Unus pro omnibus, omnes pro uno: A novel, evidence-based, unifying theory for the pathogenesis of endometriosis

The theory of retrograde menstruation as aetiopathogenesis of endometriosis formulated by John A Sampson in 1927, shows clear shortcomings: this does not explain why retrograde menstruation is a physiological process that affects 90 % of women, while endometriosis occurs in only 10 % of cases; it also does not explain the endometriotic foci distant from the pelvis, nor explains the cases of endometriosis in male patients. The immunological alterations of the peritoneal fluid explain s the effects of disease, such as the inhibition of the physiological processes of cytolysis, but does not explain the cause. There is evidence to support the hypothesis that müllerian remnants of the endometrium, and endocervix endosalpinx, ectopic, are items from the genital ridge leaked during organogenesis . It is known that tissues derived from coelomatic epithelial and mesenchymal cells have the potential to metaplas tically differentiate into epithelium and stroma . In addition, the phenotype of the ectopic endometrial cells is significantl y different from those eutopics . There is no scientific evidence that, during organogenesis, the genes of the H omeobox and Wingless family play a fundamental role in the differentiation of the ducts of Muller and development of the anatomical structure of the urogenital tract. We present here a hypothesis that deregulation of genes and the Wnt signaling pathway Wnt /β-catenin leads to aberrations and deregulation within the mesoderm, thus, may cause aberrant placement of stem cells . In addition, immune cells, adhesion molecules, extracellular matrix metalloptroeinases and pro - inflammatory cytokines activate/alter cells, creating the conditions for differentiation, adhesion, proliferation and survival of ectopic endometrial cells.

Endometriosis, Embryology, Body Patterning, Embryonic Stem Cells, Proteins homeodomain, Wingless Type Proteins. Page 3 of 40

Endometriosis is characterized by the presence, in the anomalous endometrial tissue, functionally active, with the presence of stroma and glands [1]. Numerous theories have been proposed for the pathogenesis of endometriosis , such as, retrograde menstruation, coelomic metaplasia and m üllerian remnants [2] , a hormonal disease, autoimmune disease, genetic disorder or due to an environmental stimuli [3]. Amongst the various hypotheses, the one that enjoys the greatest consensus is retrograde menstruation . Retrograde menstruation is when endometrial cells and fragments desquamate during menstruation and are transported via the fallopian tubes into the peritoneal cavity , instead of flowing out the body , and implant and proliferate onto peritoneal surfaces or pelvic orga ns [1]. The prevalence of endometriosis is estimated to be 10 % [4], with a further 11 % of women whose disease is not clinically diagnosed [5]. Endometriosis predominantly affects the ovaries (up to 88 %), the ligaments of the uterus, fallopian tubes, th e cervical-vaginal area, urinary tract and the rectum ; the involvement of the urinary tract is rare ( 1-2 % of all cases ) [6,7], of which 84 % are located within the bladder [8] ; other organs of involvement include, pancreas, spleen [9], liver, intestinal tract, gallbladder [10], wall of the abdom en and the umbilicus [11]; with brain endometriosis also being reported [12]. The foci of endometriosis distant from the pelvis can be explained as being derived from buds of the embryonic genital ridge and originate within the müllerian ducts which, during organogenesis, are located at the top. R etrograde menstruation is a physiological phenomenon which occurs in 76- 90 % of women [13], whil st disease occurs in 10 % of cases. The hypothesis of retrograde mens truation as the pathogenesis of endometriosis does not explain the gap between physiological prevalence (76-90 %) and the pathological (10 %). Interestingly, a case of endometriosis in the cul-de-sac and uterosacral ligaments was histologically confirmed in a patient undergoing pre -menarche Page 4 of 40 at 9 years of age [14]. However, this does not explain the cases of endometriosis in male individuals with normal phenotype (46, XY) , such as: endometrioma in the abdominal wall [15]; endometriosis of the bladder [16]; a case histologically indistinguishable from endometrial tissue [17]; cystic endometriosis of the epididymis [18]; paratesticular endometriosis [19]; a mass located lateral ly to the spermatic cord, removed surgically, whose histology showed tissue similar to the endometrium, a proliferation of smooth muscle, endometrial glands and stroma [20]. Such cases of male patients can only be explained by the incomplete differentiation of the müllerian ducts. Hypothesis The theory of "retrograde menstruation" , as a "cause" of endometriosis, does not correlate with the incidence among physiological event s, and prevalence of the disease does not correlate with the endometriotic foci distant from the pelvis in the reported cases in male patients. Since 1927, the year of Sampson's theory, many advances have been made in the direction of the effects but not the causes. We hypothesize that, during organogenesis, a deregulation of genes and the Wnt signaling pathway Wnt/β -catenin would produce an aberration and the axial extension of the identity of the anterior -posterior patterning, whilst a deregulation of Hox genes and cofactor Pbx1 produce s an aberration in the segmentation of the mesoderm (Fig. 1, Fig. 2) . T his may cause aberrant placement of stem cells with endometrial phenotype, ectopic, and maintain them in quiscent niche. In post -pubertal, the estrogenic activity activ ate peritoneal macrophages with consequent induction of pro- inflammatory cytokines TNF-α and IL-1β which, in turn, activate the