater than 3-fold
increase was found for these four PCB conge-
ners and their sum.
The observed absence of an increase in
TEQ values in women with endometriosis
with respect to controls confirms the results
we obtained in a study carried out on pooled
blood samples from two small groups of
Italian and Belgian women of reproductive
age (De Felip et al. 2004), in which no corre-
lation was observed between dioxin-like com-
pounds and the disease, on a country basis.
This agreement is observed although different
analytical methodologies were used: High-
resolution GC coupled with high resolution
mass spectrometry was employed to deter-
mine dioxin-like compounds in our previ-
ous study, whereas DR-CALUX bioassay was
used in this work.
The present study and the three above
have similar study designs. In fact, with the
exception (in this study) of a small minority
of non-nulliparous women who had never
breastfed, only nulliparous women were
enrolled, to avoid the confounding factor of
breast-feeding, known to determine a signifi-
cant decrease in organochlorine body burden.
All women underwent surgical confirmation
of the disease or its absence, and only women
with no visual evidence or histologic signs
of endometriosis in random peritoneal biop-
sies were included in the control group. In
the present study, women with complaints
of infertility were not enrolled as controls,
because some organochlorinated pollutants
have been hypothesized to be associated with
infertility, in particular p,p´-DDE (Korrick
et al 2001; Weiss et al. 2006). There are no
data regarding a correlation between the other
gynecologic conditions of the control group
(uterine myomas, benign adnexal mass) and
the aforesaid pollutants.
A comparison of this study with other
case–control studies designed to explore
the association between persistent organo-
halogenated pollutants and endometriosis is
complicated by the variety of study designs,
analytical methodologies used, and number
and type of compounds or congeners assessed.
Recent papers (Anger and Foster 2008;
Heilier et al. 2008) have presented a compre-
hensive overview of the studies carried out on
this topic, the vast majority focused on assess-
ing the association between dioxins and/or
PCBs and peritoneal endometriosis, and have
discussed their limited or null comparabil-
ity. On the whole, no significant correlation
was observed in case–control studies between
NDL-PCBs and/or DL-PCBs and endo-
metriosis (Buck Louis et al. 2005; Fierens
et al. 2003; Gerhard and Runnebaum 1992;
Heilier et al. 2004, 2005; Lebel et al. 1998;
Pauwels et al. 2001; Tsukino et al. 2005),
although a nonsignificant increase of the most
abundant NDL-PCB-138, PCB-153, and
PCB-180 was observed in one study (Gerhard
and Runnebaum 1992) and of the same three
congeners plus the DL-PCB-118 in another
study (Pauwels et al. 2001).
As to the sparse case–control studies avail-
able specifically focused on the AhR ligands
(PCDDs, PCDFs, and DL-PCBs), a significant
association with these chemicals and the disease
was generally not observed (Anger and Foster
2008; Pauwels et al. 2001), whereas a few stud-
ies reported increased but not significant ORs
for the disease (Buck Louis et al. 2005; Heilier
et al. 2005; Mayani et al. 1997). In one study
that considered peritoneal endometriosis and
deep endometriotic (adenomyotic) nodules
separately (Heilier et al. 2005), a significantly
increased risk was associated with dioxin and
DL-PCB serum concentrations in women with
deep endometriosis.
As to the association between endometrio-
sis and organohalogenated pollutants other
than PCBs and/or dioxins, only a few studies
are available, carried out on PBBs (Hoffman
et al. 2007) or organochlorinated pesticides,
including HCB and p,p´-DDE (Tsukino
et al. 2005). No evidence of an association
was found between endometriosis and PBB
or organochlorinated pesticide serum levels in
these studies.
In our study, no correlation was observed
between the disease and HCB, a ubiquitous
persistent pollutant identified in human tis-
sues worldwide, although its use was discon-
tinued decades ago. The effects of HCB on
ovarian function and circulating ovarian ste-
roids were demonstrated on exposed nonhu-
man primates (Foster et al. 1995).
With regard to p,p´-DDE, the main
metabolite of the pesticide p,p´-DDT char-
acterized by both immune and endocrine
toxicity (Halloway 2005; Wójtowicz et al.
2007), findings from the present investigation
confirm an increase in serum concentrations
in women with endometriosis observed in
a previous investigation. In that study, on
the basis of the reported observation of an
immunologic dysregulation in women simul-
taneously exposed to p,p´-DDE and PCBs
(Daniel et al. 2002), we evaluated the immu-
nologic status of two small groups of women
and determined the serum concentrations of
the most abundant PCBs and p,p´-DDE to
evaluate their possible role in dysregulation
of the immune function observed in patients
with endometriosis. The results we obtained
showed that peripheral blood NK cytotoxic
activity and interleukin-1 beta and interleu-
kin-12 production were significantly down-
regulated in patients with endometriosis with
respect to controls, and this matched with
higher serum concentrations of PCBs and
p,p´-DDE in the same patients (Quaranta
et al. 2006).
In addition to peritoneal endometriosis,
deep endometriosis (adenomyotic) nodules
of the recto-vaginal septum, considered a dis-
tinct clinical entity by some authors (Donnez
et al. 1996), have also been studied as to their
potential association with exposure to organo-
chlorinated pollutants. For this type of endo-
metriosis, Heilier and coworkers (Heilier et al.
2004, 2005) found a significantly increased risk
associated with both NDL-PCBs and dioxin-
like compounds in serum. When we analyzed
the association between the serum concentra-
tions of PCBs and pesticides and the differ-
ent types of disease, no differences were found
between cases with peritoneal implants, ovar-
ian endometrioma, deep lesions, or combined
lesions. Therefore, it can be hypothesized that
PCBs and p,p´-DDE are risk factors for devel-
oping any kind of endometriosis. Regarding
the relationship between the severity of disease
(according to the ASRM classification) and
exposure to organochlorinated compounds, we
did not find any significant correlation between
the pollutant serum concentrations and the dif-
ferent stages of the disease.
The reason for the observed increase in
PCB and p,p´-DDE serum concentrations in
patients with endometriosis remains to be elu-
cidated. Because no correlation with dietary
habits was found, such an increase could be
associated with a different capacity in bio-
activation and/or detoxication due to both
genetic makeup and/or induction/inhibition
phenomena in the tested population. Work
is in progress on genetic polymorphisms of

Page 6

Organochlorinated pollutants and endometriosis
Environmental Health Perspectives • volume 117 | number 7 | July 2009
1075
glutathione S-transferase and cytochrome
P-450, enzymes involved in organochlorine
compound biotransformation, to explore
the gene–environment interactions as a pos-
sible cause of the observed higher levels of the
aforesaid compounds in patients with endo-
metriosis and as a risk factor for the disease
onset/progression, as suggested by some stud-
ies (Ertunc et al. 2005; Hsieh et al. 2004;
Tsuchiya et al. 2007).
In summary, our data show that expo-
sure to PCBs and p,p´-DDE represents a risk
factor for endometriosis. In particular, the
observation that ORs increase with increas-
ing PCB concentrations strongly supports the
hypothesis of an association between exposure
to these chemicals and the disease, although
the specific mechanisms of actions remain to
be characterized.
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