{"paper_id":"270026ec-6bf9-4a0c-aad6-b1e276eafc07","body_text":"BioMed Central\nPage 1 of 5\n(page number not for citation purposes)\nJournal of Experimental & Clinical \nCancer Research\nOpen AccessResearch\nEctopic endometrium in human foetuses is a common event and \nsustains the theory of müllerianosis in the pathogenesis of \nendometriosis, a disease that predisposes to cancer\nPietro G Signorile*1, Feliciano Baldi2, Rossana Bussani3, \nMariarosaria D'Armiento4, Maria De Falco5 and Alfonso Baldi*1,2\nAddress: 1Fondazione Italiana Endometriosi, Rome, Italy, 2Dept Biochemistry, Sect Pathology, Second University of Naples, Naples, Italy, 3Dept. \nof Pathology, University of Trieste, Trieste, Italy, 4Dept. Scienze Biomorfologiche, University of Naples \"Federico II\", Naples, Italy and 5Dept \nEvolutive and Comparative Biology, University of Naples \"Federico II\", Naples, Italy\nEmail: Pietro G Signorile* - aie@endometriosi.it; Feliciano Baldi - felicianobaldi@tiscali.it; Rossana Bussani - bussani@univ.trieste.it; \nMariarosaria D'Armiento - maria.darmiento@unina.it; Maria De Falco - madefalco@unina.it; Alfonso Baldi* - alfonsobaldi@tiscali.it\n* Corresponding authors    \nAbstract\nBackground: Endometriosis is a gynecological disease defined by the histological presence of\nendometrial glands and stroma outside the ut erine cavity. Women with endometriosis have an\nincreased risk of different types  of malignancies, especially ov arian cancer and non-Hodgkin's\nlymphoma. Though there are several theories, researchers remain unsure as to the definitive cause\nof endometriosis. Our objective was to test th e validity of the theory  of müllerianosis for\nendometriosis, that is the misplacing of primitive endometrial ti ssue along the migratory pathway\nof foetal organogenesis\nMethods: We have collected at autopsy 36 human female foetuses at different gestational age. We\nhave performed a morphological and immunohi stochemical study (expression of oestrogen\nreceptor and CA125) on the pelvic organs of th e 36 foetuses included en-block and totally\nanalyzed.\nResults: In 4 out of 36 foetuses we found presence of misplaced endometrium in five different\nectopic sites: in the recto-vaginal septum, in the proximity of the Douglas pouch, in the\nmesenchimal tissue close to the posterior wall of the uterus , in the rectal tube at the level of\nmuscularis propria, and in the wall of the uterus. All these sites are common location of\nendometriosis in women.\nConclusion: We propose that a cause of endometriosis is the dislocation of primitive endometrial\ntissue outside the uterine cavity during organogenesis.\nBackground\nEndometriosis is a gynecological disease defined by the\nhistological presence of endometrial glands and stroma\noutside the uterine cavity, most commonly implanted\nover visceral and peritoneal surfaces within the female\npelvis [1,2]. The prevalence of endometriosis in the gen-\neral female population is 6–10%; in women with pain,\ninfertility or both, the frequency increases to 35–60% [3].\nPublished: 9 April 2009\nJournal of Experimental & Clinical Cancer Research 2009, 28:49 doi:10.1186/1756-9966-28-49\nReceived: 31 March 2009\nAccepted: 9 April 2009\nThis article is available from: http://www.jeccr.com/content/28/1/49\n© 2009 Signorile et al; licensee BioMed Central Ltd. \nThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), \nwhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\n\nJournal of Experimental & Clinical Cancer Research 2009, 28:49 http://www.jeccr.com/content/28/1/49\nPage 2 of 5\n(page number not for citation purposes)\nDeep infiltrating endometriosis is a particular form of\nendometriosis associated with pelvic pain symptoms,\nlocated under the peritoneal surface [4,5]. Though there\nare several theories, researchers remain unsure as to the\ndefinitive cause of endometriosis. The most commonly\naccepted mechanism for the development of peritoneal\nendometriotic lesions is the Sampson's theory claiming\nthe adhesion and growth of endometrial fragments depos-\nited into the peritoneal cavity via retrograde menstruation\n[4]. On the other hand, the coelomic metaplasia theory\nclaims that formation of deep endometriosis is caused by\nmetaplasia of the original coelomic membrane, perhaps\ninduced by environmental factors [6-8]. A different theory\npostulates that endometriosis is caused by little defects of\nembryogenesis [9,10]. Indeed, during the embryonic\nstage, the primitive cells migrate and undergo differentia-\ntion to form the pelvic organs. In particular, the Müllerian\nducts give rise to the female reproductive tract, including\nthe Fallopian tubes, uterus, cervix, and anterior vagina.