binding to DNA through the transcription factors of NFҡB; transcriptional activity, through the inflammatory cytokines IL-6 and IL -8, induces the expression of VEGF that activates the vascular endothelial cell , Page 5 of 40 while MIF induces cell endometrial, mitosis, and the survival is supported by the activation of anti-apoptotic gene Bcl-2, from the degradation of the extracel lular matrix by MMPs and the entry phone via ICAM and VCAM, creating the conditions for differentiation, adhesion, proliferation and survival of ectopic endometrial cells. U nderstanding of the biological mechanisms, genetic and epigenetic, which regulate t he differentiation and development of the urogenital tract during the fetal stage, might be a priority for research ing the aetiopathogenesis of endometriosis and understanding of our hypothesis. Evaluation of the hypothesis Embryogenesis The primordial germ cells are derived from the primitive streak ( from epiblast to caudal area); remain ing in the extra -embryonic mesenchyme to complete gastrulation and subsequently migrate along the allantois endoderm; maintain ing the feature of cell division throughout the development of the embryo and preserving all the characteristics of stem cell s. Following gastrulation, the embryonic germ cells contribute to the formation of the epithelial and mesenchymal tissues. The epithelial cell s population of the embryo have similar morphological characteristics of differentiated epithelia, whil st mesenchymal cells contribute to the basal membrane, forming the lamina and smooth muscle of tubules and differentiation into connective tissue. The space beneath the epithelium and between the mesenchymal cells is filled with extracellular matrix molecules and their receptors [21-23]. During the early stages of organogenesis, the mesoderm arises from the primitive streak and give s rise to the epithelial coelomatic. The mü llerian ducts born by invagination of coelomatic epithelium, during fetal development results in the female reproductive tract, which is further differentiated to form the uterus, oviduct and vaginal canal higher. Animal studies have Page 6 of 40 demonstrated that the coelomic epithelium form s the müllerian ducts [21]. The coelom is derived from the same lateral plate mesoderm that, in turn, are derived from the primitive streak [22]. The anatomy of the female urogenital tract arising from the mü llerian ducts is completed at the time of birth with the exception of the uterus; the histological architecture and the tissue specificity reach full development in the post- natal period w ith the full radial patterning of 3 basic histological structures: (i) endometrium, (ii) myometrium and (iii) perimetrium. This results in the structured development of endometrial glands luminal epithelium, the organization and stratification of the endometrial stroma , and, the differentiation and growth of the myometrium [23-27]. Hox - Homeobox genes In mammals, the H ox genes are well known for their crucial role during embryogenesis, and in particular the axial development of the skeleton, the hind brain, and, the limbs. Their involvement in organogenesis has been shown, in particular, during urogenital differentiation [28]. The H ox genes control the fate of cell s and the segmental embryonic formatting. The sequential arrangement of the Hox genes on its chromosome associates with the spatial distribution and protein expression along the antero- posterior axis of the embryo [29]. The biological specificity of Hox proteins derive s from cooperation with specific cofactors that contribute to modulate the binding to DNA f or the control of the expression of target genes [30]. The protein cofactors include , pre-B-cell leukemia homeobox (PBX) and m yeloid ecotropic viral integration site (MEIS) [31,32]. The sub- cellular localization of proteins PBX is highly regulated in diffe rent cellular contexts; it has been hypothesized that the binding of PBX with MEIS induces translocation to the nucleus where it associates with H ox proteins which regulate target genes; Pbx is necessary to allow the formation of heterotrimeric complex DNA binding involving Meis proteins [33, 34]. Pbx has Page 7 of 40 been shown to act as a direct regulator of expression of the target gene, and this adjustment takes place via interactions that require the cooperation of other members of the family homeobox as Meis and Hox [35]. These have been shown to be involved in malformations of the urogenital tract and its inactivation which leads to complete absence of mü llerian structures [36,37]. This plays a critical role as a regulator of the development and the absence of which leads to embryonic lethality and multiple system abnormalities of tissues and organs. Pbx1 is extensively expressed in the mesenchymal tissues during differentiation of the urogenital organs , and inadequate cell proliferation leads to total absence of the adrenal glands, whilst the formation of the gonads shows a rudimentary sexual differentiation. T he lack of expression of P bx1 greatly reduces the evolution of the urogenital ridge w hich translates into reduced differentiation of the mesonephros and kidneys and in the absence of the müllerian ducts [38- 40]. Pbx1 has been proven to be expressed in the ductus Müller but absent in the Wolff ductus during the differentiation of both sexes [40]. The clusters of Hox genes during development, are subject to transcriptional control by cofactors such as RA (Retinoic Acid) [40], FGF (Fibroblast Growth Factor) [41,42] and the genes of the Wnt signaling [43]; this loop of self -induction and/or repression of H ox genes occurs within the same cluster [44 -46], as well as the post -transcriptional regulation [47,48]. During organogenesis patterning of female genital tract is regulated by homeobox transcription factors [49]: