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Embryologic Origin of
Endometriosis: Analysis of 101
Human Female Fetuses
PIETRO G. SIGNORILE,
1
*FELICIANO BALDI,
2
ROSSANA BUSSANI,
3
ROSA VICECONTE,
1
PAMELA BULZOMI,
1
MARIAROSARIA D’ARMIENTO,
4
ALFREDO D’AVINO,
2
AND ALFONSO BALDI
1,2
*
1
Fondazione Italiana Endometriosi, Rome, Italy
2
Department of Biochemistry, Section of Pathology, Second University of Naples, Naples, Italy
3
Department of Pathology, University of Trieste, Trieste, Italy
4
Department of Morphopathology, University of Naples ‘‘Federico II’’, Naples, Italy
The etiology of endometriosis, a gynecological disease characterized by the presence of endometrial glands and stroma outside the uterine
cavity, is still unknown. Our research group has recently demonstrated the presence of ectopic endometrium in human female fetuses at
different gestational ages. In this manuscript we describe four new cases of fetal endometriosis found among a series of 52 female fetuses
analyzed at autopsy. The anatomical localization of this ectopic endometrium, and its histological and immunohistochemical characteristics
are depicted. We suggest that endometriosis is caused by dislocation of primitive endometrial tissue outside the uterine cavity during
organogenesis. The clinical and pathological implications of these findings are discussed.
J. Cell. Physiol. 227: 1653–1656, 2012. ß2011 Wiley Periodicals, Inc.
Endometriosis is defined as the growth of endometrial glands
and stroma at extra-uterine sites (Giudice and Kao, 2004; Baldi
et al., 2008; Signorile et al., 2009a). The prevalence in the
general female population is 6–10%; the frequency increases to
35–60% in women with pain, infertility, or both, (Wheeler,
1992). Endometriosis is responsible for more than 100,000
hysterectomies each year in the United States alone (Carlson
et al., 1994). Despite the fact that this disease is quite common
among women, it is frequently misdiagnosed, the pathogenesis
is unknown and the diagnostic and therapeutic protocols are
still not fully adequate (Bulun, 2009).
The most widely accepted theory for the pathogenesis of
endometriosis is the retrograde menstruation/transplantation
(Sampson, 1927). However, this theory fails to explain the
presence of endometriosis under the peritoneum (the so-called
deep endometriosis) and in remote areas outside the peritoneal
cavity (Signorile and Baldi, 2010a). Indeed, in the last years new
pathogenetic mechanisms have been proposed, such as the
coelomic metaplasia hypothesis or the involvement of
circulating stem cells originating from bone marrow (Nisolle
and Donnez, 1997; Sasson and Taylor, 2008). Interestingly, it
has been postulated by pioneer scientists of this disease in the
late 19th and early 20th century, that endometriosis is caused
by small defects of embryogenesis (Knapp, 1999; Benagiano and
Brosens, 2006). The Mu¨llerian ducts, indeed, give rise to the
female reproductive tract, and this organogenesis is controlled
by complex molecular pathways including the anti-Mu¨llerian
hormone signaling (Klattig and Englert, 2007). Aberrant
differentiation or migration of the Mu¨llerian ducts during
embryogenesis could cause spreading of cells or tracts of cells in
the migratory pathway of fetal organogenesis across the
posterior pelvic floor, thus explaining the observation that
endometriosis is commonly found in the cul-de-sac, uterosacral
ligaments, and medial broad ligaments (Mai et al., 1998).
Recently, our research group has demonstrated the presence
of ectopic endometrium in a significant number of human
female fetuses (5 over 49 cases) analyzed by autopsy in
two different works (Signorile et al., 2009b; Signorile et al.,
2010b).
Goal of this study was to significantly increase the number of
fetuses analyzed in order to better determine the real biological
impact of this phenomenon.
Materials and Methods
We collected at autopsy from three different institutions, a
series of 101 human female fetuses who died at different times
of gestation. The first 49 cases have been already described in
two precedent works (Signorile et al., 2009b; Signorile et al.,
2010b). The other 52 fetuses were analyzed essentially as
previously described. Briefly, pelvic organs were collected
en-block, fixed in paraformaldeyde and included in paraffin.
Histological analysis of the pelvic organs of the fetus was
performed using Hematoxylin/Eosin and Hematoxylin/Van
Gieson staining. For immunohistochemistry 5–7 mm specimen
sections embedded in paraffin, were cut, mounted on glass and
dried overnight at 378C. Tissue sections were quenched
sequentially in 3% hydrogen peroxide in aqueous solution and
blocked with PBS-6% non-fat dry milk (Biorad, Hercules, CA)
for 1 h at room temperature. Slides were then incubated at 48C
overnight at 1:100 dilution with the following antibodies: The
affinity-purified rabbit antibody ERafor the estrogen receptor
(Santa Cruz, Santa Cruz, CA; cat. # sc-542), the mouse
monoclonal antibody for CD10 (clone M7308) (Dako
Laboratories, Carpinteria, CA). After three washes in PBS to
remove the excess of antiserum, the slides were incubated with
*Correspondence to: Pietro G. Signorile and Alfonso Baldi,
Fondazione Italiana Endometriosi, Via E. Longoni, 81, 00159 Rome,
Italy. E-mail: research@endometriosi.it; alfonsobaldi@tiscali.it
Received 1 May 2011; Accepted 3 June 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com), 15 June 2011.