\nThis organogenesis is controlled and directed by a sophis-\nticated, but still incompletely understood, fetal system\nincluding the regulation of the anti-Müllerian hormone\nsignalling pathway [11]. It has been speculated that aber-\nrant differentiation or migration of the Müllerian ducts\ncould cause spreading of cells or tracts of cells in the\nmigratory pathway of foetal organogenesis across the pos-\nterior pelvic floor and this could conveniently explain the\nobservation that endometriosis is most commonly and\npredictably found in the cul-de-sac, utero-sacral liga-\nments, and medial broad ligaments, although location\nanywhere might be possible [12]. This theory of develop-\nmentally misplaced endometrial tissue is called mülleria-\nnosis [13]. Other theories for the genesis of endometriosis\ninclude different mechanisms such as hematogenous\nmetastasis, genetic predisposition or altered cellular\nimmunity [1,2]. Nevertheless, all these theories remain\nspeculative and no definitive evidences have been pro-\nduced to demonstrate them. We speculated that, if the\nbasis of endometriosis is an alteration during organogen-\nesis, it would be possible to see ectopic endometrial tissue\nmislocated outside the uterine cavity of human female\nfoetuses, possibly with a similar frequency found for\nendometriosis in the general population. Therefore, we\ndecided to investigate the anatomy of the pelvic organs of\na group of human female foetuses, collected at autopsy.\nMethods\nWe collected at autopsy 36 human female fetuses at differ-\nent gestational ages, that did not displayed any visible\nalteration of the pelvic organs. The characteristics of the\nfetuses are depicted in Table 1. Pelvic organs were col-\nlected en-block, fixed in paraphormaldeyde and included\nin paraffin. We performed histological analysis of the pel-\nvic organs for each fetus, using Hematoxylin/Eosin and\nHematoxylin/Van Gieson staining. For immunohisto-\nchemistry 5–7 μm specimen sections embedded in paraf-\nfin, were cut, mounted on glass and dried overnight at\n37°C. All sections were then deparaffinized in xylene,\nrehydrated through a graded alcohol series and washed in\nphosphate-buffered saline (PBS). PBS was used for all\nsubsequent washes and for antiserum dilution. Tissue sec-\ntions were quenched sequentially in 3% hydrogen perox-\nide in aqueous solution and blocked with PBS-6% non-fat\ndry milk (Biorad, Hercules, CA, U.S.A.) for 1 h at room\ntemperature. Slides were then incubated at 4°C overnight\nat 1:100 dilution with the following antibodies: the affin-\nity-purified rabbit antibody ER α for the oestrogen recep-\ntor (Santa Cruz, Santa Cruz, CA, USA; cat. # sc-542) and\nthe mouse monoclonal antibody M11 for CA125(Dako\nLaboratories, Carpinteria, CA, USA). After three washes in\nPBS to remove the excess of antiserum, the slides were\nincubated with diluted goat anti-rabbit or anti-mouse\nbiotinylated antibodies (Vector Laboratories, Burlingame,\nCA, U.S.A.) at 1:200 dilution in PBS-3% non-fat dry milk\n(Biorad) for 1 h. All the slides were then processed by the\nABC method (Vector Laboratories) for 30 min at room\ntemperature. Diaminobenzidine (Vector Laboratories)\nwas used as the final chromogen and haematoxylin was\nused as the nuclear counterstaining. Negative controls for\neach tissue section were prepared by leaving out the pri-\nmary antiserum. Positive controls constituted of tumour\ntissues expressing either the oestrogen receptor or CA125,\nwere run at the same time. All samples were processed\nunder the same conditions.\nExperiments were performed in compliance with the Hel-\nsinki Declaration and the protocols were approved by the\nethics committee of the Fondazione Italiana Endometri-\nosi.