H oxA9 is expressed in the oviduct, HoxA10 (via BMP -4 Bone Morphogenetic Protein, Wnt7a and β3-integrin) and Hoxa-11 (by Emx-2 Empty spiracles homeobox gene and IGFB1 Insulin-like Growth Factor Binding protein) are expressed in the uterus [50]. HoxA11 and 13 in the cervix and vagina [51] . HoxA genes play a role in regulating temporal and spatial expression in the formation and differentiation of the müllerian ducts [52]. Wnt - Wingless genes Page 8 of 40 Wnt4 is essential for the formation of the m üllerian ducts. [53] In fact it is involved in numerous anomalies and female genital morphology in endometrial glandular and stromal breakdown. Wnt7 is involved in the maintenance of HoxA10 and HoxA11 genes whilst Wnt5 in the development of the genital anterior -posterior axis [54,55]. Wnt5a and Wnt7a are necessary for proper glandular genesis and are expressed, respectively, in the stroma of the uterine and uterine epithelium [56]. Downstream of Wnt genes, β -catenin [57] is associated with the Foxa2 forkhead family [58]. There are 3 types of signaling pathways: Wnt/β-catenin, Wnt/JNK (c-Jun N-terminal kinases) and Wnt/Ca2 +. Wnt binds via cell surface receptors, to disable the Axin complex , consequently inhibiting the phosphorylation of β -catenin from the complex by Axin [59] . β-catenin enters the nucleus in cooperation with factors Lymphoid Enhancer Factor / Transcription Factor (LEF / TCP), which by binding to DNA activates gene transcription [60]; the absence of stimulation by Wnt causes the phosphorylation of β -catenin from t he Axin complex, which is phosphorylated and then targeted for ubiquitination and degradation in the proteasome [61]. It w as shown that an estrogenic compound may interfere with Wnt expression and/ or β-catenin target genes with a consequent alteration of t he development of the female reproductive tract [62, 63]. The signaling pat hway of canonical Wnt genes and Wnt/β-catenin, are associated in the control of different types of stem cells and can act as a factor niche to keep the embryonic stem cells ( EmSC) in a state of self -renewal [64-66]. Müllerian derivatives and remains Congenital anomalies of the urogenital tract : During organogenesis differentiation between male and female urogenital system s takes place. B etween the eighth week and the fourth month the male urogenital tract initially develops from embryological structures which are Page 9 of 40 resolved with female -specific activation of the male genome. A missing or incomplete differentiation results in disorders of se x development, chromosonic abnormalities (such as, Turner syndrome and Klinefelter's syndrome ), M üllerian agenesis , Rokitansky syndrome, developmental disorders or testicular androgen insensitivity syndrome (or Morris syndrome). Remains and Müllerian deri vatives: Sexual differentiation is, in some congenital diseases, absent or incomplete, thus, it is plausible to assume that they can co -exist in the development of müllerian remnants in asymptomatic individuals. Many mü llerian events suggest that tissues derived from the epithelium and mesenchymal cells coelomate (Secondary Müllerian System) and have the potential to differentiate directly into epithelial cells and stromal cell; possibly a metaplastic hypothesis for the pathogenesis o f endometriosis [67]. The peritone al cavity is a matrix for the benign and malignant proliferation of the secondary müllerian system where it can develop endometriosis, endosalpingiosis and endocervicosis [68]. Under immunologically “normal” conditions, the peritoneal cavity has the ability to prevent the evolution towards endometriosis, however, failure to remove fragments of endometrial tissue from the peritoneal cavity induces local inflammation, activation of macrophages which secrete cytokines and che mokines some of which c an cause metaplasia of the peritoneum or the development of m üllerian residues [69]. P elvic masses and congenital malformations associated with mü llerian have been reported at the time of diagnos is of endometriosis, comprising of smooth muscle tissue within the uterine cavity, but , pose diagnostic uncertainty between smooth muscle metaplasia or m üllerian remnant of the system [70,71]. It is speculated that in males with normal male phenotype who develop endometriosi s, have prostatic utricle as a remnant of the uterus embryo [15]. Page 10 of 40 Müllerianosis: There are considerable difficulties in the differential diagnosis between endometriosis and müllerianosi s. The main difference is that , in endometriosis , ectopic endometrial tissue cyclically executes outside of the uterine cavity invading the outer surface of other organs, whilst, in müllerianosis , there is tissue present in the endosalpinx, endometrium and endocervix, whose most common form is found in peritoneal pockets. Batt RE et al . have laid down 3 conditions for the diagnosis of müllerianosi s: 1) no e vidence of pelvic endometriosis , 2) no direct communication with endocer vix, endometrium or endosalpinx, and, 3) n o surgery to the reproductive organs. G iven the presence of the 3 components, endometrium, endocervix and endosalpinx, support s the hypothesis that müllerian remnants generated from the genital ridge leaked during organogenesis [72]. In the presence of defects in the genesis of the genital tract , differentiation and cell migration can be incomplete or aberrant. Any cells with aberrant gene expression in the migratory path through the rear pelvic floor can be implanted abnormally. Pluripotent cells can cause endometrial metaplasia or endometriosis in post pubert y. Studies on the coelomic cavity and m üllerian duct, both in the fetal period and in adulthood, suggest s that the epithelium coelomatic , fabrics and related adult epithelia m üllerian derivatives, have common embryological origin [73]. In fact, in