Physiomimetic Models of Adenomyosis

► adenomyosis ► organoids ► models of adenomyosis ► tissue engineering ► microfluidic device

Adenomyosis remains an enigmatic disease in the clinical and research communities. The high prevalence, diversity of morphological and symptomatic presentations, array of potential etiological explanations, and variable response to existing interventions suggest that different subgroups of patients with distinguishable mechanistic drivers of disease may exist. These factors, combined with the weak links to genetic predisposition, make the entire spectrum of the human condition challenging to model in animals. Here, after an overview o f current approaches, a vision for applying physiomimetic modeling to adenomyosis is presented. Physiomimetics combines a system’s biology analysis of patient populations to generate hypotheses about mechanistic bases for strati fication with in vitro patient avatars to test these hypothe- ses. A substantial foundation for three-dimensional (3D) tissue engineering of adeno- myosis lesions exists in several disparate areas: epithelial organoid technology; synthetic biomaterials matrices for epithelial –stromal coculture; smooth muscle 3D tissue engineering; and microvascular tissue engineering. These approaches can potentially be combined with micro fluidic platform technologies to model the lesion microenvironment and can potentially be coupled to other microorgan systems to examine systemic effects. In vitro patient-derived models are constructed to answer specific questions leading to target identi fication and validation in a manner that informs preclinical research and u ltimately clinical trial design. Issue Theme Adenomyosis and Endometriosis; Guest Editors, Linda C. Giudice, MD, PhD, MSc, Lisa M. Halvorson, MD, Elizabeth A. Stewart, MD, and Luk R o m b a u t s ,P h D ,F R A N Z C O G ,M D DOI https://doi.org/ 10.1055/s-0040-1719084. ISSN 1526-8004. Copyright © 2020 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 760-0888. THIEME 179 Article published online: 2020-11-09 diagnose it and assess response to therapy; and diagnostic bias toward older multiparous patients clouds true incidence. 2–5 Patients with pain, heavy menstrual bleeding, and/or infertili- ty may have adenomyosis—or something else entirely—as an underlying cause.6 About 40% of patients with symptoms, but no indication of adenomyosis on magnetic resonance imaging (MRI) or ultrasound, are found to have adenomyosis lesions upon histopathological assessment.7 Symptom-targeting ther- apies for adenomyosis, like those for endometriosis, 8 work remarkably well in some patients, but are not at all effective or tolerated in others. Moreover, there is neither reliable manner to predict who will respond nor alternative therapies (other than developing coping mechanisms around eternal suffering) for those who do not. 9,10 As such, adenomyosis is not one disease, and it is unlikely there is one model that captures all salient features for all patients. Second, the etiologies —given the prevalence and vast diversity in patient presentations, there is likely more than one—are obscure: adenomyosis appears to arise from a complex web of gene –environment interactions that may start in utero; it might be triggered perinatally via seeding of the myometrium by cells that escape the endometrium; it might arise later in life due to uterine mechanical injury; or it might arise due to invasion of the myometrium by endome- trium injured by an in flammatory insult. 2,4,11–13 This diver- sity of possible etiologies, along with the diversity of clinical presentations, underscores a shortcoming in how the disease research has traditionally been approached: adenomyosis is “a” disease. Yet, every patient is different. While there are not likely millions of individual diseases, there are likely multiple different constellations of molecular networks —immune networks, invasion networks, and metabolic networks — that go awry to instigate symptomatic and phenotypic appearance of ectopic endometrium in the myometrium, giving rise to groups of patients who could potentially be stratified molecularly as has been proposed for endometri- osis. 8,9,14–17 Animal models—where strain variation, genetic perturbations, and targeted interventions to modulate spe- cific pathways can be deployed to probe potential contribu- tions to the adenomyotic etiology —offer insights into where to look for such mechanistic strati fication in humans. This last point brings us to the third big challenge with modeling adenomyosis—the relative lack of deep clinical and pathological phenotyping to guide patient strati fication into tractable and mechanistically targetable subgroups. 5 Such clinical phenotyping and disease staging/classi fication is intertwined with therapeutic development through impacts on reimbursements for treatment 18 and evaluation of effica- cy of various forms of treatment for clusters of patients with well-defined characteristics, as is done most thoroughly for cancer. 19,20 In this way, adenomyosis is the sister, though not precisely the twin, of endometriosis, which is inadequately staged based on lesion number, size, and location. 8 Like endometriosis, adenomyosis is currently only de finitively diagnosed via surgical intervention (usually, a hysterectomy) and histopathological characterization. However, adeno- myosis, as one of several pathologies that contribute to abnormal uterine bleeding (AUB), is included in the PALM- COEIN system for classifying such disorders, as first de- scribed in 2011 6 and updated in 2018. 21 PALM-COEIN serves as a crucial tool for patient –clinician–researcher communication; hence, it is a step toward personalized medicine for the patients suffering from AUB as a symptom of adenomyosis. The state of personalized medicine for adenomyosis is still distant from as compared with cancer and other diseases. In this era of molecular strati fication, oncologists routinely tailor treatments to a combination of physical features in addition to molecular biosignatures linked to disease mech- anism and prognosis, sometimes using patient-derived orga- noids or tissue models to test drug sensitivity. 22 Mechanistic markers also inform the development of preclinical in vitro models, providing scientists an essential connection to the translational medicine. Such approaches are nascent only for endometriosis and even more primitive for adenomyosis, in part because somatic mutations, which are highly informa- tive in cancer, are at best weakly associated with ectopic endometrial diseases. Hence, classifications based on protein or metabolic network states (or possibly epigenetics), com- plemented with clinical phenotypic data incorporating pa- tient symptoms and life-long prognosis, 17,23,24 are therefore an appealing route to classi fication; such approaches will likely yield constellations of “molecular signatures ” of dis- ease state, rather than one single indicator of “diseased, or not.”14 An exemplary model for how other noncancer im- mune-mediated disease communities have spurred such phenotyping is the Juvenile Diabetes Research Foundation- sponsored Network for Pancreatic Organ Donors with Dia- betes (nPOD) program. 