Human Reproduction Vol.18, No.12 pp. 2668±2671, 2003 DOI: 10.

1093/humrep/deg484

Nitric oxide synthesis is increased in the endometrial tissue

of women with endometriosis

Ming-Yih Wu1, Kuang-Han Chao1, Jehn-Hsiahn Yang1, Tsung-Hsien Lee1, Yu-Shih Yang1
and Hong-Nerng Ho1,2,3
1
Department of Obstetrics and Gynecology and 2Department of Medical Research, College of Medicine and the Hospital, National
Taiwan University, Taipei, Taiwan
3
To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, National Taiwan University Hospital,
No. 7 Chung-Shan South Road, Taipei, Taiwan, 10063. E-mail: hnho@ha.mc.ntu.edu.tw

BACKGROUND: Previous studies have shown that peritoneal macrophages from women with endometriosis pro-
duce excess nitric oxide (NO). This study was designed to quantify the amount of NO and determine the expression

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of endothelial (eNOS) and inducible NO synthases (iNOS) in women with and without endometriosis. METHODS:
An enzyme-linked immunosorbent assay (ELISA) was performed on endometrial tissues obtained from controls
(myoma, n = 30) and on eutopic/ectopic endometrial tissues from endometriosis patients (n = 34) to evaluate eNOS
and iNOS protein concentrations in these endometrial tissues. A rapid-response chemiluminescence analyser was
used to measure NO directly in fresh endometrial tissues. RESULTS: Mean (6 6 SEM) levels of NO were signi®cantly
increased in the endometrial tissues of women with endometriosis (13.2 6 7.8 versus 19.8 6 12.6 nmol/g tissue; P =
0.016). Apparently higher levels of NO were found in ectopic compared with eutopic endometrium (P = 0.057).
Endometrial tissues of women with endometriosis appeared to contain more iNOS than those of controls (3.6 6 2.2
versus 8.6 6 12.2 pg/mg protein; P = 0.06), but no signi®cant difference was found in eNOS levels. CONCLUSIONS:
Greater amounts of NO and NOS are present in the endometrial tissues of women with endometriosis, implying a
possible role for NO in the pathogenesis of endometriosis.

Key words: endometriosis/nitric oxide/nitric oxide synthases

Introduction the pathogenesis of endometriosis (Dong et al., 2001;

Nitric oxide (NO) is an important factor in female reproductive
processes, including ovulation, menstruation, implantation,
pregnancy maintenance, labour and delivery (Yallampalli et al.,
1998; Manabe et al., 1999; Zervou et al., 1999; Chwalisz and
Gar®eld, 2000; Battaglia et al., 2002; Ekerhovd et al., 2002).
Women with endometriosis have a signi®cantly higher level of
activated peritoneal macrophages (PM) (Halme et al., 1983;
Dunselman et al., 1988) and greater PM activation leading to
increased inducible NO synthase (iNOS) expression and NO
production (Weinberg, 1998; Osborn et al., 2002). High levels
of NO are involved in antimicrobial and antitumour activities,
and an elevation of NO can be pro-in¯ammatory (Weinberg,
1998). By contrast, low levels of NO are important for several
vital physiological processes, including maintenance of smooth
muscle tone, neurotransmission and modulation of apoptosis.
The present authors' group (Ho et al., 1997a; Wu et al., 1999),
together with others (Lebovic et al., 2001; Santanam et al.,
2002), have reported pelvic pro-in¯ammatory reactions in
women with endometriosis.
Others have reported that increased NO concentration, with
altered peritoneal immune defence reaction, was involved in
2668 Human Reproduction 18(12) ã European Society of Human Reproduction and Embryology 2003; all rights reserved
Osborn et al., 2002). No difference was found in total
peritoneal ¯uid (PF) NO in frozen samples from women
with and without endometriosis (Ho et al., 1997b). However,
the PM in endometriotic samples appeared to produce more
NO after lipopolysaccharide (LPS) stimulation (Wu et al.,
1999). A similar result, derived using fresh PM cultures, has
provided further con®rmation of these ®ndings (Osborn et al.,
2002).
In order to verify the source of this PF NO increase in
women with endometriosis, NO levels were examined in
eutopic and ectopic endometrial tissues obtained from affected
women. Likewise, to compare the association between nitrite/
nitrate and endothelial NO synthase (eNOS)/iNOS, the eNOS
and iNOS protein concentrations for these samples were also
measured.
Materials and methods
Collection of eutopic and ectopic endometrial tissue
Endometrial control tissue was obtained from myoma cases (group 1,
follicular phase, n = 13; group 2, luteal phase, n = 17), while ectopic
 Increased NO and NOS in endometriosis