peritoneal biopsies of the cul -de-sac in female infants who had died from sudden infant death syndrome (SIDS), had a small whitish plaque, (~ 200μm in diameter ), which showed glandular epithelium with well -defined structures surrounding the stroma [74]. In addition, in fetal autopsies, the incidence of ectopic endometrium in 5 different locations identified in the recto -vaginal septum close to the cable Douglas near the mesenchymal tissues of the wall r ear of the uterus in the cannula at the level of the muscular wall of the uterus. Thus, one possible reason of endometriosis , is th e dislodgement of primitive endometrial tissue outside the uterine cavity during organogenesis [75,76]. Page 11 of 40 Müllerian cyst remai ns in cavitated: Accessory and Cavitated Uterine Masses (ACUM) is a sporadic condition seen in young females, which has significant clinical manifestations , in particular severe dysmenorrhea and recurring pelvic pain. The diagnosis presents considerable difficulties, so as to be placed in the differential with uterine malformations such as bicornuate uterus and segmental atresia, cystic areas or degenerate with adenomyosis, leiomyomas and degenerated primary dysmenorrhea essential [77]. ACUM is diagnosed more frequently in women aged less than 30 years and in nulliparous women (although sporadic cases are reported of women over the age of 30 years and multiparous) [78]. T he term Asian juvenile cystic adenomyoma was used for the diagnosis of cases with clinical and histopathological features similar to ACUM [77,78]. The ACUM are generally located at the level of insertion of the round ligament and is likely associated to a dysfunction of the female gubernaculum. The aetiopatogenic hypothesis classifies t his as a new variety of M üllerian anomalies [79] which may be caused by duplication or from ectopia and the persistence of müllerian duct, whose fabric is to be placed in an ectopic position at the level of the attack of the round ligament and could be related to a dysfunction of the gubernaculum [79,80]. Stems cells Human embryonic stem cells (hEmSC): hEmSC are pluripotent cells derived from various stages of embryonic development and represent the only form of stem cells able to proliferate indefinitely and to differentiate into all types of tissue -specific cells. The hEmSC are generally derived from the in ner cell mass of the blastocyst to the stage of pre -implantation embryo. hEmSC cell lines are well characterised in regards to genomic integrity and pluripotency and express high levels of telomerase activity. Telomerase (or terminal transferase) is a ribo nucleoprotein that adds telomere repeats to the chromosom al ends and Page 12 of 40 thus, maintains telomere length, and is crucial in the replication life span [81]. The expression of telomerase correlates with immortality of cell lines, and , the reintroduction of telom erase activity in some cell lines extends the ir replication activity [82]. The hEmSC, being pluripotent possess the characteristics to differentiate into the 3 germ layers which form all tissues of the embry o - (i) ectoderm, (ii) mesoderm, and, (iii) endoderm. They have specific morphological and molecular properties, they possess specific properties that epigenetic chromatin structure is open- ended to allow the entry of transcription factors , and, regulates gene expression [83] . In the promoter regions of pluripotency genes OCT4 ( octamer-binding transcription factor 4) and Nanog (homeobox transcription factor - regulator involved-in inner cell mass and embryonic stem) it denotes a marked reductio n in methylation of CpG nucleoids (cytosine-phosphate-guanine nucleotide) [84]. These properties are necessary to characterize the epigenetic hEmSC in a pluripotent state and distinctive , undifferentiated stem cell hEmSCs derived from cell lines that form both the endoderm and mesoderm. For endoderm differentiation Activin-A ligand activates transforming growth factor beta (TGF-β) [85], bone morphogenetic protein (BMP),, fibroblast growth factor (FGF) and the Wnt family of genes, which are typical modulators of the mesoderm [86]. Endometrial stem progenitor cells (hESP): Adult stem cells are found in an undifferentiated form and have the characteristics of self -renewal through cell division dependent microenvironment or niche. They are important for the regeneration and recovery of organs and tissues by ensuri ng regular functional maintenance. The human endometrium is composed of epithelium, glands and stroma , which during the menstrual cycle are subject to profound changes in tissue structure and function ; the recovery is ensured by the presence of the endometrial progenitor stem cells that are assumed to reside with in the basal layer [87]. Several lines of endometrial stem cells and progenitor cells have been characterized that show Page 13 of 40 large plastic capacity with high availability differentiation [23 ,88-90]. Endometrial stem progenitor cells ( hESP), differ for patterns of expression of cell surface markers for clonal efficiency, to the microenvironment of the niche, and endometrial localization [91,92]. In fact, in a study of clonal analysis of endometrial epithel ial cells and stromal cells derived temporally on the phases of the cycle, non- clonogenicity ranged from proliferative to secretory phase endometrium and between cycling and inactive, for both epithelial stromal cells, showing that the inactive endometrium contains clonogenic epithelial cells and stromal cells [93]. Some studies have suggested the origin of hESP from bone marrow as a source of exogenous [94,95]. Endometriotic lesions are detectable in a functionally pathological stage , and it is extremely rare to detect microscopically the phases of attachment and proliferation of endometrial tissue in the peritoneum, which is an area with