Ectopic endometrium in human foetuses is a common event and sustains the theory of müllerianosis in the pathogenesis of endometriosis, a disease that predisposes to cancer

Background: Endometriosis is a gynecological disease defined by the histological presence of endometrial glands and stroma outside the ut erine cavity. Women with endometriosis have an increased risk of different types of malignancies, especially ov arian cancer and non-Hodgkin's lymphoma. Though there are several theories, researchers remain unsure as to the definitive cause of endometriosis. Our objective was to test th e validity of the theory of müllerianosis for endometriosis, that is the misplacing of primitive endometrial ti ssue along the migratory pathway of foetal organogenesis

We have collected at autopsy 36 human female foetuses at different gestational age. We have performed a morphological and immunohi stochemical study (expression of oestrogen receptor and CA125) on the pelvic organs of th e 36 foetuses included en-block and totally analyzed.

In 4 out of 36 foetuses we found presence of misplaced endometrium in five different ectopic sites: 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. All these sites are common location of endometriosis in women.

We propose that a cause of endometriosis is the dislocation of primitive endometrial tissue outside the uterine cavity during organogenesis.

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 [1,2]. The prevalence of endometriosis in the gen- eral female population is 6–10%; in women with pain, infertility or both, the frequency increases to 35–60% [3]. Published: 9 April 2009 Journal of Experimental & Clinical Cancer Research 2009, 28:49 doi:10.1186/1756-9966-28-49 Received: 31 March 2009 Accepted: 9 April 2009 This article is available from: http://www.jeccr.com/content/28/1/49 © 2009 Signorile et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Journal of Experimental & Clinical Cancer Research 2009, 28:49 http://www.jeccr.com/content/28/1/49 Page 2 of 5 (page number not for citation purposes) Deep infiltrating endometriosis is a particular form of endometriosis associated with pelvic pain symptoms, located under the peritoneal surface [4,5]. 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 depos- ited 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 [6-8]. A different theory postulates that endometriosis is caused by little defects of embryogenesis [9,10]. Indeed, during the embryonic stage, the primitive cells migrate and undergo differentia- tion 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 sophis- ticated, but still incompletely understood, fetal system including the regulation of the anti-Müllerian hormone signalling pathway [11]. It has been speculated that aber- rant 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 pos- terior pelvic floor and this could conveniently explain the observation that endometriosis is most commonly and predictably found in the cul-de-sac, utero-sacral liga- ments, and medial broad ligaments, although location anywhere might be possible [12]. This theory of develop- mentally misplaced endometrial tissue is called mülleria- nosis [13]. Other theories for the genesis of endometriosis include different mechanisms such as hematogenous metastasis, genetic predisposition or altered cellular immunity [1,2]. Nevertheless, all these theories remain speculative and no definitive evidences have been pro- duced to demonstrate them. We speculated that, if the basis of endometriosis is an alteration during organogen- esis, 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 differ- ent 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 col- lected en-block, fixed in paraphormaldeyde and included in paraffin. We performed histological analysis of the pel- vic organs for each fetus, using Hematoxylin/Eosin and Hematoxylin/Van Gieson staining. For immunohisto- chemistry 5–7 μm specimen sections embedded in paraf- fin, 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 sec- tions were quenched sequentially in 3% hydrogen perox- ide 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 affin- ity-purified rabbit antibody ER α for the oestrogen recep- tor (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 pri- mary 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 Hel- sinki Declaration and the protocols were approved by the ethics committee of the Fondazione Italiana Endometri- osi.

In order to analyze the pelvic organs in their entirety, four sections were taken every 150 microns and stained for his- tology and for immunohistochemistry, as described in the