\nResults\nIn order to analyze the pelvic organs in their entirety, four\nsections were taken every 150 microns and stained for his-\ntology and for immunohistochemistry, as described in the\nmethod section. We have chosen, for immunohistochem-\nisitry, CA125 and the oestrogen receptor, two well defined\nmarker of epithelium of the female reproductive tract\n[1,14]. None of the selected cases displayed macroscopi-\ncal or microscopical defects of the genital system. Indeed,\nwe found in four foetuses (11% of cases), the presence of\norganoid structures outside the uterine cavity, clearly\nresembling the structure of the primitive endometrium\nand expressing both CA125 and oestrogen receptor. These\nstructures were mislocated outside the uterine cavity and\ncould not be ascribed to any normal anatomical forma-\ntion. In particular, the locations of these endometrial\nstructures were: in the recto-vaginal septum, in the prox-\nimity of the Douglas pouch, in the mesenchimal tissue\nclose to the posterior wall of the uterus, in the rectal tube\nat the level of muscularis propria, and in the wall of the\n\nJournal of Experimental & Clinical Cancer Research 2009, 28:49 http://www.jeccr.com/content/28/1/49\nPage 3 of 5\n(page number not for citation purposes)\nuterus. To note, these anatomical sites are common loca-\ntion for endometriosis in women [15]. The exact anatom-\nical distributions and the histological appearances of\nthese epithelial structures are depicted in detail in figure 1.\nWe conclude that these structures must be ascribed to dif-\nferentiated endometrial tissue, misplaced outside the uter-\nine cavity during the earlier steps of organogenesis. It is\npossible to suppose that this ectopic endometrium would\nremain quiescent and, therefore, undetectable until\npuberty, when different stimuli, and among them the hor-\nmonal inputs, would cause its re-growth (as it is the case\nfor the eutopic endometrium) and, consequently, the\nonset of the symptoms of endometriosis.\nDiscussion\nDespite the fact that Sampson's theory of retrograde men-\nstruation/transplantation is still the most popular and\naccepted pathogenetic mechanism of endometriosis, sev-\neral clinical and experimental evidence seems to contrast\nthis hypothesis. There is, for example, no evidence in vivo\nor in vitro that endometrial cells present in the peritoneal\nfluid during menstruation can attach to and invade the\nperitoneal surface [16]. Furthermore, it has been shown\nthat endometrial cells are not commonly present in peri-\ntoneal fluid [16-18]. Additionally, the fact that 90% of\nwomen have retrograde flow but less than 15% of women\ndevelop endometriosis and the presence of the disease in\nearly puberty, further contrast the validity of the theory\n[18]. Finally, this theory fails to explain the presence of\nendometriosis in such remote areas as the lungs, skin,\nlymph nodes, breasts [1,2]. Interestingly enough, there are\nsome studies showing higher prevalence of endometriosis\nin patients with Müllerian anomalies [19]; moreover, the\nexistence of choristoma composed of müllerian rests,\nnamed müllerianosis, has been postulated [13]. In recent\nyears, several evidence suggested that exposure to environ-\nTable 1: Characteristics of the foetuses enrolled in this study\nN° Gestational age Cause of death P resence of ectopic endometrium\n1 18 weeks Voluntary abortion Yes\n2 24 weeks Placental pathology Yes\n3 25 weeks Placental pathology Yes\n4 16 weeks Voluntary abortion Yes\n5 23 weeks Placental pathology No\n6 15 weeks Voluntary abortion No\n7 20 weeks Voluntary abortion No\n8 newborn Primary atypical pneumonia No\n9 newborn Acute interst itial pneumonitis No\n10 16 weeks Voluntary abortion No\n11 23 weeks Placental pathology No\n12 14 weeks Placental pathology No\n13 21 weeks Voluntary abortion No\n14 20 weeks Voluntary abortion No\n15 20 weeks Voluntary abortion No\n16 18 weeks Voluntary abortion No\n17 19 weeks Voluntary abortion No\n18 16 weeks Voluntary abortion No\n19 23 weeks Placental pathology No\n20 25 weeks Placental pathology No\n21 newborn Acute inter stitial pneumonitis No\n22 newborn Primary atypical