25 Application of programs like these would significantly improve our understanding of adenomy- otic disease. Finally, a fourth challenge is which stage(s) of disease to model. Modeling the features of disease as they present in diagnosed patients, who often have advanced varieties of disease progression, is arguably essential for identifying molecular targets and developing new therapies to treat these patients, and even more so for judicious prescription of existing therapies. Modeling proposed etiologies may yield preventive measures, or early treatment options, pre- suming a diagnostic for nascent stage disease, can be defined. The features of such models —and the throughputs required for getting useful information out of them —are somewhat but not entirely overlapping. Patients with advanced disease may also have nascent progressing disease, for example. Precise de finition of the clinical phenotype, along with mechanistic hypotheses about the features of disease being studied, as they relate to the clinic , is an essential first step in designing, and then in implementing, in vitro models of adenomyosis. A Roadmap of Phenomena to Model How do we de fine what to model? Adenomyosis studies generally lack the kind of deep clinical phenotyping that links molecular and cellular pathological findings to patient symp- toms, comorbidities, and treatment responses. However, several Seminars in Reproductive Medicine Vol. 38 No. 2-3/2020 Physiomimetic Models of Adenomyosis Gnecco et al.180 commonly observed pathological phenotypes can be modeled now using in vitro models, with a longer-term goal of more specialized patient avatars focusing on specifics u b g r o u p s .T h e overall vision for the role of in vitro models is illustrated in ►Fig. 1 .B r i efly, we envision that there are a relatively small number of subsets of patients who are phenomenologically similar, but distinct enough from each other that different therapeutic approaches are needed. Tissues and body fluids harvested from patients are used on the one hand for “omics” analysis—which can include genomics, proteomics, metabolo- mics—of molecular/cellular networks that enable stratification of patients into groups with data-informed machine learning methods, 14 based at least in part on mechanistic hypotheses that may distinguish the groups. Mechanistic strati fication offers a path to target identification and experimental hypothe- sis testing. On the other hand, tissue banks created from the patients are used to create patient avatars that can be used to test hypotheses about mechanistic strati fication and targets, with the goal of informing translational research and employing personalized therapies ( ►Fig. 1 ). In vitro models can thus potentially inform directions in clinical phenotyping, in an iterative fashion. Conceptualizing Models of Adenomyosis “Physiomimetics” is the process of conceptualizing a complex physiological process to define essential features, then building in vitro models that capture the most relevant aspects of physiology in ways that can ultimately yield clinically action- able outcomes. Physiomimetic models encompass a range of experimental complexities, from relatively standard culture of individual cell types up through complex three-dimensional (3D) microfluidic models, driven by biological questions. We start here by conceptualizing established adenomyosis lesion phenotypes and the dynamic behaviors implicated in their pathophysiology, as the etiology of nascent adenomyotic lesions is still obscure, and there is great need to treat Fig. 1 Physiomimetic approach for developing targeted therapies for adenomyosis . Hypotheses regarding different mechanisms of disease that may be operative in patient sub groups are tested with tissue and fluid samples from a large patient population containing the subgroups (1); analysis of tissue and fluid samples to re fine hypotheses about mechanism (2a and 3) are performed in tandem with development of cell banks and construction of patient avatars (2b). Mechanistic hypothesis about patient subgroups and interventions can then be tested in patient avatars representing the subgroups to de fine strati fied clinical trials, or inform more jud icious use of existing therapies. Seminars in Reproductive Medicine Vol. 38 No. 2-3/2020 Physiomimetic Models of Adenomyosis Gnecco et al. 181 established adenomyotic lesions. Furthermore, the mecha- nisms contributing to disease progression may be operative to some extent in disease onset. We focus on diffuse adeno- myosis lesions, as cystic adenomyosis lesions can in some instances be treated surgically. 26 Conceptually, here we define adenomyoticlesion as the ectopic presence of lesion-initiating cells—endometrial epithelial glands and stroma —surrounded by thelesion microenvironmentcomprising myometrial muscle cells, nerves, vasculature, and immune cells with associated extracellular matrix (ECM; ►Fig. 2 ). Dynamic behaviors of lesion-initiating cells that are im- plicated in pathologies include (1) invasion into surrounding microenvironment, potentially seeding new, distant lesions; (2) proliferation, which may drive lesion growth, invasion, or feed into a hormone-driven cycle of cell death and prolifera- tion; and (3) production of in flammatory cytokines, chemo- kines, proteases, and other modulators of signaling and remodeling of the local microenvironment 27,28 (►Fig. 2 ). The dynamic responses of the microenvironment associated with pathologies include (1) smooth muscle hyperplasia of local myometrial cells (or recruitment of fibroblasts or mesenchymal stem cells that acquire that phenotype 29); (2) in flux and activation of immune cells; (3) recruitment of vasculature and modulation of vascular permeability, possibly with local bleeding and clotting; (4) enhancement of sensory innervation and regression of sympathetic inner- vation; and (5) stiffening of the local microenvironment, via matrix deposition and cellular changes. Cells within lesions respond to local cues, including mechanical stimulation from myometrial contractions, and to systemic cues, notably sex steroids, but are also modulated by nutrition, systemic inflammation, stress hormones, and other factors. These responses play out in an orchestrated fashion, as each cell type expresses a unique repertoire of receptors for the cues, including estrogen and progesterone receptors. 