{"paper_id":"b9d53bee-fb04-4fa2-afa0-03824b61ac24","body_text":"Prime Archives in Molecular Biology \n1                                                                                www.videleaf.com \nBook Chapter \n \nPathogenomics of Endometriosis \nDevelopment \n \nVladislav Baranov*, Olga Malysheva and Maria Yarmolinskaya \n \nD.O.Ott Institute of Obstetrics, Gynecology and Reproductology, \nRussia \n \n*Corresponding Author: Vladislav Baranov, D.O.Ott Institute \nof Obstetrics, Gynecology and Reproductology, Saint-Petersburg \n199034, Russia \n \nPublished February 24, 2020 \n \nThis Book Chapter is a republication of an article published by \nVladislav Baranov, et al. at International Journal of Molecular \nSciences in June 2018. (Baranov, V.; Malysheva, O.; \nYarmolinskaya, M. Pathogenomics of Endometriosis \nDevelopment. Int. J. Mol. Sci. 2018, 19, 1852.) \n \nHow to cite this book chapter: Vladislav Baranov, Olga \nMalysheva, Maria Yarmolinskaya. Pathogenomics of \nEndometriosis Development. In: Song Guo Zheng, editor. Prime \nArchives in Molecular Biology. Hyderabad, India: Vide Leaf. \n2020. \n \n© The Author(s) 2020. This article is distributed under the terms \nof the Creative Commons Attribution 4.0 International \nLicense(http://creativecommons.org/licenses/by/4.0/), which \npermits unrestricted use, distribution, and reproduction in any \nmedium, provided the original work is properly cited. \n \nFunding: This research was funded by Russian Science \nFoundation, Grant Number 14-15-00737. \n \nConflicts of Interest: The authors declare no conflict of interest. \n \n\nPrime Archives in Molecular Biology \n2                                                                                www.videleaf.com \nAbstract  \n \nFor over 100 years, endometriosis, as a chronic, estrogen-\ndependent, inflammatory, heritable disease affecting \napproximately 5–10% of women in reproductive age has been \nthe focus of clinicians and scientists. In spite of numerous \nenvironmental, genetic, epigenetic, endocrine, and \nimmunological studies, our knowledge of endometriosis is still \nfragmentary, and its precise pathophysiology and pathogenomics \nremain a mystery. The implementation of new technologies has \nprovided tremendous progress in understanding the many \nintrinsic molecular mechanisms in the development of \nendometriosis, with progenitor and stem cells (SCs) of the \neutopic endometrium as the starting players and endometriotic \nlesions as the final pathomorphological trait. Novel data on the \nmolecular, genetic, and epigenetic mechanisms of the disease are \nbriefly outlined. We hypothesize the existence of an \nendometriosis development genetic program (EMDP) that \ngoverns the origin of endometrium stem cells programmed for \nendometriosis (1), their transition (metaplasia) into mesenchymal \nSCs (2), and their invasion of the peritoneum and progression to \nendometriotic lesions (3). The pros and cons of the recent \nunifying theory of endometriosis are also discussed. Complex \ngenomic and epigenetic interactions at different stages of the \nendometriosis process result in different forms of the disease, \nwith specific features and clinical manifestations. The \nsignificance of the EMDP in elaborating a new strategy for \nendometriosis prediction, prevention, and treatment is discussed. \n \nKeywords  \n \nEndometriosis; Developmental Pathway; Pathogenomics; \nMesenchymal Stem Cells \n \nAbbreviations  \n \nEMDP- Endometriosis Development Program; ESC- \nEndometrial Stem Cells; SC- Stem Cells; SP- Sensitive Period; \n\nPrime Archives in Molecular Biology \n3                                                                                www.videleaf.com \nBmSC- Bone Marrow Stem Cells; MeSC- Mesenchymal Stem \nCells; EMT- Epithelial–Mesenchymal Transition \n \nIntroduction  \n \nEndometriosis is a common disorder affecting 5–10% of women \nof reproductive age. By clinical manifestation, it corresponds to \nchronic, estrogen-dependent inflammation mitigated by the \ngrowth of endometrium-like tissue in sites other than the uterine \ncavity, most commonly in the pelvic cavity. Although studied for \na century, many aspects of the pathophysiology and \ndevelopmental pathogenetics of the disease still remain obscure, \nand practical achievements in the prediction, prevention or \ntreatment of endometriosis remain rather illusive to date [1,2]. A \ndetailed understanding of the molecular mechanisms underlying \nendometriosis is also far from complete. Meanwhile, spectacular \nachievements in molecular diagnostics and system genetics in \nstudies of this common