endometriotic and eutopic endometrial tissues were sampled from

women with endometriosis (group 3, follicular phase, n = 17; group 4,
luteal phase, n = 17) during the same period. Since the intention was to
obtain ectopic tissues, only women with the peritoneal type of
endometriosis were enrolled (Nisolle and Donnez, 1997). All of the
latter samples were classi®ed as revised American Fertility Society
stage III or IV. Women were excluded from the study if they had
received any hormonal treatment during the 3 months prior to surgery.
Any patients with endometrial lesions such as submucosal myoma and
endometrial polyps or an intrauterine device were excluded from the
study.
The use of these tissues and the patient protocols were approved by
the authors' institutional review board, without any requirement for
informed consent.

Measurement of nitrite/nitrate in homogenized endometrial tissue
Homogenization
The fresh endometrial tissue was weighed and carefully dissected to
remove blood, mucus and/or myometrium components. The whole
Figure 1. Nitrite/nitrate concentrations of homogenized endometrial
tissues obtained from women with and without endometriosis.
Bars represent mean values (nmol/g tissue). *Concentrations were
signi®cantly higher (P = 0.033; Mann±Whitney U-test) in
endometrial tissues from women with endometriosis (white circles)

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endometrial tissue samples were frozen in liquid nitrogen immediately
after dissection and stored at ±80°C until subsequent measurement of
NOS activity. For measurement of NO, fresh endometrial tissue
(approximate total 0.2 g) was added to 1 ml cold homogenizing buffer
(20 mmol/l HEPES-KOH, pH 7.9; 25% glycerol; 420 mmol/l NaCl;
1.5 mmol/l MgCl2; 0.2 mmol/l EDTA; 0.5 mmol/l dithiothreitol; 0.2
mmol/l phenylmethylsulphonyl ¯uoride). The buffered tissues were
homogenized in a MicrosonÔ cell homogenizer (Misonix, Inc.,
Farmingdale, NY, USA) at maximum speed for 5 s (power
consumption <4 W), and then cooled in ice-water for 30 s. This
procedure was repeated ®ve times to ensure complete tissue
destruction. Dense ®brotic components of the ectopic endometrial
tissues that were not easily dissolved were removed and discarded
after careful weighing.
Deproteinization
Two volumes of 100% cold ethanol (Merck & Co., Inc., Whitehouse
Station, NJ, USA) were added to the homogenized samples; these
were then vortexed and incubated on ice for 30 min. The homogenate
was centrifuged at 12 0003g for 5 min at 4°C, and the supernatant
transferred to a new tube on ice for NO measurement.
Measurement of nitrite/nitrate
A rapid-response chemiluminescence analyser (NOA 280; Sievers
Instruments, Boulder, CO, USA) was used to measure total gas
phase NO (nitrite/nitrate). NO gas reacts with ozone, producing
energy in the form of light, and the light emission is proportional to
the quantity of NO present. The emission can be measured using a
luminometer to determine NO concentration (Ahmed et al., 1997).
The sample tube was securely connected to a Zero Gas Filter (Sievers
Instruments) and room air passed through the device for 5 min. The
linearity of analyser response was interpolated using four repeat
calibrations (blank, 1, 10, 50, 100 and 200 mmol/l respectively; a
lower limit of <1 nmol/l was demonstrated for the present instrument).
The samples (10 ml) were injected into a helium-purged vessel
containing 0.8% vanadium chloride in hydrochloric acid to liberate
gaseous NO from the dissolved NO and nitrite. The sample gas was
then exposed to the ozone in the reaction vessel to form activated
nitrogen dioxide (NO2*), which was detected using a red-sensitive
photomultiplier tube, and the output recorded using an integrating pen
recorder. For each sample, the area under the curve was converted to
NO concentration.
