Human Reproduction Vol.23, No.3 pp. 530–537, 2008 doi:10.

1093/humrep/dem399
Advance Access publication on December 20, 2007

Endometriosis and human infertility: a new investigation

into the role of eutopic endometrium

Francesca Minici1,†, Federica Tiberi2,4,†, Anna Tropea1, Mariateresa Orlando1,

Maria Francesca Gangale1, Federica Romani1, Sebastiano Campo1, Adriano Bompiani2,
Antonio Lanzone3 and Rosanna Apa1
1
Cattedra di Fisiopatologia della Riproduzione Umana, Università Cattolica del Sacro Cuore (UCSC), 00168 Roma, Italy; 2Istituto
Scientifico Internazionale (ISI) ‘Paolo VI’, UCSC, 00168 Roma, Italy; 3Istituto di Ricerca ‘Associazione OASI Maria SS ONLUS’,
94018 Troina (EN), Italy
4
Correspondence address. Tel: þ39-06-30155297; Fax: þ39-06-30155205. E-mail: fm1810@inwind.it

BACKGROUND: Endometriosis is related to infertility even in the absence of mechanical alterations of the reproduc-

Downloaded from http://humrep.oxfordjournals.org/ by guest on January 2, 2017

tive tract. Even though the pathogenesis of this phenomenon is still unclear, an impaired endometrial receptivity has
been recently suggested. The aim of the present study was to investigate if endometriotic peritoneal fluids (EPF) could
interfere with endometrial stromal cell (ESC) decidualization and if tumor necrosis factor (TNF)-a could be involved
in the EPF effect. METHODS: Eutopic ESC were isolated from patients with or without endometriosis. ESC were
treated with 17b-estradiol 1028 M and 6a-methyl-17a-hydroxyprogesteroneacetate 231027 M for 16 days. In vitro
decidualization was morphologically and biochemically assessed. We analysed whether ESC decidualization could
be affected by EPF or peritoneal fluids from control patients (CPF), with or without soluble TNF-a receptor
1 (sTNFR-1). RESULTS: Compared with ESC from control patients, eutopic ESC from patients with endometriosis
showed an impaired decidualization. Decidualization of normal ESC was morphologically normal but biochemically
abnormal in the presence of EPF, which was able to decrease the secretion of decidualization markers. sTNFR-1 was
able to partially counteract this effect. CONCLUSIONS: In endometriosis, the milieu surrounding the uterine cavity
may be involved in impaired eutopic ESC decidualization, partially due to increased peritoneal levels of TNF-a.

Keywords: endometriosis; endometrial receptivity; decidualization

Introduction
Endometriosis is a benign estrogen-dependent gynecological
disease, which affects 10% of women of reproductive age,
is characterized by the presence of endometrial tissue outside
the uterine cavity, and is associated with pelvic pain, dysme-
norrea and infertility (Ulukus et al., 2006). Despite all the
research accumulated over the years, the etiology and the
pathogenesis of endometriosis are still unclear.
One of the most controversial aspects investigated by several
studies is the link between minimal to mild endometriosis and
infertility, particularly in patients with no mechanical alteration
of the reproductive tract (D’Hooghe et al., 2003). Whether the
endometriosis-related reduction of fertility is due to suboptimal
oocyte/embryo quality or to a compromised endometrium
remains a controversial issue (Kim et al., 2007). Several
studies have shown that gene expression in the endometrium
of women with endometriosis is aberrant, making the endome-
trium suboptimal for an implanting blastocyst (Giudice et al.,
†
These authors contributed equally to the work.
2002; Kao et al., 2003). Very recently, a role for eutopic endo-
metrium in endometriosis-related infertility has been also
suggested by Klemmt et al. (2006), who reported a reduced
decidualization capacity in endometrium from women with
endometriosis. Nevertheless, other authors did not confirm
this observation (Kim et al., 2007).
Since decidualization is a critical process for the successful
establishment and maintenance of pregnancy, we wanted to
further investigate whether eutopic endometrial stromal cells
(ESC) obtained from women affected by endometriosis
(eutopic endometriotic ESC) have a normal capacity to differ-
entiate in vitro to the decidual phenotype in presence of hormo-
nal stimuli. In vivo decidualization is a complex differentiative
process characterized by morphological and functional changes
under the influence of progesterone during the secretory phase
of the menstrual cycle.
In particular, in the present study we compared the decidua-
lization performance of eutopic endometriotic ESC with ESC
obtained from healthy subjects (normal ESC). Typical morpho-
logical changes were assessed and the levels of two important