disease have provided a huge bulk of \nuseful information regarding the genetic aspects of \nendometriosis and the molecular mechanisms of its origin and \ndevelopment [3,4]. Many theories and attractive hypotheses on \nthe pathogenesis of endometriosis are known but they are rather \ncontradictory. Genetic, endocrine, environmental, immune, and \nepigenetic factors have been studied in numerous articles to \nexplain the mechanistic basis of the origin and development of \nendometriotic lesions [5,6]. Conspicuous progress in this area \nhas been achieved during the last decade, mainly due to the \nidentification of new candidate genes and numerous SNPs \n(single nucleotide polymorphism) tightly associated with \nendometriosis [6], of genetic and epigenetic mechanisms of its \nregulation [5,7], and of endometrial stem cells [8], and to \ntranscriptome and miRNA analyses of the endometrium and \nendometriotic cells [9,10]. The contribution of epigenetic and \ngenetic factors in the pathogenesis of endometriosis has been \ndescribed in many exhaustive reviews [3,4,10-12]. \n \nStudying endometriosis as a problem of developmental genetics \nis a principal goal of the present paper. The origin of \nendometriotic cells and the genetic and epigenetic factors \ncontributing to the initiation and growth of endometriotic lesions \n\nPrime Archives in Molecular Biology \n4                                                                                www.videleaf.com \nare briefly reviewed. We hypothesize the existence of a special \nendometriosis development program (EMDP) which switches on \nin the progenitor SCs of the endometrium and in SCs descended \nfrom the Mullerian duct. EMDP suggests that the cells are prone \nto giving rise to endometriosis partly through endometrial–\nmesenchymal transition, their invasion into the peritoneum \nlining, and differentiation and growth into endometriotic lesions. \n \nClassical embryology and developmental biology postulate that \neach morphogenetic event has its own critical and sensitive \nperiod (SP) which displays a heightened sensitivity to internal \nand external stimuli [13]. According to further molecular studies, \nthe critical periods precede visible morphogenetic reactions and \ncorrespond to massive genome reprogramming [14]. The \nsuggested SPs of EMDP should be considered a suitable \ntimeframe for the prediction and treatment of endometriosis. The \nepigenetic landscape of endometriosis reflects the complex \ninteractions of genetic and epigenetic factors, which underlies \nthe pathogenomics of endometriosis [15], creates a unique \nEMDP, substantiates endometriosis clinical manifestations, and \nprovides clues for a personalized treatment of this disease. \n \nKey Stages of Endometriosis Development  \nStem Cells in the Pathogenesis of Endometriosis  \n \nSCs are defined as undifferentiated cells which possess both self-\nrenewal and differentiation abilities [16]. The possibility for \nextra-uterine SC to progress into endometriotic lesions may \nexplain endometriosis developing in distant sites such as the \nlungs. They also support the theory suggesting that SC may \ntravel via lymphovascular spaces [17]. Finding the stemness-\nrelated genes, such as OCT4, SOX2, SOX15, NOTCH1, \nTWIST1, and others, expressed in endometriotic lesions, may \nhelp show that the mechanisms determining the self-renewal \nrates and SC fates are deregulated in endometriosis, leading to \naltered SC behavior [18]. \n \nAccording to initial studies, the multi-site origin of \nendometriotic SCs was repeatedly suspected [3,6,19]. Different \ntypes of endometrial SCs were hypothesized, such as \n\nPrime Archives in Molecular Biology \n5                                                                                www.videleaf.com \nendometrial SCs in the peritoneum and pelvic cavity (1), resting \nembryonic cells descendent from the Mullerian duct (2), SCs in \nmenstrual debris (3), coelomic epithelial cells after metaplasia \n(4), and mesenchymal bone marrow SC (bmSCs) in \ninflammation sites in the peritoneum (5). It was postulated that \nSCs that originated from bone marrow SCs could also be \nattracted in the human endometrium, but their participation in \nendometriosis should be proven [3,19]. Several different types of \nSCs have been suggested in the endometrium itself, including \nprogenitor cells of the endometrium, mesenchymal stem cells, \nand endothelial stem cells [16,20]. Under appropriate conditions, \nSCs shed with menstrual blood can differentiate into typical \nmesenchymal lineages [21]. Thus, although the exact location of \nendometrial SCs still needs to be explored, some findings \nsuggest that the inner basal layer resting on the myometrium at \nthe endometrium–myometrium interface and known as the \n―junctional zone‖, should be treated as a preferential site for the \nendometrial SC niche [16,22]. Also, bmSCs in the endometrium \ncould contribute to all stem cell kinds in the endometrium \n[19,23] The existence of own SCs in the endometrium is also \npostulated, although the specific markers to identify endometrial \nSCs have not yet been established [19,24]. \n \nAs might be inferred, little doubt is left with regard to the SC \norigin of endometriosis. Whether they SCs in the endometrium \nare endometrial by origin or come from other sources like the \nbone marrow, peritoneum, or some other tissues, remains \nunknown. Meanwhile, two major sources of endometriotic SCs \nshould be considered: SCs disseminated throughout the \nperitoneum lining the pelvic cavity during embryogenesis of the \nfemale reproductive tract (endometriosis of extrauterine origin) \n(1), and SCs from the endometrial layer (endometriosis of \nintrauterine origin) (2). The hypothesis of the extrauterine origin \nof endometriosis from mesenchymal SCs disseminated during \nembryogenesis that infested the epithelium lining of the pelvic \ncavity has recently received major support in the novel ―unifying \ntheory‖ of endometriosis pathogenesis [24]. More details of this \nhypothesis will be given in the Discussion. The second \nhypothesis is in line with the well-known hypothesis by \nSampson (1927), which postulates that the endometriosis \n\nPrime Archives in Molecular Biology \n6                                                                                www.videleaf.com \noriginates from the menstrual cells of endometrial tissue \ndisseminated in the pelvic cavity [25]. \n \nInitial Stages of Endometriosis  \n \nThe most intriguing problem of endometriosis pathogenesis \nconcerns the molecular mechanisms underlying the acquisition \nof tumor-like properties by otherwise normal SCs. According to \nthe ―uterine origin‖ and the ―extrauterine origin‖ hypotheses, \nmetaplasia of the endometrial (epithelial) cells into mesenchymal \ncells (so-called epithelial–mesenchymal transition—EMT) may \nplay a key role in the pathogenesis of endometriosis [26]. \n \nEMT is a biologic process during which polarized epithelial cells \nby consecutive changes get a mesenchymal cells phenotype. \nEMT plays a role in a series of biological settings, such as \nimplantation and embryogenesis and pathogenesis of malignant \ntumors, and is also associated with wound healing, tissue \nregeneration, and organ fibrosis [27]. The molecular mechanisms \nof EMT in epithelial cells involve the functional loss of E-\ncadherin, desmoplakin, and mucin-1 and increased expression of \nsuch mesenchymal markers as N-cadherin, smooth-muscle actin \nand ohers [28]. Cells of different origin can enter EMT leading \nto development of endometriosis. These cells can be peritoneum \nepithelium cells (as according to the metaplastic theory of \ndevelopment of endometriosis), endothelial cells, and also \nepithelial cells of the endometrium [26]. The molecular \nmechanisms of EMT have now been studied in detail [18]. \n \nMain inducers of EMT are well known [27]. Chronic injury and \nsubsequent inflammation can trigger EMT through the release of \nsome cytokines, such as TGF-β, PDGF, EGF, and FGF-2. A \nnumber of authors have reported that that the TGF-β level have \nincreased in peritoneal fluid and serum of women with \nendometriosis compared to healthy women [29]. Other inducers \nof EMT are hypoxia and other factors (i.e., the Ras–MAPK \n(mitogen-activated protein kinase) pathway) leading to \nhyperexpression of hypoxia-induced factor-1 (HIF-1A) [26]. \n \n\nPrime Archives in Molecular Biology \n7                                                                                www.videleaf.com \nThe principal role in the metaplasia of the endometrial \nepithelium might be attributed to the TWIST1 gene (Twist \nfamily basic-loop-helix transcription factor 1). It was identified \nas a key regulator of mesoderm development and later have been \nimplicated in many human diseases. The expression of TWIST1 \nis closely related to tumor aggressiveness and metastatic \npotential [30]. Twist1 has also been shown to function as a key \nregulator of EMT. Driven by HIF-1, Twist1 realizes its \ndevelopmental functions by governing cell movement and tissue \nreorganization [31]. The molecular mechanisms underlying EMT \ninduced by TWIST in epithelial cells involve