compared with controls (black circles). **The Wilcoxon signed rank
test was used to compare results for eutopic (white circles) with
ectopic tissues (white circles, x-hair) (P = 0.057).
ELISA study of NOS
Protein preparation for NOS
Frozen tissues were homogenized using a method similar to that for
NO determination. The samples were diluted 403 using wash buffer
(R&D Systems, Inc., Minneapolis, MN, USA) for determination of
protein concentration.
Measurement of iNOS and eNOS
A commercially available enzyme-linked immunosorbent assay
(ELISA) kit (R&D Systems, Inc.) was used to quantitate iNOS and
eNOS levels. All samples (diluted 403) and standards were assayed in
duplicate. The iNOS and eNOS sensitivities were 50 and 25 pg/ml
respectively. In accordance with the manufacturer's instructions,
preparations of recombinant human eNOS/iNOS/nNOS (neural NOS),
and mouse eNOS/iNOS at 50 ng/ml were assayed for cross-reactivity
and interference. No signi®cant cross-reactivity or interference was
observed. The NOS contents of the endometrial tissue were derived
using the ELISA results and protein concentrations.
Statistical analysis
Comparisons of total NO and ELISA data of eNOS and iNOS were
performed using the Mann±Whitney U-test. The Wilcoxon signed
rank test was used to compare total NO between ectopic implants and
uterine endometria in cases of endometriosis.
Results
In fresh tissues, nitrite/nitrate concentrations were shown to be
signi®cantly higher in women with endometriosis (Figure 1;
P = 0.033 for the follicular phase; Table I, P = 0.016 when data
for both follicular and luteal phases were combined). Although
statistical signi®cance was not achieved, the NO level tended to
be higher in ectopic than eutopic endometrial tissues (Figure 1,
P = 0.057).
The ELISA quanti®cation of NOS proteins within the
endometrial tissues showed no difference in eNOS concentra-
2669
M.-Y.Wu et al.
Table I. Comparisons of total NO (nitrite/nitrate) and endothelial nitric
oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in
endometrial tissues from women with and without endometriosisa
Parameter Control Endometriosis Pb
(n = 22) (n = 19)
Nitrite/nitrate (nmol/g tissue) 13.2 6 7.8 19.8 6 12.6 0.016
eNOS (pg/mg protein) 6.1 6 5.6 5.8 6 4.3 0.657
iNOS (pg/mg protein) 3.6 6 2.2 8.6 6 12.2 0.060
aValues are mean 6 SEM.
bP-values obtained using the Mann±Whitney U-test.
tions between women with and without endometriosis (Table I,
P = 0.657). Rather, a slight increase in endometrial iNOS
protein concentration was seen in women with endometriosis
when compared with that in women without endometriosis (P =
0.060).
Discussion
Previous studies have reported that NO in PF from endome-
triosis cases is increased relative to that from women without
endometriosis (Dong et al., 2001; Osborn et al., 2002). A
signi®cant difference in PF levels of NO was not demonstrated,
however, in either a recent study using fresh PF (Khorram and
Lessey, 2002) or the present authors' previous report utilizing
frozen PF (Ho et al., 1997b). Another study performed by the
present authors' group determined that PM from patients with
endometriosis produced more NO after LPS stimulation
compared with controls (Wu et al., 1999), and a similar result
was reported by others (Osborn et al., 2002). In order to verify
the difference between these two previous reports, a rapid-
response chemiluminescence analyser was used in the present
study to measure total NO directly in endometrial tissue, and
this showed the endometriotic endometrium to contain more
NO. Furthermore, the ectopic tissues contained even more NO
than the eutopic tissues in affected women. Despite these
®ndings, however, Khorram's paracrine hypothesis of NO
involvement in the pathogenesis of endometriosis could not be
con®rmed absolutely (Khorram and Lessey, 2002).
Immunohistochemical (IHC) staining of iNOS and eNOS