530 # The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

biochemical decidualization markers were measured in the
culture media: prolactin (PRL) and insulin-like growth factor
binding protein-1 (IGFBP-1) (Bell et al., 1991; Daly et al.,
1983).
Beyond the intrinsically altered properties of eutopic endo-
metriotic ESC, the suggested impaired receptivity may be
due to a negative influence exerted by the uterine cavity
milieu. In order to address this issue, normal ESC were incu-
bated with peritoneal fluids from endometriotic patients
(EPF) or from control subjects (CPF), and decidualization
was morphologically and biochemically assessed. In fact, the
endometrial cavity milieu is supposed to be compromised by
peritoneal fluid diffusion through the tubes (Harada et al.,
2001) and similar immunological activation and hormonal con-
ditions have been observed in both the endometrial and perito-
neal cavities (Selam and Arici, 2000). Moreover, EPF is
characterized by high levels of cytokines, growth factors and
adhesion molecules which have been related to infertility
(Arumugam, 1994; Curtis et al., 1993; Tummon et al., 1988).
In particular, tumor necrosis factor (TNF)-a is secreted at
increased amounts in EPF (Rana et al., 1996) and a clear posi-
tive association between its content in peritoneal fluids and the
severity of endometriosis has been observed (Richter et al.,
2005). In order to verify whether TNF-a could be responsible
for EPF effects, decidualization of normal ESC was performed
in presence of peritoneal fluids with or without the soluble form
of TNF-a receptor-1 (sTNFR1), a TNF-a inhibitor.
Materials and Methods
Chemicals
Collagenase type IA, penicillin-streptomycin solution, 17b-estradiol
(E2), 6a-methyl-17a-hydroxyprogesteroneacetate (MAP) and
sTNFR-1 were purchased from Sigma Aldrich S.r.l. (Milano, Italy).
Bradford reagent was obtained from Bio-Rad Laboratories (Hercules,
CA, USA). Dulbecco Modified Essential Medium (DMEM) and
trypsin were from Invitrogen Inc. (Milano, Italy). Fetal bovine
serum was from Euro Clone (Milano, Italy). PRL enzyme-linked
immunosorbent assay (ELISA) kit was purchased from Radim SpA
(Roma, Italy) and IGFBP-1 ELISA kit was from Diagnistic
Biochem Canada Inc. (Ontario, Canada).
Samples
Participants (n ¼ 58) were women of reproductive age (23 –40 years)
undergoing laparoscopy during the secretory phase of the menstrual
cycle (19–24 days). All patients had a history of regular menstrual
cycles and did not receive any hormonal preparation in the 3
months preceding laparoscopy. Women with endometrial pathology
or with any hormonal alteration were not included. At the time of
laparoscopy, pelvic organs were carefully examined for endometrio-
sis, which was staged according to the American Society for Repro-
ductive Medicine (1997).
Endometriosis was excluded in 25 patients. At the time of laparo-
scopy, these women underwent peritoneal fluid sampling (n ¼ 10)
or uterine curetting in order to obtain eutopic endometrial specimens
(n ¼ 15).
There were 33 patients affected by endometriosis (6 with stage I
disease, 14 with stage II disease, 7 with stage III disease and 6 with
stage IV disease). In the present study, only patients with stage I
and II disease were included (minimal to mild endometriosis). At
The role of eutopic endometrium in endometriosis
the time of laparoscopy, these women underwent peritoneal fluid
sampling (n ¼ 10) or uterine curetting in order to obtain eutopic endo-
metrial specimens (n ¼ 10).
Peritoneal fluids and endometrial biopsies were obtained from
different cohorts of patients for either the normal or endometriotic
groups.
Written informed consent was obtained from all participants and the
present protocol was approved by the institutional review board of
Università Cattolica del Sacro Cuore.
Peritoneal fluid preparation
Once collected, peritoneal fluids were centrifuged (1000 rpm, 10 min,
48C), aliquoted and stored at – 808C (Rong et al., 2002). All peritoneal
fluids, 10 from women with endometriotic (endometriotic peritoneal
fluid, EPF) and 10 without endometriosis (CPF) were separately
pooled shortly before cell culture treatments.
Isolation and primary culture of normal
and eutopic endometriotic ESC
Menstrual secretory phase eutopic endometrium for each normal (n ¼
Downloaded from http://humrep.oxfordjournals.org/ by guest on January 2, 2017
15) and endometriotic (n ¼ 10) specimen was confirmed by histologi-
cal criteria of Noyes et al. (1975).
The separation of ESC from endometrial epithelial cells was
obtained following the method described by Satyaswaroop et al.
(1979) with minor modifications (Pierro et al., 2001).
Purified ESC were suspended in DMEM (supplemented with
10% heat-inactivated fetal bovine serum and 50 mg/ml penicillin –
streptomycin), plated into 25 cm2 tissue culture flasks and cultured
until confluence was reached (378C, 5% CO2).
Cell viability was assessed by Trypan Blue exclusion (85.8%).
Once grown to confluence (2–3 days later), both normal and endo-
metriotic ESC were detached by using trypsin treatment, seeded into
24-well plates (105 cells per well) and used for the in vitro deciduali-
zation experiments described below.
In vitro decidualization of normal and eutopic endometriotic ESC
Either normal or eutopic endometriotic ESC were seeded in 24-well
plates, grown until confluence (1– 2 days) and treated with E2
1028 M and MAP 2  10 – 7 M for 16 days in order to obtain
in vitro decidualization as previously described (Han et al., 1999;
Irwin et al., 1989; Kasahara et al., 2001). Each culture of ESC was
carried out in triplicate. On day 16, cells were counted in order to
assess cell viability.
In order to confirm decidual transformation of ESC, morphological
characteristics of decidualization were checked by inverted micro-
scope (Axiovert 200, Carl Zeiss S.p.A.) in all cell groups (Cornillie
et al., 1985; Wynn, 1974). Biochemical markers of ESC differen-
tiation were also assessed (Bell et al., 1991; Daly et al., 1983). To
this aim, duplicate samples of culture media from all groups of ESC
were assayed on days 7, 10, 13 and 16. Commercially available
ELISA was used to measure levels of either PRL (inter- and
intra-assay coefficients of variation below 6.99 and 8.24%, respect-
ively, sensitivity of the assay 1.5 ng/ml) or IGFBP-1 (inter- and
intra-assay coefficient of variation below 3.4 and 7.4%, respectively;
sensitivity of the assay 0.5 mg/l) in the culture media according to
the manufacturer’s instructions.
PRL and IGFBP-1 levels were normalized to the amount of total
proteins assessed by Bradford reagent in each culture medium.
In vitro decidualization of normal ESC in presence of CPF and EPF
Normal ESC seeded in 24-well plates were grown until confluence
(1–2 days).
531
Minici et al.