functional loss of \nE-cadherin (CDH1) in the eutopic endometrium of endometriosis \npatients. Reduced level of cadherins accompanied by excessive \nexpression of metalloproteases (MMP) genes provide favorable \nconditions for cell migration. A mechanosensitive transduction \npathway involving β-catenin specifies the early mesodermal \nconservation, which is required for Twist mechanical identity. \nThus, transient hypoxia and mechanical tension switch on EMT \nthrough the activation of TWIST1. The expression of \ndoublecortin- and Ca2+/calmodulin-dependent protein kinase-like \nprotein-1 (DCAMKL-1), which is known to regulate TWIST1, \nMyc, KRAS, and other factors, was also recently discovered \n[18]. Furthermore, it has been pointed out that there might also \nbe some imbalances in micro-RNAs (miRNA) in women with \nendometriosis, enhancing cell invasiveness due to impaired miR-\n145 or promoting proangiogenic factors due to the \ndownregulation of miRNA-199a-5p or extracellular matrix \nregulator miRNA 29a, significant downregulation of mir-200b in \nthe endometrium and in peritoneal lesions, and regulation of \nHOX genes family miRNA196 [10]. Over 600 different miRNAs \nassociated with endometriosis at each stage of development are \nknown so far. The available results in miRNA studies of \nendometriosis are rather contradictory and need thorough \nrevision [10]. The significant heterogenicity of endometriotic \nlesion samples is considered a major problem when analyzing \nthe miRNA signatures of whole endometriotic lesion biopsies \n[4,9,10]. \n \nThus, during the dormant stage of endometriosis, there are some \ncells of endometrial origin which might potentially contribute to \n\nPrime Archives in Molecular Biology \n8                                                                                www.videleaf.com \nthe growth of endometriotic lesions. The latter is regulated by \nthe activation of specific transcription factors induced by \ntransient hypoxia, chronic inflammation, and mechanical tension \nswitch. The cells lose their polarity and contacts and acquire the \nmigratory and invasive abilities of mesenchymal stem cells. The \nexpression of the MYC and CCND1 (cyclin D1) genes leads to \nhigh proliferative activity, while the upregulation of BCL2 \nreduces apoptosis and prolongs survival. Thus, as a consequence \nof EMT, epithelial cells lose their specific features as well as \ntheir integrity and acquire mesenchymal traits linked to increased \ninvasion and migration properties [18]. Under appropriate \nhormonal and immunological stimulation, the SCs shed into the \nperitoneal cavity during retrograde menstruation gain abilities \nfor invasion, implantation, and growth [19]. It should be \nreminded that endometriosis might also stem from the stromal \ncells of the endometrium itself, although their capacity for \nproliferation, invasion, and endometriotic lesion growth are still \nnot known. There are some data showing that SCs derived from \nthe menstrual blood debris in an endometriosis patient also \nshowed altered SC functions, which favor the establishment of \nendometriotic implants [16]. \n \nInvasion of Endometriotic SC  \n \nThe basic signs of endometriosis development include \nendometriotic SC invasion in the peritoneum, and their \nproliferation and differentiation into endometriotic lesions. \nWomen with endometriosis are known to have increased \nmacrophage activity, decreased cellular immunity, and reduced \nnatural killer cell counts [8]. Thus, following retrograde \nmenstruation, the immunodeficient condition prevents the \nclearance of the menstrual debris from the peritoneum, making \nthe ectopic endometrial cells persist [32]. The latter induce \ninflammation, recruit macrophages and leukocytes, and, thereby, \npromote the development of endometriosis [33]. \n \nThe molecular profiling of the eutopic endometrium from \nendometriosis patients suggests functional alterations in the \ngenes that facilitate proliferation, implantation, and survival of \nthe endometrial tissue in the peritoneal cavity, thus supporting \n\nPrime Archives in Molecular Biology \n9                                                                                www.videleaf.com \nendometriosis pathogenesis from the altered eutopic \nendometrium. Inflammatory, immune, and angiogenic responses \nas well as apoptosis reactions are altered in the eutopic \nendometrium of affected women, thus favoring the survival and \nthe maintenance of ethe ndometriotic tissue [34]. \n \nThe relocation of SCs from the eutopic endometrium to ectopic \nsites in the pelvic cavity