section. We have chosen, for immunohistochem- isitry, CA125 and the oestrogen receptor, two well defined marker of epithelium of the female reproductive tract [1,14]. None of the selected cases displayed macroscopi- cal 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 forma- tion. In particular, the locations of these endometrial structures were: in the recto-vaginal septum, in the prox- imity 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 Journal of Experimental & Clinical Cancer Research 2009, 28:49 http://www.jeccr.com/content/28/1/49 Page 3 of 5 (page number not for citation purposes) uterus. To note, these anatomical sites are common loca- tion for endometriosis in women [15]. The exact anatom- ical 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 dif- ferentiated endometrial tissue, misplaced outside the uter- ine 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 hor- monal 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 men- struation/transplantation is still the most popular and accepted pathogenetic mechanism of endometriosis, sev- eral 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 peri- toneal fluid [16-18]. 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 [1,2]. 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 environ- Table 1: Characteristics of the foetuses enrolled in this study N° Gestational age Cause of death P resence of ectopic endometrium 1 18 weeks Voluntary abortion Yes 2 24 weeks Placental pathology Yes 3 25 weeks Placental pathology Yes 4 16 weeks Voluntary abortion Yes 5 23 weeks Placental pathology No 6 15 weeks Voluntary abortion No 7 20 weeks Voluntary abortion No 8 newborn Primary atypical pneumonia No 9 newborn Acute interst itial pneumonitis No 10 16 weeks Voluntary abortion No 11 23 weeks Placental pathology No 12 14 weeks Placental pathology No 13 21 weeks Voluntary abortion No 14 20 weeks Voluntary abortion No 15 20 weeks Voluntary abortion No 16 18 weeks Voluntary abortion No 17 19 weeks Voluntary abortion No 18 16 weeks Voluntary abortion No 19 23 weeks Placental pathology No 20 25 weeks Placental pathology No 21 newborn Acute inter stitial pneumonitis No 22 newborn Primary atypical pneumonia No 23 20 weeks Voluntary abortion No 24 19 weeks Voluntary abortion No 25 newborn Cardiac malformation No 26 newborn Cardiac malformation No 27 20 weeks Voluntary abortion No 28 23 weeks Placental pathology No 29 19 weeks Voluntary abortion No 30 newborn Cardiac malformation No 31 newborn Cardiac malformation No 32 19 weeks Voluntary abortion No 33 newborn Acute inter stitial pneumonitis No 34 20 weeks Voluntary abortion No 35 newborn Cardiac malformation No 36 21 weeks Placental pathology No Journal of Experimental & Clinical Cancer Research 2009, 28:49 http://www.jeccr.com/content/28/1/49 Page 4 of 5 (page number not for citation purposes) mental toxicants possessing estrogenic activity, the so- called endocrine disruptors, resulted in endometriosis [20]. Although the epidemiological evidences are not con- clusive to date, animal and experimental investigations have provided a basis for the proposed association between estrogenic contaminants exposure and endome- triosis [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 direc- tion 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 uter- Histological and immunohistochemical appearance of ectopic endometrium in four female human foetusesFigure 1 Histological and immunohistochemical appearance of ectopic endometrium in four female human foetuses. Panel A: A 25 weeks foetus showing an endometrial structure in the recto-vaginal septum; in the inset named A', the immuno- histochemical expression of CA-125 of this structure at higher magnification is depicted. Panel B: A 24 weeks foetus showing an endometrial structure in the proximity of the Douglas poutch; in the inset named B', the immunohistochemical expression of oestrogen receptor of this structure at higher magnification is depicted. Panel C: A 18 weeks foetus showing an endometrial structure in the rectal tube at the level of muscularis propria; in the inset named C', the immunohistochemical expression of CA-125 of this structure at higher magnification is depicted. Note that the epithelium of the rectum is negative for CA-125. Panel D: A 16 weeks foetus showing an endometrial structure in the mesenchimal tissue close to the posterior wall of the uterus; in the inset named D', the immunohistochemical expression of CA-125 of this structure at higher magnification is depicted. Note that in the wall of the primitive miometrium is present a little group of endometrial cells positive for CA-125 (indicated by an asterisk), that could represent a primitive nest of adenomyosis. Abbreviations used: an (anus); co (coccyx); dp (Douglas' pouch); re (rectum); rvs (recto-vaginal septum); sc (spinal column); ut (uterus); bl (bladder). u bl re re A A’ B B’ C C’ D D’ * Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Journal of Experimental & Clinical Cancer Research 2009, 28:49 http://www.jeccr.com/content/28/1/49 Page 5 of 5 (page number not for citation purposes) ine wall [21]. To the best of our knowledge, this observa- tion is the first direct evidence in human female foetuses of the presence of ectopic endometrium outside the uter- ine 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 toxi- cants. 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 [1-3]. 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 defini- tive conclusions. Further studies are urgently required in order to better define the molecular mechanisms underly- ing 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 gen- ital 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-Hodg- kin's lymphoma [1], the implications of these findings could be very important also in the oncology field.