DOI: 10.1002/jcp.22888
ORIGINAL RESEARCH ARTICLE 1653
Journal of
Journal of
Cellular
Physiology
Cellular
Physiology
ß2011 WILEY PERIODICALS, INC.
diluted goat anti-rabbit or anti-mouse biotinylated antibodies
(Vector Laboratories, Burlingame, CA) at 1:200 dilution in PBS-
3% non-fat dry milk (Biorad, Milan, Italy) for 1 h. All the slides
were then processed by the ABC method (Vector
Laboratories) for 30 min at room temperature.
Diaminobenzidine (Vector Laboratories) was used as the final
chromogen and haematoxylin was used as the nuclear
counterstaining. Negative controls for each tissue section were
prepared by leaving out the primary antiserum. Positive
controls of breast, intestinal, and uterine tumor tissues
expressing each of the antigens analyzed, were run at the same
time. All samples were processed under the same conditions.
Experiments were performed in compliance with the Helsinki
Declaration and the protocols were approved by the ethics
committee of the Italian Endometriosis Foundation.
Results
Pelvic organs were analyzed in their entirety. To this end, four
sections were taken every 150 microns and stained for
histology and for immunohistochemistry, as described in the
methods section. Indeed, we did not find any evidence of
macroscopical defects of the genital system in the fetus analyzed
(data not shown). We found in four out of 52 fetuses (7.7% of
cases), the presence of glandular structures outside the uterine
cavity, clearly resembling the structure of the primitive
endometrium and expressing estrogen receptor. Moreover,
the stroma surrounding these glandular structures expressed
both CD10 and estrogen receptor. The anatomical locations of
these endometrial structures were: Two cases in the recto-
vaginal septum, one case in the proximity of the Douglas pouch,
and one case in the mesenchimal tissue close to the posterior
wall of the uterus. To note, these anatomical sites are common
location for endometriosis in women (Baldi et al., 2008). To
note, the four fetuses were also screened for the most common
chromosomal abnormalities and found to have a normal
cariotype. Table 1 summarizes the characteristics of the four
fetuses and the anatomical locations of the glands. The
histological and immunohistochemical appearances of these
epithelial structures are depicted in detail in Figure 1. We
conclude that these structures must be ascribed to endometrial
tissue, misplaced outside the uterine cavity during the earlier
steps of organogenesis and displaying identical phenotype to the
endometrium present in the uterus.
Discussion
The data of this manuscript give evidences for an embryological
origin of endometriosis, suggesting alterations in the fine tuning
of female genital structures organogenesis. Indeed, considering
all the 101 human female fetuses analyzed in this and in previous
works form our research group (Signorile et al., 2009b;
Signorile et al., 2010b), we found a total of nine cases of ectopic
endometrium (9% of the total). This incidence is very close to
the one found in the adult female population (Bulun, 2009).
Nevertheless, the existence of choristoma composed of
mu¨llerian remains in adult has been codified and named
mu¨llerianosis, even if this phenomenon has been interpreted,
but not demonstrated, as different from endometriosis (Batt
et al., 2007). In particular, we have carefully analyzed the
molecular phenotype of this ectopic endometrium, showing
that it expresses characteristic markers of the epithelium and of
the stroma of the genital tract, such as CA125, estrogen
receptor, and CD10. The histological and
immunohistochemical analysis of the eutopic and ectopic
endometrium shows a very similar phenotype, as already
described in our previous works (Signorile et al., 2009b;
Signorile et al., 2010b). This observation argues against the
hypothesis that this ectopic endometrium could disappear
during the final steps of organogenesis. We propose that this
ectopic endometrium would remain quiescent and
asymptomatic until puberty, when the hormonal inputs, would
cause its growth and, consequently, the onset of the symptoms
of endometriosis.
Interestingly, several data from the scientific literature
underline the fact that the uterus of women with endometriosis
displays some congenital alterations. Parker et al. (2006),
indeed, described alterations in the muscular characteristics of
the innermost myometrium, such as the thickness and fiber
orientation and abnormal JZ morphology, securely due to the
congenital alteration of uterine wall in patients with
endometriosis. Similarly, Kunz et al. (2000) have described that
infertile women with endometriosis show alterations of the
myometrial wall with an archimetral significantly expanded.