In order to evaluate the effect of CPF and EPF on decidualization, a

first group of ESC was treated with E2 1028 M and MAP 2  10 – 7 M
for 16 days as described in the previous paragraph (BASAL group).
Similarly, a second and a third group of ESC were cultured for 16
days in presence of E2 and MAP together with 10% (Rong et al.,
2002) of CPF or EPF, respectively (CPF and EPF groups). Finally, a
fourth group of ESC was cultured for 16 days without E2 and P
(CONTROL group). Each group of ESC was carried out in triplicate.
On day 16, cells were counted in order to assess cell viability. Each
culture from an individual donor was tested simultaneously for sensi-
tivity to the CONTROL, BASAL, CPF or EPF treatments.
As described in the previous paragraph, all groups of ESC were
morphologically and biochemically (PRL and IGFBP-1 levels)
characterized in order to assess decidual transformation. PRL and
IGFBP-1 levels were normalized to the amount of total proteins.
Prior to cell treatment, EPF and CPF were assayed for IGFBP-1 and
PRL content.

In vitro decidualization of normal ESC in presence of sTNFR-1

In order to evaluate the effect of sTNFR-1 on stromal decidualization,

Downloaded from http://humrep.oxfordjournals.org/ by guest on January 2, 2017

each group of normal ESC from the same donor was cultured in pre-
sence of sTNFR-1 (1 ng/ml). The concentration of sTNFR-1 was
chosen in order to largely exceed TNF content of both CPF and
EPF (Richter et al., 2005). sTNFR1 was dissolved in PBS with
0.1% BSA and the solvent was tested on ESC as a negative control.
On day 16, at the end of the decidualization protocol, cell viability
was checked and culture media from all groups of ESC were collected
in order to assess total proteins, PRL and IGFBP-1 levels as previously
described.
Statistical analyses
Statistical analysis was performed using ANOVA followed by the
‘Tukey–Kramer’ test for comparisons of multiple groups or by
paired Student’s t-test for comparison of data derived from two
groups. Values with P , 0.05 were considered statistically significant.
Results
In vitro decidualization of normal ESC
As expected, the incubation of normal ESC with E2 1028 M
and MAP 2  10 – 7 M for 16 days (BASAL group) was able
to induce stromal differentiation. In particular, in the presence
of E2 and MAP, ESC were transformed into large polygonal
cells resembling in vivo decidual cells, as previously described
(Kasahara et al., 2001).
Moreover, ESC decidualization was evaluated by biochemi-
cal markers assays. Fig. 1 shows PRL (panel A) and IGFBP-1
(panel B) secretion in the BASAL group of normal ESC on
days 7, 10, 13 and 16 of the treatment with E2 and MAP. In
our in vitro system, on days 10, 13 and 16 both PRL and
IGFBP-1 release was increased in the culture media compared
to day 7, confirming the occurrence of stromal differentiation.
A time-dependent increase was observed for PRL and IGFBP-1
release until days 13 and 16, respectively. PRL and IGFBP-1
values were normalized to the total proteins from cultured
cells. At day 7 the mean value was 2.5 + 0.3 mg, and in the
following time points total protein amount didn’t change in a
statistically significant manner (data not shown).
As expected, normal ESC incubated for 16 days without E2
and MAP (CONTROL group) retained a fibroblast-like
532
Figure 1: Time dependent secretion of PRL (panel A) and IGFBP-1
(panel B) in BASAL group of normal ESC on days 7, 10, 13 and 16 of
treatment with E2 and MAP (in vitro decidualization)
PRL and IGFBP-1 are expressed in ng/mg total protein. Each value
represents the mean + SEM of 15 independent experiments.
***P , 0.001 respect to day 7; 888P , 0.001 respect to day 10;
þþ þ
P , 0.001 respect to day 13
spindle-shaped appearance (Kasahara et al., 2001). Moreover,
in the CONTROL group, PRL and IGFBP-1 release did not
show any increase compared to day 7 (data not shown).
In vitro decidualization of eutopic endometriotic ESC
We wanted to test whether eutopic endometriotic ESC retain
the capacity for differentiation in response to E2 1028 M and
MAP 2  10 – 7 M for 16 days as assessed by morphology
and measurement of PRL and IGFBP-1 secretion.
As previously demonstrated by Klemmt et al. (2006) in
response to 8-Br-cAMP, from day 7 onward eutopic endome-
triotic ESC treated with E2 1028 M and MAP 2  10 – 7M
were able to differentiate into polygonal decidual cells, and
IGFBP-1 secretion by eutopic endometriotic ESC was signifi-
cantly lower than from normal ESC (Fig. 2B).
Eutopic endometriotic ESC secreted significantly lower
levels of PRL on days 7, 10 and 13 with respect to normal
ESC, however from day 16, eutopic endometriotic ESC were
able to secrete higher levels of PRL with respect to normal
ESC (Fig. 2A).
PRL and IGFBP-1 values were normalized to the total pro-
teins from cultured cells. At day 7 the mean value was 2.3 +
0.4 mg, and in the following time points the total protein
amount didn’t change in a statistically significant manner
(data not shown).