potentiates the release of several \nchemokines and cytokines which favor revascularization and \nthus allow the development of endometriotic lesions [17]. \nComparisons between SCs in the eutopic endometrium and \nectopic SCs in the peritoneal cavity by analyzing their \nphenotypes and gene expression of pro-inflammatory cytokines, \nmigration markers, and angiogenic factors proved the increased \nlevels of these molecules, accompanied by the reduced levels of \nanti-inflammatory cytokines such as TGFβ. The increased levels \nof pro-inflammatory cytokines such as interleukin-6 (IL-6) and \ninterferon-γ (IFNγ) and the presence of the migration markers \nmatrix metalloproteases (MMP)-2, -3, -9 and of the \nproangiogenic vascular endothelial growth factor (VEGFA) in \nectopic tissue indicate that the abnormal behavior of ectopic \nmesenchymal SCs may suppress the immune system and \nenhance angiogenesis [35]. The increased expression of MMPs \nwould also be useful for the ectopic endometrial tissue to \nactivate invasion. \n \nThe processes of implantation of endometriotic SC onto the \nperitoneum and endometriotic lesion growth obviously require \nangiogenesis. Several studies have reported an increase in \nVEGFA level in the serum and peritoneal fluid of endometriosis \npatients in comparison with women without the disease [36]. \nEndometrial expression of interleukin-8 (IL-8) is responsible for \nthe chemotaxis of neutrophils and partly for angiogenesis. The \ndensity of IL-8 receptors is significantly higher in women with \nendometriosis, as this molecule is involved in endometrial cell \nproliferation and attachment [17,23]. In a systematic review of \ndifferent chemokines as markers of endometriosis, IL-8 appeared \nto be the most significant [9]. \n \n\nPrime Archives in Molecular Biology \n10                                                                                www.videleaf.com \nThe anti-apoptotic BCL-2 gene, upregulated in the eutopic \nendometrium of women with endometriosis, enhances cell \nsurvival and thus plays a major role in the pathogenesis of \nendometriosis. Increased proliferation and decreased apoptosis \nrates in the eutopic endometrium correlate with the expression \nprofile of the BCL-2 gene in endometriosis patients [37]. \n \nThe endometriotic lesion cells express high levels of P450 \naromatase–a protein which allows estrogen overproduction and \ndecreases the expression of 17β-HSD2 (17β-Hydroxysteroid \ndehydrogenase), thus inhibiting the response to progesterone \n(―progesterone resistance‖) [16]. This is considered a key \nprocess through which the maintenance and growth of \nendometriotic lesions are promoted. It is not known, however, \nwhether these processes are a necessary cause of endometriosis \nor rather its consequence [32]. These results support the notion \nthat intrinsic abnormalities in the eutopic endometrium cells in \nwomen with endometriosis predispose the endometriotic SCs \ncells to survive in the pelvic cavity, attach, invade, and establish \na blood supply in the peritoneum or other areas. \n \nEndometriotic lesions provoke local inflammation of the \nperitoneum, which attracts bmSCs through the expression of the \nC-X-C chemokine receptor type 4 (CXCR4) and of the \nchemokine ligand 12 (CXCL12) which plays a role of \nchemoattractant in the migration of bmSC towards the \nendometrial stromal cells. Thus, the deregulation of estrogen \ncombined with local peritoneal injuries may be important in the \npathogenesis of endometriosis [23]. Also, bmSCs may migrate \nfrom the peripheral circulation and provoke the formation of \nendometriosis foci in remote sites as well as infiltrate the \nendometrium of endometriotic lesions [19]. \n \nThe endometriosis implant can also result from the outgrowths \nof the dormant SCs disseminated in the pelvic lining during \nembryogenesis of the female reproductive system [19] (see also \nSection 1). \n \nThus, pelvic and extrapelvic endometriosis implants are \nhypothesized, each with a distinctive epigenetic expression \n\nPrime Archives in Molecular Biology \n11                                                                                www.videleaf.com \nprofile. Epigenetics plays a major role in modulating steroid \naction, and the inflammatory reaction is a key factor for the \nrecruitment of bmSCs [5,38-40]. Whether gene expression \nprofiles in endometriosis cells of the endometrium or bone \nmarrow are similar or different remains unknown. Clarifying this \npuzzle is important to understand the pathogenetics of \nendometriosis. \n \nDiscussion  \n \nGenetic and epigenetic