high incidence of injury [96]. The origin of the cells within ovarian endometriomas are monoclonal, whilst peritoneal lesions are polyclonal [97- 99]. The cells that give rise to ectopic endometrial implantation must necessarily possess the ability to migrate, the angiogenic potential for proliferation and pluripotency to form glandular tissue and the hESP cells demonstrate all the requirements [87]. Inded a hypothesis was formulated in that repeated physical and biochemical injuries caused by inflammatory cytokines and reactive oxygen species are able to trigger the cell cycle of quiescent stem cells that may be involved in the development of benign and malignant endometrial aberrations as endometrial hyperplasia, endometriosis and endometrial cancer [100]. Stem/progenitor cells residing in adult uterus (SP) : The mucosal lining of the uterus remarkably regenerates during the reproductive years of a woman and this plasticity of the endometrium has been attributed to a small population of stem/progenitor cells, known as side population (SP). In fact, SP cells reside in the adult basal endometrium and is assumed to be Page 14 of 40 the remains of the original epithelial cells , the Müller Duct (MD) [101]. The SP ha s all the features that define poorly differentiated stem cells that are able to divide asymmetrically and quiescently [102]. The stem cells, to maintain the pool of progenitors from which arise the differentiated cells , are programmed to have a long lifespan; in order to activate the mechanisms of protection from senescence and stress of DNA, including the activation of several signalin g pathways such as Shh (Sonic hedgehog), Wnt/ β-catenin, Bmi -1 (B lymphoma Mo-MLV insertion region 1 homolog) the expression of Bcl -2 anti-apoptotic and the increased capacity of the repair of DNA damage [103 -108]. The SP are characterized by high expressi on of stem cell markers and low levels of differentiation markers, high expression of genes that are part of some of the signal transduc tion pathways such as the Wnt/β-catenin [109] and of genes involved in regulation of cell cycle [110]. Compared with other stem cells, the SP are small, even smaller than those fr om non SP [111,112] and ha ve endoplasmic reticulum s with ribosomes which indicates a lack of metabolic activity [113]. The SP are generated in the embryo, and , persist in specific niches, where the y can remain mitotically quiescent for long periods of time maintaining the capacity for self - renewal,symmetric division and the ability to rapidly produce progenitors for asymmetric division [114]. The microenvironment surrounding stem cells contribute to a number of functions, such as, physical anchorage for stem cells as well as cell-cell communiation mediated by direct contact and/or indirect extracellular factors . In as such, Wnt ligands are secreted by both stem cells and niche cells, BMPs are released from the cells and niche Shh epithelial cells, which interact between neighboring cells through the Notch signal transmembrane. This microenvironment also provides signaling through the cellular recepto r integrin [115] and its co- expression with CD133 (prominin-1) in basal cell lysophospholipids [116] as well as through signaling m ediated by metalloproteinases [ 117]. The identification and characterization of SP cells will further aid in our understanding of normal human Page 15 of 40 endometrial regenerative cyclic processes and the pathophysiology of human endometrial proliferative diseases, including endometriosis, endometrial hyperplasia and cancer [118,119]. Mesenchymal stem cells from bone marrow : Mesenchymal stem cells (MSC) are multipotent stromal cells which have the ability to differentiate into a variety of specialized cell types. Cells derived from bone marrow, known as bone marrow stromal cells ( BMSC) have been used in a number of studies. It has been hypothesized that endometrial stem cells may originate from mesenchymal stem cells of the bone marrow. Stem cells derived from bone marrow are able to differentiate into hematopoietic cells and contribute to the maintenance of different tissues; cells of the bone marrow donor -derived have been identified in the uterine human endometrium [120]. In fact, CD45 + hematopoietic progenitor cells colonize within the epithelial layer of the uterus , and. during pregnancy over 80 % of epithelial cells are derived from these cells [121]. In addition, in intravenous transplantation of bone marrow stem cells , the epithelial (0.02÷48 %) and stromal (0.03÷ 52 %) compartments arose from the donor [122]. Furthermore, endometrial regenerative cells ( ERC) compared to BMSC cells are similar but not identical in regards to , their morphology, the production of cytokines, the inhibition of mixed leukocyte reactions, the expression micro RNA (miRNA) and global gene expression. However, ERC are affected by over -expression of gene immune path, whil st BMSC are affected by over -expression of gene path stem/ tumor; ERC also show greater inhibition of proliferation [123]. In other studies , ERC have been isolated from menstrual blood, which are distinct from the MSC as they do not express the BMSC marker STRO -1 (cell surface protein expressed by bone marrow stromal cells and erythroid precursors) [124,125]. It is not known whether the transplanted cells retain all the characteristics of stem cells, and whether they behave like those for the physiological endometrial cyclicity ; the Page 16 of 40 mechanism of physiological recruitment of stem cells from the bone marrow into the uterus is not clear. Stem cell niche: In adults, stem cells reside in a physiologically limit ed and specialized microenvironment, called a niche, which supports stem cells but changes in nature and position according to the type of fabric [126,127]. The niche is a collection of cells in