Figure 2: Time dependent secretion of PRL (panel A) and IGFBP-1
(panel B) from normal (empty circles) and eutopic endometriotic
(black circles) ESC on days 7, 10, 13 and 16 of treatment with E2
and MAP (in vitro decidualization)
PRL and IGFBP-1 are expressed in ng/mg total protein. Each value
represents the mean + SEM of 15 (normal ESC) or 10 (endometriotic
ESC) independent experiments. ***P , 0.001, **P , 0.01 and *P ,
0.05 respect to normal ESC
Effects of CPF and EPF on PRL and IGFBP-1 release
during normal ESC decidualization
In order to assess the effects of CPF and EPF on PRL and
IGFBP-1 release by stromal differentiating cells, normal ESC
were cultured for 16 days in the presence of E2 and MAP
together with 10% of CPF or EPF (CPF and EPF groups,
respectively). With this aim on days 7, 10, 13 and 16, PRL
and IGFBP-1 were assayed in the culture media of both groups.
On culture day 7, no effect was observed on PRL and
IGFBP-1 release for either the CPF or EPF groups with
respect to BASAL group (Figs. 3A and 4A).
We demonstrated that CPF was able to significantly reduce
IGFBP-1 levels respect to the BASAL group from day 10
onward (Fig. 4B – D). On the contrary, PRL levels were signifi-
cantly decreased only on day 16 by CPF with respect to the
BASAL group (Fig. 3D).
Our results show the ability of EPF to significantly reduce
PRL and IGFBP-1 levels with respect to the BASAL group
on day 10 (Figs. 3B and 4B), on day 13 (Figs. 3C and 4C)
and on day 16 (Figs. 3D and 4D). Moreover, on days 13 and
16, EPF was able to reduce PRL (Fig. 3C and D) or IGFBP-1
(Fig. 4C and D) release with respect to the CPF group in a sig-
nificant manner.
Preliminary assays were performed in order to assess
IGFBP-1 and PRL content in EPF and CPF. The levels of
The role of eutopic endometrium in endometriosis
both IGFBP-1 and PRL were under the lower detection limit
of the assay in both CPF and EPF.
During in vitro decidualization, normal ESC underwent the
typical morphological modifications in both the CPF and
EPF groups, despite the above mentioned differences in
secretion of biochemical markers.
Effect of CPF and EPF on PRL and IGFBP-1 release
during normal ESC decidualization in the
presence of sTNFR-1
In order to evaluate the effect of sTNFR-1 on stromal decidua-
lization, sTNFR-1 1 ng/ml was added to the culture media of
normal ESC belonging to the BASAL, CPF and EPF groups.
At the end of the differentiation protocol (day 16), PRL and
IGFBP-1 were assayed in the culture media.
No difference in PRL or IGFBP-1 release was found when
sTNFR-1 was added to the BASAL group of ESC (data not
shown).
Similarly, no difference in IGFBP-1 release was found when
Downloaded from http://humrep.oxfordjournals.org/ by guest on January 2, 2017
sTNFR-1 was added to the CPF and EPF groups of ESC (data
not shown).
On the contrary, sTNFR-1 was able to counteract EPF
effects on PRL release with respect to BASAL group
(Fig. 5A and B). The addition of sTNFR-1 was able to signifi-
cantly modify the inhibitory effect on PRL release previously
described for the EPF group. No significant effect was
observed on CPF-treated cells.
Discussion
Decidualization is a complex differentiative process character-
ized by morphological and functional changes under the influ-
ence of progesterone during the secretory phase of the
menstrual cycle (Hess et al., 2007). It is well known that decid-
ualization is critical for the successful establishment and main-
tenance of pregnancy (Kim et al., 2007). Very recently, a
reduced decidualization capacity has been reported in endome-
trium from women with endometriosis (Klemmt et al., 2006).
Kim et al. (2007) did not confirm this observation, invoking
differences in experimental design as possible explanations
for the conflicting results. Klemmt et al. elicited in vitro decid-
ualization using 8-Br-cAMP alone without including pro-
gesterone, i.e. known to be critical in the decidualization
process. Indeed, the use of cAMP alone has been described
as being able to ‘prime’ cells for decidualization, but unable
to up-regulate all the genes required for optimal differentiation
without progesterone cooperation (Brosens and Gellersen,
2006; Kim et al., 2007). Moreover, endometrial cells used in
the Klemmt study were obtained randomly at different stages
of the menstrual cycle (Kim et al., 2007).
In order to contribute to debate on impaired decidualiza-
tion in endometriosis, we compared in vitro decidualization
of eutopic endometriotic ESC with normal ESC by using E2
and MAP as previously reported (Han et al., 1999; Irwin
et al., 1989; Kasahara et al., 2001). Either normal or
eutopic endometriotic ESC were obtained exclusively from
women during the secretory phase of the menstrual cycle,
when cells are appropriately primed to respond to a
533
Minici et al.