These alterations, moreover, are identical in patients with
adenomyosis, thus supporting the concept that endometriosis
and adenomyosis are the same diseases and that the defect is
primarily at uterine level. Nevertheless, it has also been shown
that endometriosis is more frequent in patients with Mu¨llerian
anomalies (Nawroth et al., 2006) and other genital anomalies
(Acie
´n, 1986).
Moreover, the presence of the disease in early puberty and
exceptionally also in newborns (Diez Garcia et al., 1996; Batt
and Mitwally, 2003; Marsh and Laufer 2005; Ebert et al., 2009),
as well as in women affected by the Mayer–Rokitansky–Ku¨ ster–
Hauser, a syndrome characterized by congenital aplasia of the
uterus and the upper part of the vagina (Enatsu et al., 2000; Yan
and Mok, 2002; Balci et al., 2008), further supports the validity
of an embryological origin for endometriosis.
Increasing experimental evidences are showing that
exposure to toxicants during critical periods of pre- and peri-
natal development can have long-lasting effects. In particular,
the ability of endocrine disruptors to alter reproductive
function and health in females are quite well characterized,
thanks especially to the numerous works on the effects of
endocrine disruptors exposition in utero (Newbold et al.,
2009). Interestingly enough, there are several studies in humans
linking exposition to endocrine disruptors with insurgence of
endometriosis (Foster, 2008). In particular, a robust
epidemiological study on a wide cohort of patients with
endometriosis has shown that the rate of endometriosis is 80%
greater among women exposed to the endocrine disruptor
diethylstilbestrol in utero (Missmer et al., 2004). In very recent
works, indeed, we have described in mice exposed in utero to
the endocrine disruptor bisphenol A the presence of
endometriosis-like structures and premature ovarian failure
(Signorile et al., 2010c; Signorile et al., 2011), a phenotype, that
strictly recapitulates the clinical picture seen in women suffering
of endometriosis. This observation is in agreement with the
work by Huseby and Thurlow (1982), that have described a
similar phenotype in mice exposed prenatally to low dose of
diethylstilbestrol: Alterations in the genital tract consisting of
adenomyosis and enlargement of the cervix, and reduced
fecundity.
In conclusion, it is possible to claim that endometriosis is a
multi-factorial disease with multifaceted features.
Nevertheless, the demonstration of the presence of ectopic
endometrium in the female fetus in same anatomical locations
TABLE 1. Characteristics of the fetuses with endometriosis
No. Gestational age Cause of death
Location of ectopic
endometrium
1 21 weeks Placental pathology Recto-vaginal septum
2 23 weeks Placental pathology Recto-vaginal septum
3 24 weeks Placental pathology Douglas pouch
4 24 weeks Placental pathology Posterior wall of the uterus
JOURNAL OF CELLULAR PHYSIOLOGY
1654 SIGNORILE ET AL.
found in the adult patients affected by endometriosis and with a
frequency very similar, makes the embryogenetic theory on
endometriosis the only one scientifically proved and suggests
that this pathogenetic mechanism is prevalent in the genesis of
this disease. Several epidemiological and animal studies, finally,
suggest an important role for an abnormal estrogenic signaling
during embryogenesis in causing the endometriosis phenotype.
The clinical and therapeutic implications are clear-cut.
Endometriosis could still be regarded as a recurrent disease;
nevertheless recurrence could not be ascribed to the
retrograde menstruation, but to an incomplete surgical
intervention, since it is demonstrated that endometriosis
lesions could be also made up of microscopic foci (Redwine,
2003), and or to different timing of growth of the lesions in the
same patient, probably due to individual susceptibility that is a
typical phenomenon of the diseases inducted by endocrine
disruptors (Mori et al., 2003). Therefore surgery, if complete in
exhausted growth disease can be considered curative.
Contrarily, exposition to endocrine disruptors such as
synthetic estrogens or SERM chemical compounds, though
reducing the symptoms, could increase the growth of
endometriosis. Such studies shed new light on the pathogenesis
of this disease and, possibly, suggest suitable therapeutic targets
for both the typical phenotypes of endometriosis: Ectopic
endometrial tissue and infertility.
Acknowledgments
This work was supported by a grant from the Italian
Endometriosis Foundation.
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Fig. 1. Histological and immunohistochemical appearance of ectopic endometrium. Panel A: Case 1: Endometriosis in the recto-vaginal septum.
Histological appearance of the endometriotic gland; the vagina is visible in the upper right corner of the picture (Haematoxylin and Eosin, original
magnification T20). Panel B: Case 2: Endometriosis in the recto-vaginal septum. Histological appearance of the endometriotic gland
(Haematoxylin and Eosin, original magnification T20). Panel C: Case 3: Endometriosis in the proximity of the Douglas pouch. Histological
appearance of the endometriotic gland (Haematoxylin and Eosin, original magnification T20). Panel D: Case 4: Endometriosis in the mesenchimal
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receptor (ABC, original magnification T40). Panel F: Higher magnification of one of the glandular structures displaying strong
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