a specific anatomic location which together aid in the maintainance (number, proliferation and fate) of stem cells via secretion of extrinsic factors [128- 130]. The morphological configuration of the dimensional niche can define the number of stem cells within a tissue. The asymmetric cell division of stem ce lls allows the self-renewal and differentiation of the cell produced by providing a simple method for tissue homeostasis; divisions are dependent on cell polarity within the cell and are influenced by cell niche. Most of the asymmetric divisions determine a stem cell, and a cell differentiation in which the daughter cell is placed outside of the niche. The self-preservation given to the daughter cell allows it to keep features such as stem cell proliferation and maintenance of undifferentiated state [131]. The ability of cells to divide asymmetrically to produce 2 different cell types provides the cellular diversity proper to each multicellular organism . T he asymmetric localization of cell- cell junctions and/or the intrinsic cells is crucial to the fate and position within the niche and is used to specify cell polarity and asymmetric divisions that determine the polarity of the cell fate; the asymmetric divisions are directly regulated by genes that control the process of division and determine different fates for the two daughter cells [132]. The molecular signaling Shh, BMP, FGF and Notch are implicated in the control of stem -cell self-renewal and regulation of the fate of the lineage in different systems [128 -130]. Reactive oxygen species (ROS), a natural byproduct of metabolism of oxygen plays an important role homeostastis. However, during stress, the levels of ROS increases as well as the number of free radicals , such as, superoxide Page 17 of 40 radical anion, hydrogen peroxide and hydroxyl radical, which cause DNA damage. The levels of intracellular ROS plays a crucial role in the control of self-renewal capacity of stem cells in the long term as they may involve signaling of JNK (c -Jun N -terminal kinases) and FoxO (trigger for apoptosis through up -regulation of gene s) and sub -regulation of Polycomb (protein Able to remodel chromatin and Hox gene silencing) [133]. Marking of endometrial cells The phenotype of SP cells is similar to that of adult stem cells and is detected with fluoro-cytometric analysis using Hoech st 33342 dye (H33342 Bisbenzimide trihydrochloride), through the expression of ABC transporters, Brand gene expression Bcrp1 (ABCG2) that characterizes the phenotype of SP [134]. It was shown that the upper fraction is composed mainly of epithelial cells w hile the lower fraction contains both the epithelial and stromal cells; popula tions expressing epithelial CD9 + and E -cadherin while t he portion stromal express CD13+ demonstrated the presence of endometrial progenitor s tem cells [135]. Cunha GR et al . obtained the differentiation of hEmSC into mesodermal cells; the line of hESCs with genetic characterization of Forkhead protein- green fluorescent protein that regulates cell regionalization, placed under the control of MIXL1 (Mix paired- like), homeobox protein that acts as a transcription factor for the regulation of cell fate, demonstrating that FRT ( female reproductive tract) arises from embryon ic bodies characterized by MIXL + ; have also observed the expression of Hoxa -10 and Pax2 during development of hESCs epithelial FRT [136]. The use of cell surface markers was used for the isolation of endodermal progeny of hESCs. SOX17, FOXA1, FOXA2, HNF1β , HNF4 α, KITL, SHH and HB9 were used as markers expressed in cells CD49e + CD141+ CD238+ ; Page 18 of 40 OCT4, NANOG, and ME OX1 SOX7 were used to mark the pluripotency expressed in cells CD49e-/low CD141-CD238 [137]. Consequences and discussion John A Sampson, publi cation in 1921, reported observations i n 14 patients with cysts in that the co ating was similar to that in hematomas in the uterine lining with both having content similar to the phase of the menstrual cycle [138]; whilst the study of 1927, of 293 cases in a period of 5 years, presented at the "American Gynecological Society", adopted the theory of retrograde menstruation as the aetiopathology of endometriosis [139]. The problem- related histogenesis of endometriosis does not accept or reject the theory of Sampson JA, but provided a direction for research . The basic question is why retrograde menstruation is a physiological process that affects 90 % of women and endometriosis occurs only in 10 % of cases? How do we explain the endometriotic foci away from the pelvi s? How are we to explain the cases of endometriosis in male patients? The theory of endometriosis, s uch as endometrial cells from functional retrograde menstruation, since its formulation, has shown gaps. Endometriotic lesions are detectable at a pathological stage, and it is extremely rare to be able to detect microscopically the phases of attachment an d proliferation of endometrial tissue in the peritoneum [96]. Numerous studies have attempted with rigorous methods, to give answers as to why the eutopic plant develops resistance to the elimination by the immune system, demonstrating the altered function of macrophages and natural killer cells: that in the early stages of the disease there is a prevalence of pro -inflammatory cytokines (Th1 profile), whilst in late stages this changes to a Th2 profile [140]; that alterations of immune peritoneal exert an i mmunosuppressive effect on the activity of phagocytic and cytotoxic immune cells infiltrating the endometrial tissue, promoting immunoescaping, survival and growth of Page 19 of 40 endometrial cells [141 -144]. Therapeutic strategies can be improved through the use of non - steroidal anti-inflammatory drugs [145], combination oral contraceptives [146,147], progestin [148], selective progesterone