The clinical and therapeutic implications of this observa- tion are straightforward. Endometriosis could not be regarded as a recurrent disease, therefore surgery, if com- plete can be considered curative and it would be not justi- fied post-operative hormonal treatments. Nevertheless, it must be underlined the fact that other pathogenetic mech- anisms for the genesis of endometriosis can not be com- pletely ruled out by these observation, even if, to date, there are no direct evidence of their validity. Competing interests The authors declare that they have no competing interests. Authors' contributions PGS and AB conducted the work, analyzed the data and wrote together the manuscript. FB performed the histolog- ical and immunohistochemical analysis. RB, FB and MDA performed the autopsies. MDF performed the immuno- histochemical staining.

This work was supported by a grant from "Fondazione Italiana Endometri- osi".

1. Baldi A, Campioni M, Signorile PG: Endometriosis: pathogenesis, diagnosis, therapy and association with cancer. Oncol Reports 2008, 19:843-846. 2. Giudice LC, Kao LC: Endometriosis. The Lancet 2004, 364:1789-1799. 3. Houston DE: Evidence for the risk of pelvic endometriosis by age, race, and socioeconomic status. Epidemiol Rev 1984, 6:167-191. 4. Koninckx PR, Martin D: Treatment of deeply infiltrating endometriosis. Curr Opin Obstet Gynecol 1994, 6:231-234. 5. Signorile PG, Campioni M, Vinc enzi B, D'Avino A, Baldi A: Rectovag- inal septum endometriosis: an immunohistochemical analy- sis of 62 cases. In Vivo 2009 in press. 6. Nap AW, Groothuis PG, Demir AY, Evers JL, Dunselman GA: Pathogenesis of endometriosis. Bet Pract Res Clin Obstet Gynaecol 2004, 18:233-244. 7. Brosens I: Endometriosis and the outcome of in vitro fertiliza- tion. Fertil Steril 2004, 81:1198-1200. 8. Nisolle M, Donnez J: Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities. Fertil Steril 1997, 68:585-595. 9. Fujii S: Secondary mullerian system and endometriosis. Am J Obstet Gynecol 1991, 165:219-225. 10. Redwine DB: Was Sampson wrong? Fertil Steril 2002, 78:686-693. 11. Klattig J, Englert C: The mullerian duct: re cent insight into its development and regression. Sex Dev 2007, 1:271-278. 12. Mai KT, Yazdi HM, Perkins DG, Parks W: Development of endometriosis from emb ryonic duct remnants. Hum Pathol 1998, 29:319-322. 13. Batt RE, Smith RA, Buck Louis GM, et al.: Mullerianosis. Histol His- topathol 2007, 22:1161-1166. 14. Scharl A, Crombach G, Vierbuchen M, Musch H, Bolte A: CA 125 in normal tissues and carcinom as of the uterine cervix, endometrium and fallopian tube. Immunohistochemical detection. Arch Gynecol Obstet 1989, 244:103-112. 15. Rubin J, Farber A: Pathology 2nd edition. JB Lippincott Company; 1994. 16. D'Hooghe TM: Invisible microscopic endometriosis; how wrong is the Sampson hypothes is of retrograde menstrua- tion to explain the pathog enesis of endometriosis? Gynecol Obstet Invest 2003, 55:61-62. 17. Redwine DB: Invisible microscopic endometriosis: a review. Gynecol Obstet Invest 2003, 55:63-67. 18. Batt RE, Mitwally MF: Endometriosis from thelarche to midteens: pathogenesis and prognosis, prevention and peda- gogy. J Pediatr Adolesc Gynecol 2003, 16:337-347. 19. Nawroth F, Rahimi G, Nawroth C, Foth D, Ludwig M, Schmidt T: Is there an association between septate uterus and endometri- osis? Hum Reprod 2006, 2:542-544. 20. Anger DL, Foster WG: The link between environmental toxi- cant exposure and endometriosis. Front Biosci 2008, 13:1578-1593. 21. McLachlan JA, Simpson E, Martin M: Endocrine disrupters and female reproductive health. Best Pract Res Clin Endocrinol Metab 2006, 20:63-75.