Downloaded from http://humrep.oxfordjournals.org/ by guest on January 2, 2017

Figure 3: Effect of CPF and EPF on PRL released by decidualizing normal ESC on day 7 (panel A), on day 10 (panel B), on day 13 (panel C) and
on day 16 (panel D)
Results are expressed as percentage of PRL released by a BASAL group set equal to 100. Each value represents the mean + SEM of 15
independent experiments. ***P , 0.001 and **P , 0.01 respect to BASAL; 88P , 0.01 respect to CPF

Figure 4: Effect of CPF and EPF on IGFBP-1 release by decidualizing normal ESC on day 7 (panel A), on day 10 (panel B), on day 13 (panel C)
and on day 16 (panel D)
Results are expressed as percentage of IGFBP-1 released by a BASAL group set equal to 100. Each value represents the mean + SEM of 15
independent experiments. ***P , 0.001 respect to BASAL; 8P , 0.05 respect to CPF

534

Figure 5: Effect of CPF and EPF on PRL release by decidualizing
normal ESC on day 16 in the presence (‘þsTNFR-1’) or absence
(‘2sTNFR-1’) of sTNFR-1
Results are expressed as percentage of PRL released by the BASAL
group set equal to 100. Each value represents the mean + SEM of
15 independent experiments. ***P , 0.001 and **P , 0.01 respect
to BASAL; 88P , 0.01 respect to ‘2sTNFR-1’
decidualization stimulus (Kim et al., 2007). Our results
demonstrated a reduced decidualization capacity for
eutopic endometriotic ESC, since PRL and IGFBP-1 levels
released were significantly lower with respect to normal
ESC starting from day 7 until day 13. These data seem to
suggest that the decidualization process of eutopic endome-
trium could be impaired in endometriosis, potentially affect-
ing endometrial receptivity, as previously suggested by
Klemmt et al. (2006).
However, contrary to Klemmt results, in the present study on
day 16 PRL levels were significantly higher in eutopic endome-
triotic cells than in normal ESC. The PRL increase on day 16
could suggest a sort of hyper-response to E2 and MAP, prob-
ably reflecting the ability of eutopic endometriotic ESC to
differentiate when they are rescued from negative environ-
mental influences after several days of hormonal stimulation.
Alternatively, the PRL surge on day 16 could be due to a
dysregulation in the response of eutopic endometriotic ESC
to hormonal stimuli. Indeed, an aberrant distribution of
progesterone and E2 receptors has been previously described
in eutopic endometrial tissue obtained by patients affected by
endometriosis (Attia et al., 2000; Bulun et al., 2006; Jackson
et al., 2007).
The role of eutopic endometrium in endometriosis
Interestingly, our results demonstrated that IGFBP-1
secretion by eutopic endometriotic ESC was significantly
lower than from normal ESC even on day 16, when PRL
levels were higher than from normal ESC. We can hypothesize
that in eutopic endometriotic ESC, the amount of PRL secreted
by cells after 16 days could autocrinally determine the lack of
IGFBP-1 surge. Very recently, Eyal et al. (2007) demonstrated
an autocrine mechanism by which PRL could regulate the
extent of differentiation in human uterine fibroblast cells by
negatively affecting the expression of IGFBP-1, as well as of
other genes known to be induced in decidualization.
On the basis of these in vitro results, it is not possible to con-
clude whether the impaired decidualization of eutopic endome-
triotic ESC reflects a negative environmental influence or an
intrinsic cell dysregulation. In order to investigate whether
endometriotic milieu surrounding the uterine cavity could
affect ESC differentiation, we compared the effects of EPF
to CPF on normal ESC decidualization.
We demonstrated that EPF, but not CPF, can negatively
Downloaded from http://humrep.oxfordjournals.org/ by guest on January 2, 2017
affect decidualization of normal ESC by decreasing PRL
secretion compared with basal decidualization (BASAL
group). Interestingly, IGFBP-1 release was strongly decreased
by both EPF and CPF starting from day 10, even though on day
13 and 16 EPF exerted a higher negative effect on IGFBP-1
than CPF did. We can hypothesize that peritoneal fluids
could interfere with IGFBP-1 determination by affecting post-
secretion IGFBP-1 levels. Indeed, it is well known that either
urokinase-type plasminogen activator (uPA) or metalloprotei-
nases (MMPs) are able to proteolyze IGFBPs and that both
of them are components of peritoneal fluids (Coppock et al.,
2004; Lembessis et al., 2003). Whether or not this IGFBP-1