30–32 Many of the same phenomena are the subject of substantial investi- gation in carcinomas, but the spotlight on dynamic changes spurred by cyclic variation in sex steroids is omnipresent in Fig. 2 Conceptualization of an adenomyosis lesion, showing the biological components and pathological processes to consider in building an in vitro model. Seminars in Reproductive Medicine Vol. 38 No. 2-3/2020 Physiomimetic Models of Adenomyosis Gnecco et al.182 adenomyosis. After a review of the known features of the myometrium and its changes in adenomyosis, we then consider how each of these dynamic phenomena can be modeled in vitro. The Uterus in Adenomyosis The presence of adenomyosis in the myometrium induces changes throughout the uterus in ways that suggest interest- ing hypotheses that can be tested using well-designed in vitro models. The myometrium is composed of the inner myometrium (or junctional zone, JZ), characterized as having a relatively greater cell density with relatively less ECM, and the outer myometrium, which is more ECM-rich and has a lower cell density. 33 On a cellular level, the myometrium comprises layers of smooth muscle cells (SMCs) arranged into fibular bundles approximately 0.3 mm in diameter, interwoven with blood vessels and connective, lymphatic, and nerve tissue. 33,34 These layers of SMC bundles are oriented in different directions around the uterus to enable complex contractile patterns that drive fluid flow in the nonpregnant uterus. 33 In adenomyosis, these contractile functions can become dysregulated. 35,36 Like the endome- trium, the inner myometrium/JZ exhibits molecular and cellular changes in response to cyclic hormones through the menstrual cycle —such as tissue thickening and modula- tion of estrogen and progesterone receptor expression —but the outer myometrium appears less sensitive to menstrual cycle hormonal changes at both the gross anatomical level and the molecular level. 37–39 How these differences translate into the propensity for adenomyosis lesions to drive symp- toms is unknown, but it is a potential facet that could be modeled in vitro. The healthy human myometrium is typi- cally well delineated from the endometrium, but this clear delineation is blurred in adenomyosis, suggesting invasion of cells from the endometrium into the myometrium, attribut- ed by one theory to microtraumas at the endometrial – myometrial interface during uterine peristalsis. 11,38,40–43 Changes in ECM architecture and composition, as well as smooth muscle hyperplasia of the region immediately surrounding lesions, characteristically affect multiple cell types and may involve chemical and mechanical cues. Patients with adenomyosis have been shown to have in- creased levels of matrix metalloproteinases (MMP)-2 and -9, suggesting abnormal matrix remodeling may be present in these patients. 44 Inflammation may be driven by repeat- ed local bleeding in the lesion, 45 which may further en- hance contractile phenotypes that in turn drive fibrogenic processes.46 Endometrial cells in adenomyotic tissue exhibit many aber- rant behaviors, including apparent resistance to progesterone, epithelial–mesenchymal transitions, decreased apoptosis re- gardless of menstrual phase, and other processes.11 Although adenomyosis-targeting therapeutic approaches are intended to suppress aberrant proliferation of endometrial-derived tis- sues by targeting proestrogenic pathways or inducing proges- terone action, 11 these approaches fail in some patients, a finding consistent with dysregulation of speci fich o r m o n e receptors ESR1, ESR2, and PGR isoforms A and B in adenomyo- sis.47,48 The downstream consequences of estrogenic signaling and acquired progesterone resistance that may contribute to the etiology of disease are still not well understood, and in vitro models offer an opportunity to parse the individual mechanisms. Adenomyosis is associated with both pain and heavy men- strual bleeding. Several studies have reported an increased sensory nerve fiber density in both ectopic lesions and eutopic endometrium in women withendometriosis, along with reduc - tion in sympathetic nerves, suggesting a possible diagnostic criterion. 49–52 Nerve fibers are increased in the fibrotic endo- metriosis nodules in the rectovaginal space and greater nerve fiber in filtration of lesions is found in patients with greater pain.53–55 Increased nervefiber density is also associated with an increase in macrophages,56 with some evidence of estrogen modulation of the crosstalk. 57 Similar analysis of nerve fiber and macrophage density inadenomyosisis relatively scarce, but one study examined nerve fiber density in the endometrium and myometrium of women with adenomyosis and fibroids, and found no difference.58 The immune environment in the uterus is dynamic, with dramatic increases in the number of macrophages, uterine natural killer (NK) cells, and other immune components during the secretory phase. The extent to which the increased number of immune cells arises from in situ prolif- eration of tissue-resident cells or from recruitment of circu- lating monocytes is still unclear. 59–61 Furthermore, whether CD45þ intraepithelial lymphocytes (IELs) that are character- istically observed in the epithelial layer in adenomyosis lesions 62 derive from locally recruited cells (and are there- fore part of the microenvironment) or derive from IELs present in the epithelial layer of the eutopic endometrium (and are therefore lesion-initiating cells) is unknown. The crosstalk between nerves and macrophages or other immune cells may contribute to pain and/or bleeding. An intriguing clinical finding, albeit from a small study, examined macro- phage density in the uteri of 54 patients undergoing hyster- ectomy for adenomyosis due to pain as the primary symptom, with a comparison group of 20 women undergo- ing hysterectomy for adenomyosis with heavy menstrual bleeding as the primary symptom, 63 where adenomyosis was tentatively diagnosed by both transvaginal ultrasound and MRI prior to surgery. Greater leukocyte in filtration was seen in both the myometrium and endometrium of patients with pain, and groups of macrophages were seen near lesions and interspersed in the muscle in the patients with pain; in both groups, there were macrophages around blood vessels, as macrophages are known to regulate vessel permeability. 64 Although the detailed interactions between the vasculature and adenomyotic lesions are not fully appreciated in the establishment and progression of adenomyosis, increased microvessel density has been observed in adenomyosis, 65,66 and a recent meta-analysis of studies examining vascular morphology and marker expression in the myometrium and endometrium suggests increased angiogenesis in the endo- metrium of women with adenomyosis. 67 Early studies that explored immune responses in adenomyosis showed that the disruption of the JZ at the endometrial –myometrial Seminars in Reproductive Medicine Vol. 38 No. 2-3/2020 Physiomimetic Models of Adenomyosis Gnecco et al. 183 interface may result from the abnormal aggregation of macrophages in the myometrium, similar to the increased number of macrophages previously observed in endometri- osis patients. 68 Finally, intriguing data on sex hormone responsiveness of mast cells, which are among the most common immune cell types in the myometrium and impli- cated in the pain associated with interstitial cystitis, may contribute to adenomyosis symptoms. 69 These observations suggest that recapitulating immune –epithelial–neuronal interactions in vitro using patient-speci fic cells may yield insights between symptomology and molecular and cellular behaviors characteristic of distinct patient populations. Interestingly, the eutopic endometrium of patients with adenomyosis confer dysregul ated angiogenesis, innerva- tion, immune cell presence, and gene and protein expres- sion that suggest greater invasive and survival potential at ectopic sites and contribution to symptoms. 70–74 Global changes in the myometrium outside the peri-lesion envi- ronment, including smooth muscle hypertrophy, increased stiffness, and immune cell in filtration,38,75 raise questions about whether these global changes precede, or are the