pneumonia No\n23 20 weeks Voluntary abortion No\n24 19 weeks Voluntary abortion No\n25 newborn Cardiac malformation No\n26 newborn Cardiac malformation No\n27 20 weeks Voluntary abortion No\n28 23 weeks Placental pathology No\n29 19 weeks Voluntary abortion No\n30 newborn Cardiac malformation No\n31 newborn Cardiac malformation No\n32 19 weeks Voluntary abortion No\n33 newborn Acute inter stitial pneumonitis No\n34 20 weeks Voluntary abortion No\n35 newborn Cardiac malformation No\n36 21 weeks Placental pathology No\n\nJournal of Experimental & Clinical Cancer Research 2009, 28:49 http://www.jeccr.com/content/28/1/49\nPage 4 of 5\n(page number not for citation purposes)\nmental toxicants possessing estrogenic activity, the so-\ncalled endocrine disruptors, resulted in endometriosis\n[20]. Although the epidemiological evidences are not con-\nclusive to date, animal and experimental investigations\nhave provided a basis for the proposed association\nbetween estrogenic contaminants exposure and endome-\ntriosis [21]. Nevertheless, the mechanism(s) underlying\nthis potential association are poorly understood. The\nproper function of the normal human endometrium\nrelies on well organized cell-cell interactions regulated\nlocally by cytokines and growth factors under the direc-\ntion of steroid hormones. The onset and progression of\nthe disease processes of endometriosis may result from\ndisruptions of this well balanced cellular equilibrium,\nthat would cause the interruption of some organizational\nevents associated with development of the neonatal uter-\nHistological and immunohistochemical appearance of ectopic endometrium in four female human foetusesFigure 1\nHistological and immunohistochemical appearance of ectopic endometrium in four female human foetuses. \nPanel A: A 25 weeks foetus showing an endometrial structure in the recto-vaginal septum; in the inset named A', the immuno-\nhistochemical expression of CA-125 of this structure at higher magnification is depicted. Panel B: A 24 weeks foetus showing \nan endometrial structure in the proximity of the Douglas poutch; in the inset named B', the immunohistochemical expression \nof oestrogen receptor of this structure at higher magnification is depicted. Panel C: A 18 weeks foetus showing an endometrial \nstructure in the rectal tube at the level of muscularis propria; in the inset named C', the immunohistochemical expression of \nCA-125 of this structure at higher magnification is depicted. Note that the epithelium of the rectum is negative for CA-125. \nPanel D: A 16 weeks foetus showing an endometrial structure in the mesenchimal tissue close to the posterior wall of the \nuterus; in the inset named D', the immunohistochemical expression of CA-125 of this structure at higher magnification is \ndepicted. Note that in the wall of the primitive miometrium is present a little group of endometrial cells positive for CA-125 \n(indicated by an asterisk), that could represent a primitive nest of adenomyosis. Abbreviations used: an (anus); co (coccyx); dp \n(Douglas' pouch); re (rectum); rvs (recto-vaginal septum); sc (spinal column); ut (uterus); bl (bladder).\nu\nbl\nre\nre\nA\nA’\nB\nB’\nC\nC’\nD\nD’\n*\n\nPublish with BioMed Central   and  every \nscientist can read your work free of charge\n\"BioMed Central will be the most significant development for \ndisseminating the results of biomedical research in our lifetime.\"\nSir Paul Nurse, Cancer Research UK\nYour research papers will be:\navailable free of charge to the entire biomedical community\npeer reviewed and published immediately upon acceptance\ncited in PubMed and archived on PubMed Central \nyours — you keep the copyright\nSubmit your manuscript here:\nhttp://www.biomedcentral.com/info/publishing_adv.asp\nBioMedcentral\nJournal of Experimental & Clinical Cancer Research 2009, 28:49 http://www.jeccr.com/content/28/1/49\nPage 5 of 5\n(page number not for citation purposes)\nine wall [21]. To the best of our knowledge, this observa-\ntion is the first direct evidence in human female foetuses\nof the presence of ectopic endometrium outside the uter-\nine cavity. Our data sustain the müllerianosis hypothesis\nof an embryological origin for endometriosis, suggesting\nalterations in the fine tuning of female genital structures\norganogenesis, possibly caused by environmental toxi-\ncants. Interestingly, the percentage of foetuses analyzed in\nour study, that displayed the presence of ectopic\nendometrium is very similar to the prevalence of women\nsuffering for this disease in the general population [1-3].