proteolytic activity is increased in endometriosis is highly
controversial in the literature (Gilabert-Estelles et al., 2003;
Laudanski et al., 2005; Szamatowicz et al., 2002). Neverthe-
less, very recently, the peritoneal fluid from women with endo-
metriosis has been demonstrated to show a significant increase
in uPA and MMP-3 levels compared to that of controls
(Gilabert-Estelles et al., 2007). This last finding could
explain our results, either the low levels of IGFBP-1 in pre-
sence of both peritoneal fluids or the effect exerted by EPF
being stronger than that of CPF.
Despite the difference in biochemical markers secretion, in
our system eutopic endometriotic ESC as well as normal
ESC under all tested conditions (BASAL, CPF, EPF) under-
went the typical morphological modifications. This peculiarity
has been already demonstrated by previous papers, suggesting
that the endometrium from women with endometriosis displays
morphologically normal, but biochemically abnormal, decid-
ualization responses (Giudice et al., 2002; Klemmt et al.,
2006).
Finally, we tested whether sTNFR-1 could modify the effect
of EPF on normal ESC differentiation, since TNF-a is secreted
at increased amounts in EPF (Rana et al., 1996) and is able to
decrease PRL secretion from stromal cells during in vitro
decidualization (Inoue et al., 1994). Our data demonstrated
that the negative influence of EPF on PRL secretion was
limited by adding sTNFR-1, suggesting that TNF-a could be
involved in the effects exerted by EPF. These in vitro results
535
Minici et al.
may contribute to the current research on the possible thera-
peutic targets of TNFa-inhibitors on the treatment of endome-
triosis. Actually, promising animal research using TNF
inhibitors provide evidence of the potential effectiveness of
such an anti-inflammatory drug in the management of endome-
triosis and confirm the role of TNF in the development of this
disease (D’Antonio et al., 2000; D’Hooghe, 2003).
Moreover, Braun et al. have demonstrated for endometrial
cells obtained from patients affected by endometriosis a
higher sensitivity to the effects of TNF than for endometrial
cells from healthy subjects (Braun et al., 2002). We could
speculate that the effects of EPF and sTNFR-1 on eutopic endo-
metriotic ESC could be even stronger if we consider their
intrinsically higher ability of utilize factors in peritoneal
fluids, such as TNF. New experiments would be very useful
to directly test the sensitivity to TNF of eutopic endometriotic
ESC during decidual differentiation.
Surprisingly, sTNFR-1 was not able to affect IGFBP-1
secretion when added in combination with either CPF or
EPF. We can hypothesize that any possible sTNFR-1 effect
on IGFBP-1 levels could be masked by strong post-secretion
IGFBP-1 proteolysis in presence of peritoneal fluids, as we pre-
viously hypothesized.
In conclusion, on the basis of our results, we can speculate
that in endometriosis either intrinsic defects of ESC differen-
tiation or the biochemical environment of the uterine cavity
could concur to compromise the normal decidualization
required for optimal implantation. In particular, a role for
TNF-a is suggested by the capacity of sTNFR-1 to attenuate
the inhibitory effects of EPF on PRL secretion by ESC.
Funding
This work was supported by grants from Italian Health
Ministry. Current Research 2006; title project: ‘La prevenzione
dell’handicap mentale: ruolo dei fattori paracrini ovarici ed
endometriali nelle prime fasi di gestazione’.
References
American Society for Reproductive Medicine. Revised American Society for
Reproductive Medicine classification of endometriosis: 1996. Fertil Steril
1997;67:817– 821.
Arumugam K. Endometriosis and infertility: raised iron concentration in the
peritoneal fluid and its effect on the acrosome reaction. Hum Reprod
1994;9:1153– 1157.
Attia GR, Zeitoun K, Edwards D, Johns A, Carr BR, Bulun SE. Progesterone
receptor isoform A but not B is expressed in endometriosis. J Clin
Endocrinol Metab 2000;85:2897– 2902.
Bell SC, Jackson JA, Ashmore J, Zhu HH, Tseng L. Regulation of insulin-like
growth factor-binding protein-1 synthesis and secretion by progestin and
relaxin in long term cultures of human endometrial stromal cells. J Clin
Endocrinol Metab 1991;72:1014– 1024.
Braun DP, Ding J, Shen J, Rana N, Fernandez BB, Dmowski WP. Relationship
between apoptosis and the number of macrophages in eutopic endometrium