indicated that, in comparison with controls, the endometria
from endometriosis patients showed a more pronounced eNOS
staining in the glandular portion during both the follicular and
luteal phases (data not shown). These results were similar to
those reported previously (Ota et al., 1998; Hatazawa et al.,
2000; Khorram and Lessey, 2002). Another IHC study of
eNOS in endometrial tissue from endometriosis patients
revealed no difference compared with that found in normal
controls (Kamada et al., 2000), although these results may have
been affected by factors such as inter-laboratory staining
variation, inter-observer differences in estimation of staining
intensity, and the scoring system used (Regitnig et al., 2002).
Hence, the decision was made to evaluate the protein content of
eNOS and iNOS directly in endometrial samples from
endometriosis patients and controls. Using ELISA, an eleva-
tion in eNOS could not be demonstrated for the affected
patients, and only increased quantities of iNOS were found.
2670
An earlier Northern blot analysis of endometrial tissues
showed a predominance of eNOS mRNA (Tseng et al., 1996),
but only a small quantity of iNOS mRNA was identi®ed during
menstruation. A subsequent study using RT-PCR of the normal
endometrium (Telfer et al., 1997) showed that both eNOS and
iNOS expression occurred in the glandular epithelial cells
throughout the entire cycle, though these differences in results
may have been due to variations in technique sensitivity. By
using immunoblotting, one group (Osborn et al., 2002) showed
that, compared with normal controls, iNOS activity was
increased in freshly isolated PM from endometriosis cases,
and these results were similar to those of the present study.
Furthermore, the results agreed with the ELISA ®ndings of
increased levels of iNOS protein in the endometrium of women
with endometriosis.
The inducible isoform of NOS (iNOS) produces large
quantities of NO, while the constitutive isoforms (nNOS and
eNOS) produce lower levels (Osborn et al., 2002). The
majority of studies, including those of the present authors
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(Punnonen et al., 1996; Harada et al., 1997; Ho et al., 1997a;
Odukoya et al., 1997) have indicated that activation of PM is
greater in endometriosis cases. The results from the present
study demonstrated through ELISA that the iNOS isoforms
were elevated in tissues from endometriosis patients compared
with those from women without endometriosis.
In endometriosis, measurements of NOS and NO production
may depend on the method selected, as well as the tissues
targeted. In the present study, a rapid-response chemilumines-
cence analyser was used to measure NO production in fresh
endometrial tissues, with NO overproduction in endometriosis
con®rmed. This was the ®rst study to use ELISA to evaluate
iNOS/eNOS protein levels in the endometriotic endometrium.
An attempt to elucidate the role of NO in the pathogenesis of
endometriosis may generate more speci®c data, though the
activity of these enzymes and the associated results may not be
re¯ected by total enzyme quantity. The present study showed
there to be higher levels of NO and NOS in the eutopic/ectopic
endometrial tissues of women with endometriosis. Hence, in
conjunction with the results of previous studies wherein
signi®cantly higher levels of PF NO could not be demon-
strated, the hypothesis is favoured that NO may produce a
paracrine effect in the pathogenesis of endometriosis.
Acknowledgements
These studies were supported by grant no. NTUH 91-S027 from the
National Taiwan University Hospital and grants NSC 91-2314-B-002-
313 and NSC 91-2314-B-002-378 from the National Science Council.
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Submitted on March 14, 2003; resubmitted on June 17, 2003; accepted on
August 20, 2003
2671