of, lesion establishment —a hypothesis that could potentially be tested in vitro. Designing, Building, and Interpreting In Vitro Models The growing evidence that animal models do not capture essential features of human diseases involving chronic inflam- mation, sex steroid signaling, or other complex, molecular phenomena is driving development of sophisticated in vitro models that capture complex tissue architecture, and even dynamic perfusion of microvascular networks, using human patient-derived cells. While these approaches are just now emerging, especially driven by applications in cancer, cardio- vascular, and liver diseases, they offer promise for modeling adenomyosis. In this section, we review basic approaches to tissue engineering of the uterine environment, including

of cell sourcing and 3D model design. Then, in the next section, we turn to modeling specific phenotypic facets of adenomyosis including invasion, vascular and systemic inter- actions, mechanics, and innervation, drawing from impactful work in disparate tissue systems. Basic Approaches in Tissue Engineering of the Endometrium and Myometrium The functions of the uterus arise from the integrated actions of epithelia, stromal fibroblasts, myometrial smooth muscle, immune cells, and endothelial cells, which are each directly and indirectly responsive to variations in sex hormones. Thus, many responses of various cell types to hormone changes are indirect and governed by dynamic communica- tions with other cells. The endometrial epithelial response to progesterone, for example, is governed by signaling proteins produced by the progesterone-responsive stromal fibro- blasts, which in turn respond to endothelial-derived signals to enhance the decidualization process. 31,76 While parsing responses of individual cell types to hormonal and other perturbations in vitro is an essential first step to understand- ing the integrated responses, building models that capture the dynamic interlinked hormone responsiveness in vitro is ultimately essential for illuminating complex phenomena like adenomyosis. Cell Sources: Explants, Cell Lines, Primary Cells For decades, explant cultures of the endometrium and myome- trium were the only external window into the functions of the human uterus in vitro 36,77–81 and, until recent advances in primary cell culture, they remained preferred models for anal- ysis of complex primary tissue functions. 31,81,82 Endometrial explants in the absence of ECM quickly deteriorate,83 but can undergo outgrowth and remodeling when embedded in afibrin matrix.84 For the endometrium —the presumed instigator of adenomyosis lesions—definition of protocols for isolating and culturing epithelial cells and stromalfibroblasts from endome- trial biopsies85 enabled mechanistic studies on patient-derived samples, allowing diverse healthy and diseased donors to be combined in different ways to parse contributions of each cell type to health and disease. Primary cells remain the gold standard for mechanistic disease studies, but permanent epi- thelial 86,87and stromal88–90 cell lines that exhibit many (but not all) tissue-speci fic hormone-responsive features, and some disease states, have been routinely used for protocol develop- ment, pilot studies, and investigations of general features of endometrial cell behavior. The carcinoma-derived Ishikawa cell line is most commonly used for “normal” epithelia, 91–93 the h-TERT-immortalized endometriosis-derived“12Z” line is used for invasive endometrial epithelia,15,87,94 and the h-TERT- immortalized stromal cell line “tHESC”89,91 is used for endo- metrial stromal cells. (Caution: cell line contamination was reported for the endometrial epithelial cell line“HES” 86;h e n c e , routine SNP profiling of cell lines is recommended for these, as it i sw i t ha l lc e l ll i n e s ,t ov a l i d a t ep r o v e n a n c e . )W h i l ec e l ll i n e s offer convenience and relatively good reproducibility, including from laboratory to laboratory (subject to the typical cell line drift), they have some shortcomings for reproducing primary cell behaviors, 91 including greatly skewed cytokine and growth factor production profiles.95 Pilot studies with cell lines can, however, offer insights that can be investigated further in more difficult-to-culture primary cells.15 The majority of studies on primary endometrial cells focus on stromal cells, as they can readily be expanded in standard culture and frozen to create tissue banks, allowing repeated studies from the same donor within limits of passage num- ber. 96,97 For years, however, investigators were limited by the lack of robust protocols for expansion and cryopreservation of endometrial epithelial cells, with few laboratories reporting success. 98 The landscape changed dramatically in 2017 with publication of the first two studies describing robust expan- sion and cryopreservation of human endometrial epithelial cells as organoids ( ►Fig. 3a ), with retention of hormone responsiveness, cellular architecture, polarity, heterogeneity, and other functions, 99,100 using modi fications of protocols first described by Clevers and coworkers for expansion of human intestinal epithelia.101 In the Clevers approach, epithe- lial cells are either dispersed as individual cells or fragments Seminars in Reproductive Medicine Vol. 38 No. 2-3/2020 Physiomimetic Models of Adenomyosis Gnecco et al.184 and cultured in Matrigel, a basement membrane-rich isolate of a tumor cell line propagated in mice. 101 While Matrigel had long been used as an ECM for 3D culture of endometrial glands99,102 and many other types of epithelial cells, enabling remarkable retention of tissue phenotype, Clevers de fined a cocktail of growth factors and other medium supplements that spur dramatic proliferation of the stem cell compartment, allowing the cells in an initial biopsy to expand orders of magnitude in culture and withstand cryopreservation, while preserving their ability to differentiate into proper tissue phenotypes upon a change in medium composition to remove stem cell cues. 101 This organoid approach has also been applied to derive continuously propagated organoid cultures from ectopic endometrium and endometrial tumors. 