data analysis revealed significant \ndifferences in various tissues and cell types undergoing the \nEMDP compared to the normal ones. Complex molecular \ngenetic and epigenetic features constitute the pathogenomic \narchitecture of endometriosis and include gene polymorphisms, \npeculiarities of their expression, numerous interactions of gene \nnets, complex combinations of functional protein modules, as \nwell as different metabolic pathways which are altered by sever \nimbalances in the hormonal and immunologic systems [3,5,32]. \nEach of these factors is affected at different levels during \nendometriosis depending on the specific EMDP. On the other \nhand, common clinical manifestations indicate the existence of \nsome crucial molecular pathways common to all clinical types of \nendometriosis. Irrespective of the obvious differences in the \nintermediate events, the EMDP ultimately ends in the typical \nendometriotic lesions. Thus, the EMDP should be roughly \nsubdivided into three parts: transition of mesodermal embryonic \ncells into cells of the endometrium within Muller ducts \nrudiments (1), acquisition of endometrial cells abnormalities and \ncell transition into endometriotic SCs (2), invasion of the SCs \ninto the peritoneum lining and their differentiation into \nendometriotic lesions (3). \n \nAs it was indicated (see 1), any developmental event should be \nattributed to a massive genome reprogramming which follows \nthe short critical phases (the epigenetic crises after Waddington) \nof higher sensitivity to any inducers or noxious triggers [14,41]. \nThus, at least three critical phases, corresponding to each of the \nmorphogenetic events described above, should be recognized in \nthe EMDP. The first one corresponds to the initial stages of the \n\nPrime Archives in Molecular Biology \n12                                                                                www.videleaf.com \ndevelopment of the reproductive tract in female embryos, while \nthe second and third stages take place in postnatal life (Figure 1). \n \n \n \nFigure 1: Sensitive periods in the Endometriosis Development Program. SC, \nstem cells, MD, Mullerian ducts, EE, eutopic endometrium, EMT, epithelial–\nmesenchymal transition, EML, endometriotic lesions, w.g., weeks of gestation.  \n \nThe dislocation of the primitive endometrial tissue in female \nfetuses coincides with human embryonic developmental stages \nXVII–XX (5–8 weeks of gestation) and lasts into the early \npostnatal period [42]. Both the coelomic epithelium of the \nperitoneum and the Mullerian ducts giving rise to all parts of the \nfemale reproductive tract generate from the mesoderm layer in \nthe early human embryo. The development of the female \nurogenital tract is completed only at birth. The genes responsible \nfor female reproductive tract development are well known, and \nmany of them have already been identified [24]. The \ntranscription factors of the HOX family, in particular HOXA10, \nare the principal coordinators and regulators of the expression of \nthese genes [3], being responsible for mesoderm segmentation \nand its axial extension. The next important contributor to the \nformation of the Mullerian ducts is the WNT gene family, with \nWNT4 as a key regulator of female sex development. It is \nlocated at the 1p36 chromosomal region, wich variants may \ncontribute to endometriosis susceptibility through abnormal \ndifferentiation of the female reproductive tract [24]. WNT4 was \nshown to be expressed in the normal peritoneum, suggesting that \nendometriosis can arise through a reversible transformation of \n\n\nPrime Archives in Molecular Biology \n13                                                                                www.videleaf.com \nthe epithelium cells to endometriotic cells (metaplasia) through \nthe developmental pathways associated with the HOXA9 and \nCDKN1A genes [43]. These data are in line with a recently \nsuggested ―unifying hypothesis‖ of endometriosis [24]. \nAccording to this, Müllerian remnants of the endometrium may \nleak into the peritoneal cavity during embryogenesis of the \nurogenital system as a result of the deregulation of WNT genes \nand of the Wnt–β-catenin signaling pathway. The latter can lead \nto aberrations and deregulation within the mesoderm, thus \ncausing the aberrant placement of SCs. Deregulation in the \nhormonal and immune systems, abnormalities of adhesion, \nextracellular matrix metalloproteinases, and pro-inflammatory \ncytokines activate or alter the peritoneal microenvironment, \ncreating the conditions for the differentiation, adhesion, \nproliferation, and