Endometriosis is a gynecological disease defined by the histological presence of endometrial glands and stroma outside the uterine cavity, most commonly implanted over visceral and peritoneal surfaces within the female pelvis 12. The prevalence of endometriosis in the general female population is 6–10%; in women with pain, infertility or both, the frequency increases to 35–60% 3. Deep infiltrating endometriosis is a particular form of endometriosis associated with pelvic pain symptoms, located under the peritoneal surface 45. Though there are several theories, researchers remain unsure as to the definitive cause of endometriosis. The most commonly accepted mechanism for the development of peritoneal endometriotic lesions is the Sampson's theory claiming the adhesion and growth of endometrial fragments deposited into the peritoneal cavity via retrograde menstruation 4. On the other hand, the coelomic metaplasia theory claims that formation of deep endometriosis is caused by metaplasia of the original coelomic membrane, perhaps induced by environmental factors 678. A different theory postulates that endometriosis is caused by little defects of embryogenesis 910. Indeed, during the embryonic stage, the primitive cells migrate and undergo differentiation to form the pelvic organs. In particular, the Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, uterus, cervix, and anterior vagina. This organogenesis is controlled and directed by a sophisticated, but still incompletely understood, fetal system including the regulation of the anti-Müllerian hormone signalling pathway 11. It has been speculated that aberrant differentiation or migration of the Müllerian ducts could cause spreading of cells or tracts of cells in the migratory pathway of foetal organogenesis across the posterior pelvic floor and this could conveniently explain the observation that endometriosis is most commonly and predictably found in the cul-de-sac, utero-sacral ligaments, and medial broad ligaments, although location anywhere might be possible 12. This theory of developmentally misplaced endometrial tissue is called müllerianosis 13. Other theories for the genesis of endometriosis include different mechanisms such as hematogenous metastasis, genetic predisposition or altered cellular immunity 12. Nevertheless, all these theories remain speculative and no definitive evidences have been produced to demonstrate them. We speculated that, if the basis of endometriosis is an alteration during organogenesis, it would be possible to see ectopic endometrial tissue mislocated outside the uterine cavity of human female foetuses, possibly with a similar frequency found for endometriosis in the general population. Therefore, we decided to investigate the anatomy of the pelvic organs of a group of human female foetuses, collected at autopsy.

We collected at autopsy 36 human female fetuses at different gestational ages, that did not displayed any visible alteration of the pelvic organs. The characteristics of the fetuses are depicted in Table 1. Pelvic organs were collected en-block, fixed in paraphormaldeyde and included in paraffin. We performed histological analysis of the pelvic organs for each fetus, using Hematoxylin/Eosin and Hematoxylin/Van Gieson staining. For immunohistochemistry 5–7 μm specimen sections embedded in paraffin, were cut, mounted on glass and dried overnight at 37°C. All sections were then deparaffinized in xylene, rehydrated through a graded alcohol series and washed in phosphate-buffered saline (PBS). PBS was used for all subsequent washes and for antiserum dilution. Tissue sections were quenched sequentially in 3% hydrogen peroxide in aqueous solution and blocked with PBS-6% non-fat dry milk (Biorad, Hercules, CA, U.S.A.) for 1 h at room temperature. Slides were then incubated at 4°C overnight at 1:100 dilution with the following antibodies: the affinity-purified rabbit antibody ERα for the oestrogen receptor (Santa Cruz, Santa Cruz, CA, USA; cat. # sc-542) and the mouse monoclonal antibody M11 for CA125(Dako Laboratories, Carpinteria, CA, USA). After three washes in PBS to remove the excess of antiserum, the slides were incubated with diluted goat anti-rabbit or anti-mouse biotinylated antibodies (Vector Laboratories, Burlingame, CA, U.S.A.) at 1:200 dilution in PBS-3% non-fat dry milk (Biorad) 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 constituted of tumour tissues expressing either the oestrogen receptor or CA125, 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 Fondazione Italiana Endometriosi.