103 An additional advance essential to building and character- izing physiologically relevant 3D models is the identification of both mesenchymal and epithelial stem and progenitor cell compartments within the human endometrium. 96,104–106 Intriguingly, recent data suggest that stem or at least progeni- tor cells are present in the endometrial functional layer and luminal regions of the endometrium, as evidenced by in situ hybridization for expression of the epithelial stem cell marker LGR5. 107 The tools to identify putative stem and progenitor cells are valuable in assessing the phenotypic states of cells that are used to initiate models, and to assess their phenotypes under long-term culture conditions, especially to test hypoth- eses regarding possible contributions of these stem cells to ectopic adenomyosis lesions. 108 Related to efforts to identify Fig. 3 Development of 3D in vitro models using synthetic hydrog els and endometrial epithelial organoids technologies .( a)E n d o m e t r i a l epithelial organoids (EEOs) promote the culture, expansion, and propagation of epithelial cells using 3D hydrogel systems. EEOs retain epithelial structure, heterogeneity, and function of the native tissue glands. ( b) Bio-labile, synthetic extracellular matrices (ECMs) can be designed to establish EEOs cultures and cocultures th at include additional cell populations, such as endometrial stromal cells (ESCs). ( c) Polyethylene glycol (PEG)-derived hydrogels are fully de fined, modular, and can be tuned by modifying their molecular and biophysical properties to mimic key features of the adenomyotic and endometriotic phenotype. Seminars in Reproductive Medicine Vol. 38 No. 2-3/2020 Physiomimetic Models of Adenomyosis Gnecco et al. 185 and characterize functions of stem and progenitor cells in postnatal endometrium are efforts to create uterine tissue de novo from induced pluripotent stem cells (iPSCs), which could potentially advance patient-specific disease modeling as they have for other diseases. Encouragingly, a protocol for creation of human stromal cells with responses to progesterone char- acteristic of endometrial stromal cells undergoing deciduali- zation in vitro has been developed, 109 providing a foundation for future efforts to reproduce other uterine tissues. To model the pathogenesis of adenomyosis, it is important to consider its ectopic environment in addition to the lesion- initiating cells. The myometrium comprises several cell types, but the major focus in in vitro models is on myometrial SMCs, which have different properties in the inner (JZ) and outer myometrium and express different receptor and sig- naling repertoires. 33,37,110 Protocols for deriving and cultur- ing primary SMCs developed for many other tissues (vascular, intestinal, etc.) facilitated development of robust protocols for isolation and culture of myometrial SMCs, which are commonly obtained from hysterectomy or caesar- ean section specimens. Cell origin (geographic location, pregnant or nonpregnant uterus) is an important consider- ation in de fining primary cell source for myometrium. Per- manent myometrial cell lines are also widely used for circumscribed studies such as screening drug actions. 111–113 Finally, all studies with patient-derived cells must carefully consider the donor pro files in design and interpretation of experiments. Age and parity may influence donor cell pheno- type. Unlike endometriosis lesions, which can be collected from even very young patients, almost all myometrial tissue, along with adenomyosis lesion tissue, is derived from hyster- ectomy specimens. Although protocols for resecting and biop- sying adenomyotic tissue have been described, 7,26,114 these procedures are not widely practiced. Thus, significant barriers exist in creating tissue banks from young patients who suffer from adenomyosis. General Approaches to 3D Tissue Engineering of Endometrium and Myometrium Using Natural and Synthetic ECMs An enduring challenge at present is to de fine suitable con- ditions to sustain long-term cocultures of multiple endome- trial cell types that each has their own ECM and growth factor requirements. Matrigel comprises a mix of proteins and proteoglycans typical of the epithelial basement membrane, which filters proteins from the stromal compartment that requires a type I collagen and fibronectin-rich ECM with a very different composition of proteoglycans and associated matrix-bound growth factors. 115 The first 3D primary cell endometrial coculture model, tailored for studying blasto- cyst implantation, addressed this by embedding stromal fibroblasts in a collagen gel, mimicking features of the stromal matrix, which was coated by a thin layer of Matrigel onto which the epithelial cells were seeded, resulting in a confluent well-differentiated epithelia monolayer compris- ing ciliated and secretory (glandular) epithelia. 116 Versions of this model using cell lines, with stromal cells in collagen topped with Matrigel, were adapted to mimic the upregula- tion of MMPs and accompanying matrix degradation follow- ing progesterone withdrawal as a trigger of menstruation 28,117 as well as some features of the menstrual response to hormones, and other shorter-term aspects of stromal epithelial communication. 91,118 In a simpler version, in which stromal cells were embedded in Matrigel with epithelial cells on top, epithelial cells underwent 3D mor- phogenesis and proliferated, but stromal cells were relatively nonproliferative within the gel, 119 suggesting the need for cell-type–tailored ECM. An alternate approach, adapted from a successful clinical matrix for healing full-thickness (der- mal–epidermal) wounds in skin, is to seed stromal cells in a porous collagen matrix fabricated by freezing and lyophiliz- ing a type I collagen solution, and overlaying the matrix with epithelial cells, which produce their own basement membrane. 120 These models employing natural ECMs have shortcomings, in part because there are no “one-size-fits-all” ECMs for both epithelia and stroma (and potentially other cell types), and also because natural proteins are impure (Matrigel has myriad growth factors); are substantially variable from lot-to-lot; are relatively rapidly degraded by cells; and are subject to variation in structural and mechanical properties depending on how quickly or slowly the gels are polymerized. 