survival of ectopic endometrial cells, thus \ngiving rise to endometriosis in adults. The growth of \nendometriotic lesions may occur by inclusion and transformation \nof the mesothelium cells of the peritoneal lining. \n \nStructural variations (polymorphisms) or functional insufficiency \nof the HOXA10 and WNT4 genes and of the genes of their \ngenetic cascade (MIF, VEGFA, MMPs, VCAM, BMP, etc.) may \nderegulate highly balanced genetic and epigenetic mechanisms \nof female reproductive tract embryogenesis, causing \ndisorganization of the endometrium as well as dissemination of \nmesoderm cells, including SCs, outside the uterine cavity; this \ninitiates an inborn predisposition to endometriosis in postnatal \nlife. Mullerian embryogenesis-related genes in the uterine \nendometrium in early life might be associated with \nendometriosis in the adults. \n \nDirect association of the HOX and WNT families as well as of \n10 other genes with endometriosis was repeatedly confirmed \n[3,32]. By means of genome-wide association studies (GWAS), \n12 single nucleotide polymorphisms at 10 independent genetic \nloci associated with endometriosis have also been identified [4]. \nObviously, mesoderm cells with epigenetic or inborn defects \nincorporated both in the peritoneal lining and the uterine \nrudiments are suspected to be associated with the risk of \ndeveloping endometriosis in adulthood [32]. \n\nPrime Archives in Molecular Biology \n14                                                                                www.videleaf.com \n \nThus, endometriosis might be provoked by the failure of the \nexpression of HOXA10 or WNTs genes regulating the initial \nstages of reproductive tract development in female embryos or \nalso induced by the direct harmful effects of some toxins during \nembryonic development, which result in the dislocation of the \nprimitive endometrial tissue outside the uterine cavity during \nearly organogenesis [44]. \n \nIt also might be suspected that endometriotic SCs with inherited \ndisorders of WNT4 or HOXA10 genes give rise to clinically \nforms of endometriosis more severe than those of mostly \nepigenetic origin [2]. \n \nThus, the first sensitive period (SP) of the EMDP most probably \ncorresponds to the embryonic stages of the female reproductive \ntract development. An unfavorable combination of endometriosis \npredisposition genes (predominantly of WNT and HOX families) \nand noxious agents (oxidative stress, pesticides, endocrine \ndisruptors) might create conditions for the differentiation, \nadhesion, proliferation, and survival of eutopic and ectopic \nendometrial SCs. The direct association of the unfavorable \nWNT4 allele with endometriosis has been recently demonstrated \n[45]. This finding deserves further studies to establish if this \nallele can be a predictive biomarker of endometriosis. \n \nThe second SP of the EMDP concerns the presence of dormant \nendometriotic cells in the endometrium. The duration of this \nperiod is unknown, as progenitors of endometriotic cells may \nstay dormant for many years until some provocative stimuli \ntrigger their metaplasia into endometriotic SCs. Numerous \ngenetic and epigenetic factors are involved. It was suspected and \nrecently shown that eutopic endometrium cells in endometriosis \npatients contain aberrantly expressed genes and exhibit \nderegulated pathways that predispose them to implantation, \ninvasion, and migration outside the uterus [34]. Dysfunctional \nexpression of the genes related to the Mullerian embryogenesis \n(see SP1) as well as epigenetic immuno-endocrine deregulation \nof genes in endometrium (IL11, LIF, TGF-β, FKBP4, COX2, \n\nPrime Archives in Molecular Biology \n15                                                                                www.videleaf.com \nPGs, FOXO1, and C/EBPβ) might appear critical to the \ndevelopment of endometriotic lesions [3,32]. \n \nThe involvement of external triggers, such as transient hypoxia, \nchronic inflammation, and mechanic transduction, is also \nsuspected. Transient hypoxia and inflammation induce the HIF-\n1A gene and mechanic transduction upregulate the expression of \nthe TWIST1 gene. Thus, any measures reducing hypoxia and \nmechanical stretch of the uterus might be useful in endometriosis \nprevention. The search for other genes and epigenetic factors in \neutopic endometrium cells predisposing to endometriosis should \nbe encouraged. \nThe third SP of the EMDP includes adhesion, proliferation, \ninvasion, angiogenesis, and growth of endometriotic stem cells \ninto endometriotic lesions. The genes highly