from women with and without endometriosis. Fertil Steril 2002;78:830–835.
Brosens JJ, Gellersen B. Death or survival-progesterone-dependent cell fate
decisions in the human endometrial stroma. J Mol Endocrinol
2006;36:389– 398.
536
Bulun SE, Cheng YH, Yin P, Imir G, Utsunomiya H, Attar E, Innes J, Julie KJ.
Progesterone resistance in endometriosis: link to failure to metabolize
estradiol. Mol Cell Endocrinol 2006;248:94– 103.
Coppock HA, White A, Aplin JD, Westwood M. Matrix metalloprotease-3 and
-9 proteolyze insulin-like growth factor-binding protein-1. Biol Reprod
2004;71:438–443.
Cornillie FJ, Lauweryns JM, Brosens IA. Normal human endometrium. An
ultrastructural survey. Gynecol Obstet Invest 1985;20:113–129.
Curtis P, Lindsay P, Jackson AE, Shaw RW. Adverse effects on sperm
movement characteristics in women with minimal and mild endometriosis.
Br J Obstet Gynaecol 1993;100:165–169.
D’Antonio M, Martelli F, Peano S, Papoian R, Borrelli F. Ability of
recombinant human TNF binding protein-1 (r-hTBP-1) to inhibit the
development of experimentally-induced endometriosis in rats. J Reprod
Immunol 2000;48:81 –98.
D’Hooghe TM. Immunomodulators and aromatase inhibitors: are they the next
generation of treatment for endometriosis? Curr Opin Obstet Gynecol
2003;15:243–249.
D’Hooghe TM, Debrock S, Hill JA, Meuleman C. Endometriosis and
subfertility: is the relationship resolved? Semin Reprod Med 2003;21:
243–254.
Daly DC, Maslar IA, Riddick DH. Prolactin production during in vitro
decidualization of proliferative endometrium. Am J Obstet Gynecol
1983;145:672– 678.
Downloaded from http://humrep.oxfordjournals.org/ by guest on January 2, 2017
Eyal O, Jomain JB, Kessler C, Goffin V, Handwerger S. Autocrine prolactin
inhibits human uterine decidualization: a novel role for prolactin. Biol
Reprod 2007;76:777–783.
Gilabert-Estelles J, Estelles A, Gilabert J, Castello R, Espana F, Falco C,
Romeu A, Chirivella M, Zorio E, Aznar J. Expression of several
components of the plasminogen activator and matrix metalloproteinase
systems in endometriosis. Hum Reprod 2003;18:1516–1522.
Gilabert-Estelles J, Ramon LA, Espana F, Gilabert J, Vila V, Reganon E,
Castello R, Chirivella M, Estelles A. Expression of angiogenic factors in
endometriosis: relationship to fibrinolytic and metalloproteinase systems.
Hum Reprod 2007;22:2120– 2127.
Giudice LC, Telles TL, Lobo S, Kao L. The molecular basis for implantation
failure in endometriosis: on the road to discovery. Ann NY Acad Sci
2002;955:252– 264.
Han SW, Lei ZM, Rao CV. Treatment of human endometrial stromal
cells with chorionic gonadotropin promotes their morphological
and functional differentiation into decidua. Mol Cell Endocrinol
1999;147:7–16.
Harada T, Iwabe T, Terakawa N. Role of cytokines in endometriosis. Fertil
Steril 2001;76:1–10.
Hess AP, Hamilton AE, Talbi S, Dosiou C, Nyegaard M, Nayak N,
Genbecev-Krtolica O, Mavrogianis P, Ferrer K, Kruessel J et al. Decidual
stromal cell response to paracrine signals from the trophoblast:
amplification of immune and angiogenic modulators. Biol Reprod
2007;76:102–117.
Inoue T, Kanzaki H, Iwai M, Imai K, Narukawa S, Higuchi T, Katsuragawa H,
Mori T. Tumour necrosis factor alpha inhibits in-vitro decidualization of
human endometrial stromal cells. Hum Reprod 1994;9:2411–2417.
Irwin JC, Kirk D, King RJ, Quigley MM, Gwatkin RB. Hormonal regulation of
human endometrial stromal cells in culture: an in vitro model for
decidualization. Fertil Steril 1989;52:761– 768.
Jackson KS, Brudney A, Hastings JM, Mavrogianis PA, Kim JJ, Fazleabas AT.
The altered distribution of the steroid hormone receptors and the chaperone
immunophilin FKBP52 in a baboon model of endometriosis is associated
with progesterone resistance during the window of uterine receptivity.
Reprod Sci 2007;14:137– 150.
Kao LC, Germeyer A, Tulac S, Lobo S, Yang JP, Taylor RN, Osteen K,
Lessey BA, Giudice LC. Expression profiling of endometrium
from women with endometriosis reveals candidate genes for
disease-based implantation failure and infertility. Endocrinology
2003;144:2870–2881.
Kasahara K, Takakura K, Takebayashi K, Kimura F, Nakanishi K, Noda Y.
The role of human chorionic gonadotropin on decidualization of
endometrial stromal cells in vitro. J Clin Endocrinol Metab
2001;86:1281– 1286.
Kim JJ, Taylor HS, Lu Z, Ladhani O, Hastings JM, Jackson KS, Wu Y,
Guo SW, Fazleabas AT. Altered expression of HOXA10 in
endometriosis: potential role in decidualization. Mol Hum Reprod
2007;13:323–332.