115,121 Variation is further exacerbated as some investigators use atelocollagen, which yields very different outcomes than whole collagen.122 These shortcomings have prompted a sustained effort in the biomaterials community to create synthetic alternatives to Matrigel and collagen.123,124 A popular approach exploits the relative inertness of poly(ethylene glycol) (PEG), which is commercially available in multi-arm star/branched configura- tions activated with cell-compatible reactive groups, to create modular cell-encapsulating hydrogels crosslinked with short matrix metalloprotease (MMP)-sensitive peptides and incor- porating synthetic integrin-binding motifs. 121,123–126 Other types of synthetic gel ECMs based entirely on synthetic proteins or semisynthetic gels based on modi fied hyaluronic acid are also widely used.124,127 The local and bulk mechanical modu- lus, permeability, degradation properties, and biochemical recognition motifs—all of which have been correlated to cell responses115,121,125—can be tuned independently to match the needs of individual cells and tissues. Recently, a locally responsive,“one-size-fits-all” PEG-based modular synthetic ECM was developed speci fically for cocul- ture of human endometrial epithelial and stromal cells, incor- porating synthetic integrin-binding peptides for both cell types, and synthetic peptides that capture and sequester the different ECM proteins produced by epithelial and stromal cells. 92,95,121 A version of this synthetic ECM also supports expansion of human endometrial organoids121 and formation of networks by human microvascular endothelial cells,125 thus providing a foundation for engineering complex 3D cocultures ( ►Fig. 3b). This matrix can be dissolved by a microbial enzyme to release cells and local cell –cell signaling molecules for analysis.92,121 A relatively unsolved problem, however, is formulation of culture media compositions that support mul- tiple different cell types. Solution of this problem may require finer speci fication and validation of “physiological” cell Seminars in Reproductive Medicine Vol. 38 No. 2-3/2020 Physiomimetic Models of Adenomyosis Gnecco et al.186 phenotypes, particularly for stromal cells which are often grown in serum-containing media optimized for maximal cell proliferation rates, rather than specific tissue functions. Tissue engineering of the myometrium has bene fitted ex- tensively from the intense activity in using 3D models to illuminate arterial smooth muscle pathophysiology and regen- erate blood vessels, where the roles of basement membrane, 3D architecture, cell density, and biomechanical stimulation on cell and tissue phenotype have been analyzed. 128 Understanding and modulating contractions in the pregnant uterus, to inter- vene therapeutically in preterm birth, has been the major application focus of in vitro 3D myometrial explant and tis- sue-engineered models. 36,111–113,129–132 Tissue-engineered models employing myometrial SMCs embedded in 3D collagen gels, adapted from successful models for vascular applications, offer advantages over explants by enabling a reproducible cell source from a tissue bank to be employed in multiple experi- ments, 131 and providing control over which cell types are present, to parse contributions of various immune cell pop- ulations, for example. An innovation involving an ECM-free magnetic cell aggregation 112 has been adapted to the myome- trial contraction assays, but approaches involving synthetic ECMs are still only nascent for other SMC applications.133 To summarize this section, the essential cell-based founda- tion for creating complex, 3D models of myometrial–endome- trial interactions has been established through a combination of advances in basic methods of cell culture, combined with technologies for controlling the ECM microenvironment with synthetic biomaterials ( ►Fig. 3c ). These tools can be further combined with micro fluidic devices to model pathologic phenotypes in a physiomimetic sense. Modeling the Dynamic Processes of the Adenomyotic Microenvironment The fundamental tools for building 3D tissue models are the constituent cells, scaffolds, matrices, platforms, and soluble cues that facilitate reconstruction or morphogenesis of physiologically relevant mimics of tissues. These fundamen- tal tools are then deployed in specialized ways to model specific dynamic processes and phenotypes. Here, we describe the processes illustrated in ►Fig. 2 ,a n dd r a w from applications in other disease research areas to illustrate how fundamental tissue engineering tools are often com- bined with other devices to further control elements of the culture microenvironment, such as mechanical stimulation, innervation, or fluid flow. Invasion and Survival Whether adenomyosis initially arises from a true invasion of the myometrium from the endometrium or through trans- port of endometrial cells into the myometrium by other means is debated, but once present, the displaced endome- trial cells form lesions that grow and invade the surrounding tissue as the lesion enlarges. Cancer, wound healing, immune response to infection, and myriad other pathophysiological behaviors—including endometriosis and adenomyosis —have motivated a vast array of mechanistic experimental studies and biophysical models of cell migration and inva- sion. 15,122,134–136 Although various authors often inter- change the terms migration and invasion, here we will refer to migration as a phenomenon of movement along a two-dimensional (2D) surface (e.g., endothelial cells moving across a denuded region of a vessel) or a quasi-2D surface (highly porous 3D matrix such as a large-pore transwell membrane), where no ECM degradation is required for movement. In contrast, we de fine invasion as a 3D process where breakdown of matrix or cell–cell junctions is required. Migrating and invading cells inherently integrate an array of individual molecular processes to generate biophysical forces resulting in cell movement, which may be random or influenced by chemotactic (chemical-based), durotactic (matrix stiffness- based), or haptotactic (adhesion-based) gradients. 134,136 This integration can result in nonintuitive outcomes, necessitating careful attention to quantitative experimental parameters and metrics. For example, experimentally, cell migration speeds exhibit maxima for intermediate values of ECM-coating densi- ties, across many different cell types and ECM coatings, as predicted by a biophysical model describing the balance between cell-matrix adhesion forces and cell-generated con- tractile forces. 134 Growth factors, which modulate both cell- matrix adhesion and cell contractile force generation, can appear to increase or decrease cell speed for a given ECM- coating density, depending on which side of the biphasic curve is operative for the chosen condition. 137 3D invasion assays inherently integrate more complex processes. The diversity in types of 3D movement, together with cell-mediated matrix degradation, results in different biophysical phenomena gover- nance compared with that of 2D migration assays. 122,138 Although there are numerous studies focusing on migration and invasion of cells derived from eutopic or ectopic tissue in patients with endometriosis,15,139 studies on adenomyosis are more limited and focused on stromal cells. 