expressed at this \nstage include cell cycle regulators (cyclins and CDKs), \nangiogenesis factors (VEGFA, ANGPTs, and TIEs), immuno-\ninflammatory factors (COX2), matrix metalloproteinases \n(MMP3, MMP9), and integrins. Their protein products play a \ncritical role in the establishment, maintenance, and development \nof the endometriotic lesions. Theoretically, interference with the \nexpression of any of this gene might be sufficient for the active \nprevention and treatment of endometriosis. Clinical practice, \nhowever, contradicts these assumptions and favors the view that \nthe EMDP is a well-canalized process, buffered against curative \nintrusions. At a definite stage of progression, the EMDP \nbecomes irreversible and proceeds to its final stage producing \nthe endometriotic lesions. It should be mentioned that in women \nreceiving a hormonal contraceptive treatment that prevents the \nimplantation, the frequency of endometriotic lesions on the \nperitoneum is comparable with that of the controls [46]. In \nagreement with this, hormonal treatment did not prevent the \ninvasion and implantation of endometriotic SCs. On the other \nhand, to the best of our knowledge, the implantation of \nendometriotic SCs per se as well as their invasion into the pelvic \nlining was never registered, thus giving some credit to the extra \nuterine origin of endometriosis from the mesenchymal stem cells \n(meSC) disseminated during embryogenesis of the female \nreproductive tract (See part 1). \n\nPrime Archives in Molecular Biology \n16                                                                                www.videleaf.com \nConclusions  \n \nAs might be inferred from the reviewed studies and suggested \nhypothesis, each of the three sensitive stages in the EMDP \ndeserves special attention. Intrinsic and external factors \ninterfering with the embryogenesis of the female reproductive \ntract should be subjected to thorough studies. Of special interest \nare the inherited forms of endometriosis and their correlation \nwith relevant mutations or polymorphisms of the genes involved \nin the differentiation of the Mullerian duct and in the \ndevelopment of the urogenital tract, such as WNT, HOXA10, \nHOXA11, and their signaling pathways, as well as other genes \nregulating mesoderm differentiation and SC trafficking. The \nsearch for teratogenic agents affecting the development of the \nfemale reproductive tract should also be encouraged. \nMore knowledge of SP2 should be drawn from the data on the \nheterogeneity of eutopic endometrium cells, with special \nemphasis on the cells prone to induce endometriotic lesions \ngrowth. The significance of EMT as a trigger of epigenetic \nchanges amenable to launch the EMDP should be also \nconsidered. Both SP1 and SP3 need further global molecular \nstudies of gene expression and its regulation by methylation and \nmicroRNA analysis. There are still few reports on these topics, \nwith rather contradictory results for both endometrial \ntranscriptome [9,47] and microRNAs [10]. Large differences \nbetween studies can be explained by differences in the study \ndesign, subject characteristics, procedures for tissue collection, \nstorage, and processing, assay platforms and data analysis \nmethods. The necessity for the unification of these variables was \nrecently supported by the World Endometriosis Research \nFoundation initiative that issued the Endometriosis Phenome and \nBiobanking Harmonization Project, which developed standards \nfor tissue collection, processing, and storage in endometriosis \nresearch [48]. It looks very awarding that only –omics analysis \nof massive endometriosis data stratified according system \ngenetics architecture and collected according to International \nConference on Bioinformatics and Biomedicine regulations [7] \nmay pave a reliable way to ultimate solution of endometriosis \nmystery and maybe give more credit to existence of special \ndevelopmental program in pathogenomics of endometriosis. \n\nPrime Archives in Molecular Biology \n17                                                                                www.videleaf.com \nReferences  \n \n1. Batt RE. A History of Endometriosis, 1st edn. London: \nSpringer. 2011; 202. \n2. Shubina AN, Egorova AA, Baranov VS, Kiselev AV. Recent \nadvances in gene therapy of endometriosis. Recent Pat. DNA \nGene Seq. 2013; 7: 169–178.    \n3. Borghese B, Zondervan KT, Abrao MS, Chapron C, Vaiman \nD. Recent insights on the genetics and epigenetics of \nendometriosis. Clin. Genet. 2017; 91: 254–264.    \n4. 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