Klemmt PA, Carver JG, Kennedy SH, Koninckx PR, Mardon HJ. Stromal cells
from endometriotic lesions and endometrium from women with
endometriosis have reduced decidualization capacity. Fertil Steril
2006;85:564–572.
Laudanski P, Szamatowicz J, Ramel P. Matrix metalloproteinase-13 and
membrane type-1 matrix metalloproteinase in peritoneal fluid of women
with endometriosis. Gynecol Endocrinol 2005;21:106–110.
Lembessis P, Milingos S, Michalas S, Milingos D, Creatsas G, Sourla A,
Koutsilieris M. Urokinase-type plasminogen activator and insulin-like
growth factor-binding protein 3 mRNA expression in endometriotic
lesions and eutopic endometrium: implications for the pathophysiology of
endometriosis. Ann NY Acad Sci 2003;997:223–228.
Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet
Gynecol 1975;122:262–263.
Pierro E, Minici F, Alesiani O, Miceli F, Proto C, Screpanti I, Mancuso S,
Lanzone A. Stromal-epithelial interactions modulate estrogen responsiveness
in normal human endometrium. Biol Reprod 2001;64:831–838.
Rana N, Braun DP, House R, Gebel H, Rotman C, Dmowski WP. Basal and
stimulated secretion of cytokines by peritoneal macrophages in women
with endometriosis. Fertil Steril 1996;65:925–930.
Richter ON, Dorn C, Rosing B, Flaskamp C, Ulrich U. Tumor necrosis factor
alpha secretion by peritoneal macrophages in patients with endometriosis.
Arch Gynecol Obstet 2005;271:143– 147.
The role of eutopic endometrium in endometriosis
Rong R, Ramachandran S, Santanam N, Murphy AA, Parthasarathy S.
Induction of monocyte chemotactic protein-1 in peritoneal mesothelial
and endometrial cells by oxidized low-density lipoprotein and
peritoneal fluid from women with endometriosis. Fertil Steril
2002;78:843– 848.
Satyaswaroop PG, Bressler RS, de la Pena MM, Gurpide E. Isolation and
culture of human endometrial glands. J Clin Endocrinol Metab
1979;48:639– 641.
Selam B, Arici A. Implantation defect in endometriosis: endometrium or
peritoneal fluid. J Reprod Fertil Suppl 2000;55:121–128.
Szamatowicz J, Laudanski P, Tomaszewska I. Matrix metalloproteinase-9 and
tissue inhibitor of matrix metalloproteinase-1: a possible role in the
pathogenesis of endometriosis. Hum Reprod 2002;17:284–288.
Tummon IS, Maclin VM, Radwanska E, Binor Z, Dmowski WP. Occult
ovulatory dysfunction in women with minimal endometriosis or
unexplained infertility. Fertil Steril 1988;50:716– 720.
Ulukus M, Cakmak H, Arici A. The role of endometrium in endometriosis.
J Soc Gynecol Investig 2006;13:467– 476.
Wynn RM. Ultrastructural development of the human decidua. Am J Obstet
Gynecol 1974;118:652– 670.
Submitted on July 12, 2007; resubmitted on October 4, 2007; accepted on
November 21, 2007
Downloaded from http://humrep.oxfordjournals.org/ by guest on January 2, 2017
537