\nThis further suggests a strict link between embryological\nabnormalities and onset of the disease, even if the number\nof foetuses analyzed is too small in order to reach defini-\ntive conclusions. Further studies are urgently required in\norder to better define the molecular mechanisms underly-\ning this phenomenon. In particular, ad hoc in vitro and in\nvivo models should be set up to analyze the effects on cell\nhomeostasis and on the morphogenesis of the female gen-\nital system of different endocrine disruptors. Considering\nthat, based on epidemiological studies, women with\nendometriosis have an increased risk of different types of\nmalignancies, especially ovarian cancer and non-Hodg-\nkin's lymphoma [1], the implications of these findings\ncould be very important also in the oncology field.\nConclusion\nThe clinical and therapeutic implications of this observa-\ntion are straightforward. Endometriosis could not be\nregarded as a recurrent disease, therefore surgery, if com-\nplete can be considered curative and it would be not justi-\nfied post-operative hormonal treatments. Nevertheless, it\nmust be underlined the fact that other pathogenetic mech-\nanisms for the genesis of endometriosis can not be com-\npletely ruled out by these observation, even if, to date,\nthere are no direct evidence of their validity.\nCompeting interests\nThe authors declare that they have no competing interests.\nAuthors' contributions\nPGS and AB conducted the work, analyzed the data and\nwrote together the manuscript. FB performed the histolog-\nical and immunohistochemical analysis. RB, FB and MDA\nperformed the autopsies. MDF performed the immuno-\nhistochemical staining.\nAcknowledgements\nThis work was supported by a grant from \"Fondazione Italiana Endometri-\nosi\".\nReferences\n1. Baldi A, Campioni M, Signorile PG: Endometriosis: pathogenesis,\ndiagnosis, therapy and association with cancer.   Oncol Reports\n2008, 19:843-846.\n2. Giudice LC, Kao LC: Endometriosis.  The Lancet  2004,\n364:1789-1799.\n3. Houston DE: Evidence for the risk of pelvic endometriosis by\nage, race, and socioeconomic status.   Epidemiol Rev  1984,\n6:167-191.\n4. Koninckx PR, Martin D: Treatment of deeply infiltrating\nendometriosis.  Curr Opin Obstet Gynecol 1994, 6:231-234.\n5. Signorile PG, Campioni M, Vinc enzi B, D'Avino A, Baldi A: Rectovag-\ninal septum endometriosis: an immunohistochemical analy-\nsis of 62 cases.  In Vivo 2009 in press.\n6. Nap AW, Groothuis PG, Demir AY, Evers JL, Dunselman GA:\nPathogenesis of endometriosis.  Bet Pract Res Clin Obstet Gynaecol\n2004, 18:233-244.\n7. Brosens I: Endometriosis and the outcome of in vitro fertiliza-\ntion.  Fertil Steril 2004, 81:1198-1200.\n8. Nisolle M, Donnez J: Peritoneal endometriosis, ovarian\nendometriosis, and adenomyotic nodules of the rectovaginal\nseptum are three different entities.   Fertil Steril  1997,\n68:585-595.\n9. Fujii S: Secondary mullerian system and endometriosis.   Am J\nObstet Gynecol 1991, 165:219-225.\n10. 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Redwine DB: Invisible microscopic endometriosis: a review.\nGynecol Obstet Invest 2003, 55:63-67.\n18. Batt RE, Mitwally MF: Endometriosis from thelarche to\nmidteens: pathogenesis and prognosis, prevention and peda-\ngogy.  J Pediatr Adolesc Gynecol 2003, 16:337-347.\n19. Nawroth F, Rahimi G, Nawroth C, Foth D, Ludwig M, Schmidt T: Is\nthere an association between septate uterus and endometri-\nosis?  Hum Reprod 2006, 2:542-544.\n20. Anger DL, Foster WG: The link between environmental toxi-\ncant exposure and endometriosis.   Front Biosci  2008,\n13:1578-1593.\n21. McLachlan JA, Simpson E, Martin M: Endocrine disrupters and\nfemale reproductive health.  Best Pract Res Clin  Endocrinol Metab\n2006, 20:63-75.","source_license":"CC0","license_restricted":false}