136,140,141 Consid- eration of how the individual molecular components in the extracellular environment (e.g., adhesion molecules, growth factors, porosity and stiffness of the matrix, matrix remodel- ing) and intracellular environment (e.g., integrin–cytoskeletal links, actin –myosin contractile forces, signaling pathways governing protein–protein associations) are integrated bio- physically provides insight into why literature reports com- paring adenomyotic and healthy cell migration and invasion are con flicting. 136 When eutopic endometrial stromal cells from adenomyosis patients and controls were compared in an assay format involving migration/invasion of cells across a thin porous membrane coated with Matrigel for 24 hours, no differences between the two groups were observed. 140 How- ever, when eutopic endometrial stromal cells from patients with adenomyosis were compared with controls in an assay that involved invasion for 10 days into a 1 cm-thick slab of type I collagen gel, adenomyotic cells invaded further than controls; invasion of both control and adenomyotic cells was further enhanced when myometrial cells from control donors were included in the gel, and even further enhanced again if the donor myometrial cells were from an adenomyosis patient. 141 Furthermore, this same assay format revealed that estradiol and tamoxifen drive additional increases in the invasion depth Seminars in Reproductive Medicine Vol. 38 No. 2-3/2020 Physiomimetic Models of Adenomyosis Gnecco et al. 187 of cells in each condition, while progesterone blunts the effects.142 The 24-hour Matrigel invasion assay may not have captured differences between control and diseased popula- tions, due to a combination of the relatively short assay time, use of an epithelial basement membrane-type matrix (Matri- gel) rather than a stromal-like matrix (collagen I), and the potential for the many growth factors in Matrigel to stimulate chemokinesis (random motility) in a way that drove maximal invasion and obscured the differences. Still, whether these endometrial cells would invade a true smooth muscle struc - ture, and if so, whether the factors regulating such invasion are the same as those regulating migration in ECM, is an interest- ing question for future models to address. The enduring challenge, as noted at the outset of this article, is whether new targets for therapeutics are revealed, and if so, whether drugs that treat them (as well as existing drugs) can be circumscribed to certain groups of patients who might be identified by accessible clinical tests. Answering these related questions, especially in a manner that captures the multiple phenotypes leading to symptomatic pain and bleeding in adenomyosis, is still at an early stage of physiomimetic analysis for any type of therapeutic. With respect to identifying new targets, a study employing a high-throughput 3D invasion assay for the 12Z endometriotic cell line in collagen gels that examined invasion as a function of growth factor/cytokine stimulation, intracellular kinase signaling pathway response, and proteolytic shedding of cell surface receptors and growth factors 15 might be considered relevant for adenomyosis, as the 12Z line likely shares features with adenomyotic epithelia. As noted earlier, experiments with cell lines offer insights that can be followed up with primary cells. In the study, a Bayesian analysis linking the recursive protease-growth factor-kinase signaling loops revealed potential vulnerabilities in the net- work involving, among others, JUN kinase (JNK), and this prediction was experimentally veri fied with inhibitors. 15 In- terestingly, a JNK inhibitor was also identi fied as a driver of inflammation in a separate study of peritonealfluid in patients with surgically veri fied endometriosis, where a subset of patients exhibited a constellation of signature cytokines. 14 Two preclinical studies indicated effectiveness of a JNK inhibi- tor against endometriosis,143,144 yet a clinical study in a non- stratified patient population (NCT01630252, NCT01631981) failed to meet endpoints. (None of the patients in either study were evaluated for possible adenomyosis, which is often comorbid with endometriosis and may contribute to symp- toms and which may also respond to therapies for endometri- osis.) Further exploration of this hypothesis with the types of models now being developed that include multicellular com- partments and robust metrics may provide insights into how to design a clinical trial to treat subsets of patients with certain characteristics. Adenomyosis invasion phenocopies features of carcino- ma invasion into smooth muscle. Although the overwhelm- ing majority of in vitro invasion studies have investigated invasion into ECM-rich tissues, the conceptual framework for building 3D invasion models in muscle has been mapped out over the past decade by observations in clinical speci- mens and intravital microscopy of carcinoma invasion in muscle in mouse models. 135 Epithelial invasion into muscle is associated with changes in multiple facets of cell cyto- skeletal and mechanosensing behaviors, driving a prefer- ence for stiff environments. 135 Cells can invade into muscle as individual cells, or as chains, clusters, or collective strands, following the collagenous matrix between bundles or migrating along individual SMCs. 135 Features of collec - tive cell invasion have been observed in deep in filtrating endometriosis lesions in the rectocervical space, 55 suggest- ing the possible invasion from adenomyotic lesions ema- nating from the cervical region of the uterus. The role of the regular smooth muscle contractions in the invasion process is still speculative, as there are consequences for mechanical damage, as well as promoting mitosis. 135 Hence, the devel- opment of models that experimentally recapitulate these multiple features is the goal o f adenomyosis physiomimetic modeling. Vascularization and Immune Interactions With a vision toward building an adenomyosis lesion model that incorporates large-scale features of the lesion microenvi- ronment, including a vascular bed, creation of a microvascular network becomes essential for survival and homeostasis of the engineered tissue. First, the microvascular system in the uterus, as in other organs, is essential for providing oxygen and nutrients. The dimensions of 3D in vitro models are inherently limited by diffusion of oxygen and nutrients to approximately 0.2 mm for metabolically active tissue like liver and muscle, and slightly larger for relatively acellular connec - tive tissues such as dermis. 115 Second, the microvascular barrier also regulates trafficking of immune cells (and possibly circulating stem cells) and plays essential roles in tissue homeostasis through paracrine signaling to tissues, serving as the nidus for commencing decidualization of the endome- trium. 