receptor modulators [149], GnRH agonists [150], and, aromatase inhibitors [151,152]. Ultimately we are able to demonstrate the pa thophysiological mechanisms that allow grafting of endometriotic cells and the inhibition of the physiological processes of cytolysis, but the origin of these cells remains unknown. In 1987, Redwine and colleagues examined peritoneal biopsies of the cul -de-sac of female infants who died from SIDS, reported a case with well -defined structure glandular epithelium surrounded by stroma [74]. Fujii S in 1991, suggested that the tissues derived from epithelial and mesenchymal cells coelomatic accompaniment, called " Secondary M üllerian S ystem" have the potential to differentiate into epithelium and stroma, metaplastic, and that this potential is a basic concept in the pathogenesis endometriosis [153]. Batt RE et al. in 2007, concluded that the presence of endometrium and endocervix endosalpinx, which supports the hypothesis of m üllerian remnants generated from the genital ridge leaked during organogenesis [72]. Master PG et al . in 2012, investigat ed fetal autops ies and noted the presence of ectopic endometrium, assuming that one possible cause of endometriosis was the dislocation of primitive endometrial tissue outside the uterin e cavity during organogenesis [76] . Bouquet de Jolinière J et al . in 2012, demonstrated that reproductive organs derived from autopsies of female fetuses (via immunohistochemical analysis) were identified ectopic, and, concluded that endometriosis may develop from misplaced endometrial glands and/ or residues of embryonic cells [154] . hESCs, ident ified in the basal layer of the endometrium, appear to possess the phenotype that contains all the characteristics of self-renewal and differentiation that occurs in the context of the niche in which they exist, or in those in which they migrate. hESCs pos sess a potential immunomodulatory triggered by hypoxic stimuli, proteolytic, inflammatory, in order to induce angiogenesis, intercellular communication, migration, and capacity to Page 20 of 40 differentiate into cells of the same lineage (Fig. 1, Fig. 2) [155]. It was also hypothesized that the hESCs may have originated from mesenchymal stem cells of the bone marrow; studies on the expression of miRNA and global gene expression, showed that they could be considered similar but not identical, and that the endometrial reg enerative cells were affected by over - expression of gene immune path, whil st bone marrow stromal were characterized by over - expression of gene path stem/tumor . I n addition, the regenerative cells showed a greater inhibition of endometrial proliferation (Fi g. 1, Fig. 2) [123]. It ha s not been demonstrated whether the transplanted cells retain the characteristics of stem cell, if they behave as physiological ones for endometrial cyclicity, but, above all, mechanism of physiolog ical recruitment in the uterus has not been shown. Moreover, Delbandi and colleagues evaluated characteristics of the cells and ectopic endometrial stromal eutopics in women with respect to controls eutopics of healthy women and noted that ectopic endometrial stem c ells differ from eutopics, with a greater capacity for proliferation, greater adhesion to the extracellular matrix, increased invasiveness and higher levels of pro-inflammatory cytokines, IL-6 and IL-8 [156]. The H ox genes control cell fate and segmental embryonic patterning along the antero - posterior axis of the embryo [29] in cooperation with specific cofactors and in particular, through the Notch signaling pathway, determini ng positional identity and the activities of the genetic cascade of somitogenesis [157]. The morphogenetic processes axial extension, the segmentation of the mesoderm and anterior -posterior patterning are regulated by the interaction between Hox genes and Wnt: while Wnt, RA and FGF regulate the a xial extension and the identity of the anterior-posterior patterning, Hox, Cdx (paraHox genes) and Notch are involved in the segmentation of the mesoderm [158]. PBX and MEIS contribute to modulate the binding to DNA for the control of the expression of target genes and Pbx1, in particular, is widely expressed in mesenchymal tissues during the differentiation of the urogenital organs [38-40]. Wnt7 has been shown to be involved in the maintenance of the genes HoxA10 and Page 21 of 40 11, and Wnt5 in the development of the genital anter ior-posterior axis [54,55]. Three members of the family Wnt (Wnt4, Wnt5a and Wnt7a) have proved to be fundamental for uterine development: inactivation of Wnt4 causes sex reversal; Wnt7 causes inactivation of stratified epithelium, stroma and the absence of thin gland; inactivation of Wnt5 inhibits the development of the corr ect anatomy of the uterus [119] . In the presence of defects of adjustment, on the part of the genes responsible, during organogenesis differentiation of the urogenital tract and/or the migration of the cells may be aberrant or incomplete , and any cells with aberrant gene expression during the migratory path may implant themselves in the anomalous [73]. Tissues derived from the epithelium and mesenchymal cells accompanying coelomatic have the potential to differentiate directly into e pithelium and stroma and [67] . Hoang Ngo c and colleagues studied embryonic finds, and came to the conclusion that the myometrium is derived from the primitive mesenchyme, and the endometrium is derived from mesoltelio coelomatic [159]. The signaling pathwa y of canonical Wnt, the Wnt/ β-catenin, is implicated in the control of various types of stem cells and can act as a factor to maintain the niche hESP in a state of self -renewal [64-67]. Targeted r esearch on the coelomic cavity and the müllerian duct epithelium suggest that coelomatic and associated tissues, epithelia adults and müllerian derivatives have a common embryological origin, and that pluripotent cells can cause endometrial metaplasia or endometriosis in post pubertal stage [73]. The estrogenic activity active peritoneal macrophages with consequent induction of pro- inflammatory cytokines TNF -α and IL -1β which, in their turn, activate the binding to DNA through the transcription factors of N FҡB; through inflammatory cytokines IL -6 and IL -8, induces the expression of VEGF that activates the vasculature endothelial cell, while MIF induces cell mitosis endometrial, and survival is supported by the activation of anti -apoptotic gene Bcl-2, from the degradation of the extracellular matrix by MMPs and the entry phone via ICAM and VCAM (Fig. 1, Fig. 2) [160]. Page 22 of 40