In order to analyze the pelvic organs in their entirety, four sections were taken every 150 microns and stained for histology and for immunohistochemistry, as described in the method section. We have chosen, for immunohistochemisitry, CA125 and the oestrogen receptor, two well defined marker of epithelium of the female reproductive tract 114. None of the selected cases displayed macroscopical or microscopical defects of the genital system. Indeed, we found in four foetuses (11% of cases), the presence of organoid structures outside the uterine cavity, clearly resembling the structure of the primitive endometrium and expressing both CA125 and oestrogen receptor. These structures were mislocated outside the uterine cavity and could not be ascribed to any normal anatomical formation. In particular, the locations of these endometrial structures were: in the recto-vaginal septum, in the proximity of the Douglas pouch, in the mesenchimal tissue close to the posterior wall of the uterus, in the rectal tube at the level of muscularis propria, and in the wall of the uterus. To note, these anatomical sites are common location for endometriosis in women 15. The exact anatomical distributions and the histological appearances of these epithelial structures are depicted in detail in figure 1. We conclude that these structures must be ascribed to differentiated endometrial tissue, misplaced outside the uterine cavity during the earlier steps of organogenesis. It is possible to suppose that this ectopic endometrium would remain quiescent and, therefore, undetectable until puberty, when different stimuli, and among them the hormonal inputs, would cause its re-growth (as it is the case for the eutopic endometrium) and, consequently, the onset of the symptoms of endometriosis.

Despite the fact that Sampson's theory of retrograde menstruation/transplantation is still the most popular and accepted pathogenetic mechanism of endometriosis, several clinical and experimental evidence seems to contrast this hypothesis. There is, for example, no evidence in vivo or in vitro that endometrial cells present in the peritoneal fluid during menstruation can attach to and invade the peritoneal surface 16. Furthermore, it has been shown that endometrial cells are not commonly present in peritoneal fluid 161718. Additionally, the fact that 90% of women have retrograde flow but less than 15% of women develop endometriosis and the presence of the disease in early puberty, further contrast the validity of the theory 18. Finally, this theory fails to explain the presence of endometriosis in such remote areas as the lungs, skin, lymph nodes, breasts 12. Interestingly enough, there are some studies showing higher prevalence of endometriosis in patients with Müllerian anomalies 19; moreover, the existence of choristoma composed of müllerian rests, named müllerianosis, has been postulated 13. In recent years, several evidence suggested that exposure to environmental toxicants possessing estrogenic activity, the so-called endocrine disruptors, resulted in endometriosis 20. Although the epidemiological evidences are not conclusive to date, animal and experimental investigations have provided a basis for the proposed association between estrogenic contaminants exposure and endometriosis 21. Nevertheless, the mechanism(s) underlying this potential association are poorly understood. The proper function of the normal human endometrium relies on well organized cell-cell interactions regulated locally by cytokines and growth factors under the direction of steroid hormones. The onset and progression of the disease processes of endometriosis may result from disruptions of this well balanced cellular equilibrium, that would cause the interruption of some organizational events associated with development of the neonatal uterine wall 21. To the best of our knowledge, this observation is the first direct evidence in human female foetuses of the presence of ectopic endometrium outside the uterine cavity. Our data sustain the müllerianosis hypothesis of an embryological origin for endometriosis, suggesting alterations in the fine tuning of female genital structures organogenesis, possibly caused by environmental toxicants. Interestingly, the percentage of foetuses analyzed in our study, that displayed the presence of ectopic endometrium is very similar to the prevalence of women suffering for this disease in the general population 123. This further suggests a strict link between embryological abnormalities and onset of the disease, even if the number of foetuses analyzed is too small in order to reach definitive conclusions. Further studies are urgently required in order to better define the molecular mechanisms underlying this phenomenon. In particular, ad hoc in vitro and in vivo models should be set up to analyze the effects on cell homeostasis and on the morphogenesis of the female genital system of different endocrine disruptors. Considering that, based on epidemiological studies, women with endometriosis have an increased risk of different types of malignancies, especially ovarian cancer and non-Hodgkin's lymphoma 1, the implications of these findings could be very important also in the oncology field.

The clinical and therapeutic implications of this observation are straightforward. Endometriosis could not be regarded as a recurrent disease, therefore surgery, if complete can be considered curative and it would be not justified post-operative hormonal treatments. Nevertheless, it must be underlined the fact that other pathogenetic mechanisms for the genesis of endometriosis can not be completely ruled out by these observation, even if, to date, there are no direct evidence of their validity.

The authors declare that they have no competing interests.

PGS and AB conducted the work, analyzed the data and wrote together the manuscript. FB performed the histological and immunohistochemical analysis. RB, FB and MDA performed the autopsies. MDF performed the immunohistochemical staining.