It is n cessary to understand the biological and genetic mechanisms that regulate the differentiation of the urogenital tract during the phase of embryonic organogenesis, in the period of completion of development of the urogenital tract, and in post -puberty. It is important to study the signaling pathways of Hox genes and cofactors Pbx and Meis genes and Wnt signaling pathway Wnt/ β-catenin. It is necessary to deepen the knowledge on embryonic stem cells, niches and the functioning of the regulatory mecha nisms of the state s of quiscenze , self -renewal, proliferation and functional specialization. T he research on immuno-phenotype, proliferation capacity, invasiveness and adhesion to the extracellular matrix of the endometrial stem cells (hESCs) eutopic and ectopic should be implemented. The study of the causal mechanisms of endometriosis involves in- depth knowledge of embryology, genetics, biology, histology, immunology and specific expertise in medical research with multidisciplinary team s which together, will lead to underst anding our hypothesis and etiology of endometriosis.. Page 23 of 40

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The epithelial cell populations of the embryo have similar morphological characteristics of differentiated epithelia, whilst mesenchymal cells contribute to the basal membrane, forming the lamina and smooth muscle of tubules and differentiation into connective tissue. During the early stages of organogenesis, the mesoderm emerges from the primitive streak and give s rise to coelomic epithelium. The müllerian ducts (Wnt4 is essential) arise from invagination of the coelomic epithelium during fetal development resulting in the female reproductive tract , which further differentiates to form the oviduct, uterus and vaginal canal higher. The clusters of Hox genes during development, undergo the transcriptional control by cofactors such as retinoic acid RA [40], FGF and Wnt signaling; during organogenesis patterning of the female genital tract is regulated by homeobox transcription fact ors: HOXA9 is expressed in the oviduct , Hoxa-10 (by BMP-4, Wnt7a and β3-integrin) and Hoxa-11 (via Emx-2 and IGFB1 ) are expressed in the uterus, Hoxa -11 and 13 cervix and vagina. The Wingless genes are implicated in endometrial glandular and stromal morp hology: Wnt7 has been shown to be involved in the maintenance of the genes HoxA10 and HoxA11, while in the development Wnt5 genital anteroposterior axis: Wnt5a and Wnt7a are both necessary for proper glandular genesis , and Wnt5a, in particular, is a critical element in the endometrial glandular formation which entails the role of epithelial-mesenchymal interaction required for uterine development. As a downstream effector of the Wnt genes , it has been demonstrated that the involvement of β -catenin and FoxA2, in the absence of stimulation by Wnt, causes the phosphorylation of β - catenin which is phosphorylated and then targeted for ubiquitination and degradation in the proteasome. In t he signaling pathway of the canonical Wnt genes, Wnt/β -catenin, is Page 39 of 40 implicated in the control of various types of stem cells and can act as a niche factor to keep the EmSC (Embryonic Stem Cell) in a state of self-renewal. Fig. 2. Fetal development: the morphogenetic processes of the axial extension, of the segmentation of mesoderm and anterior -posterior patterning are regulated by the interaction between Hox genes and Wnt signaling network in a gene that involves Wnt/β -catenin, in the extension and axial the identity of the anterior -posterior patterning, while the cofactors of H ox gene, Pbx1 and Meis1 are involved in the segmentation of the mesoderm; Wnt7 has been shown to be involved in the maintenance of the genes HoxA10 and HoxA11, while in the development Wnt5 genital anterior -posterior axis; Wnt4 is involved in the sexual wa y, Wnt7 in the epithelium, stroma and glands, Wnt5 in the anatomy of the uterus. Postnatal development: molecular signaling of Shh, BMP, FGF and Notch are implicated in the control of stem cell self-renewal and in regulating the fate of the lineage; the es trogenic activity active peritoneal macrophages with consequent induction of pro- inflammatory cytokines TNF -α and IL -1β which, in turn, activate the binding to the DNA through the transcription factors of NFҡB; the transcriptional activity through inflammatory cytokines IL-6 and IL-8, induces the expression of VEGF that activates the vasculature endothelial cell, while MIF induces cell mitosis endometrial and survival is supported by the activation of the anti-apoptotic gene Bcl-2 , from the degradation of the extracellular matrix by MMPs and the entry phone via ICAM and VCAM. Post -pubertal development: the eutopic plant develops resistance to elimination by the immune system, demonstrating altered function of macrophages and natural killer peritoneal cells; in the early stages of the disease there is a prevalence of proinflammatory cytokines (Th1 profile), while in the late stages cytokines predominantly fibrogenic and angiogenic action (Th2 profile) prevails. Page 40 of 40