145 Vascular function is modulated by systemic and local levels of sex hormones and inflammatory cues.146,147 In vitro, cultures of endometrial stromal and epithelial cells in the presence of estrogen regulate angiogenesis, 148 and signals from flow-activated endothelial cells enhance hormone re- sponse in endometrial stromal cells.145 Encouragingly, a fusion of tissue engineering and micro- fluidic cell culture technologies has spurred development of devices and protocols that could be applied to modeling adenomyosis lesions. Micro fluidic devices with planar cul- tures that probe endothelial –stromal–immune interactions have already been applied to study the role of environmental chemicals on facets of endometriosis phenotypes. 149 Micro- fluidic and mesofluidic devices incorporating 3D tissue struc - tures that can be perfused continuously with culture media, imaged, and sampled in situ are endemic tools in the emerging field of “microphysiological systems (MPSs)”—in vitro repre- sentations of complex physiological phenomena involving multiple cell types and dynamic behaviors in 3D. A crucial first step was to de fine protocols for the creation of stable, perfusable microvascular networks in a collagen or fibrin gel situated between two channels perfused with cell culture media ( ►Fig. 4a ).150–152 Stable microvessels from endothelial cells andfibroblasts seeded in the central gel region (►Fig. 4b), Seminars in Reproductive Medicine Vol. 38 No. 2-3/2020 Physiomimetic Models of Adenomyosis Gnecco et al.188 enabling phenomena involving traf ficking of immune (►Fig. 4c ) and tumor cells across the endothelial barrier to be observed in detail ( ►Fig. 4d ).153 These foundational approaches have now been widely adapted to support appli- cations as diverse as in vitro tumor and islet microvasculari- zation and formation of functional blood –brain barriers. 154–157 These studies establish principles for how a microvascularized endometrial–stromal lesion could be mod- eled; indeed, the authors are currently leveraging them to build multicellular microvascularized models of endometri- osis lesions, employing primary patient-derived endometrial epithelial, stromal, and immune cells for analysis of how microvascular permeability and immune cell recruitment are regulated as a function of circulating sex steroids. A model of vascularized muscle developed for analyzing fibrosis and muscle damage 158 could potentially be adapted for creating a model of vascularized adenomyosis in the host tissue. One significant technical challenge relevant for modeling repro- ductive tissues is that the material most commonly used for fabricating micro fluidic devices, polydimethylsiloxane (PDMS), absorbs lipophilic compounds like estrogen and pro- gesterone, making it almost impossible to control their con- centrations and thus tissue exposure. 159 Among fabrication materials, PDMS is uniquely permeable to oxygen—a limiting nutrient in most cultures; hence, significant design changes to provide oxygenation are required in transitioning to more suitable materials, but these shifts are gradually occurring as commercial chips based on thermoplastics are coming into use. 160 Biomechanical Stimulation It is now well recognized that the mechanical compliance of the ECM microenvironment—“stiffness” or “softness”—dramatical- ly influences the phenotypes of epithelial cells, stem cells, and other cell types.161 Matrices that are relatively stiff compared with the normal mechanical properties that a cell experiences can drive transition of fibroblasts to myofibroblasts and epi- thelial cells to a tumor-like phenotype. 161 Similarly, tumor cells accustomed to a stiff environment may exhibit poor survival and growth when placed into a more normal mechanical environment—a factor that may contribute to the dif ficulty in growing cells from endometriosis and adenomyosis lesions. These adaptations occur through a compendium of mechanical signaling pathways that are integrated in part by the intracel- lular translocation of transcriptional regulators YAP and TAZ to the nucleus, allowing differences in mechanical signaling be- tween two different tissue environments in vivo to be inferred from immunohistochemistry. 162 An elegant in vivo –in vitro mechanical signaling study involving the myometrium first demonstrated links between the mechanical properties of uterine leiomyomas (fibroids) relative to surrounding myome- trium and the corresponding relative amounts of nuclear YAP/ TAZ (via immunohistochemistry), and then showed that isolat- ed myometrial SMCs or fibroid cells cultured in 2D on a set of synthetic hydrogel substrates with systematically varied me- chanical stiffness recapitulated the trends in nuclear YAP/TAZ s e e nf o re a c hc e l lt y p ei nv i v o ,a l o n gw i t he n h a n c e dE C M deposition by fibroid cells as seen in vivo. 162 This in vivo –in vitro correspondence suggests that the in vitro model may be a useful proxy for analyzing potential therapeutic interventions into mechanical signaling pathways. Other behaviors, such as invasion/survival, may require a 3D environment to capture these in vivo behaviors. Toward this goal, we have observed that normal human endometrial epithelial cells, which exhibit a prototypical spherical organoid morphology when cultured in soft synthetic hydrogels, 121 adopt a lesion-like morphology with invasive protrusions and epithelial-mesenchymal transi- tion (EMT)-like cellular morphology when cultured in stiff Fig. 4 Micro fluidic model of immune and tumor cell traf ficking between the microvasculature and tissues .( a)Am i c r ofluidic device comprising a central tissue-containing channel, flanked by two channels for flow of culture medium, is inoculated with a mixture of fibrin containing endothelial and stromal cells. Over the initial few days, ce lls undergo morphogenesis to form perfusable microvascular networks which are stable for weeks in culture as shown in ( b) for a day 23 culture (actin stain). ( c) Immune cells (green) can be perfused through the microvasculature (red) to model peripheral cell recruitment. ( d) The dynamic cell-level phenomena in the devices can be imagined by confocal or two-photon microscopy to observe phenomena such as neutrophil –tumor cell interactions in the extrav asation of tumor cells through the vascular wall into tissue. (Images from Zhang et al, 110 permission is in progress.) Seminars in Reproductive Medicine Vol. 38 No. 2-3/2020 Physiomimetic Models of Adenomyosis Gnecco et al. 189 synthetic hydrogels (Gnecco et al, unpublished data Aug, 15, 2020; ►Fig. 3c ). These type of reductionist models may help parse the relative contributions of in flammatory cues, hor- mones, and other microenvironment factors reported in adeno- myosis lesions. However, the myometrium is not just a static microenvironment. The nonpregnant myometrium undergoes constant contractions to movefluid, in ways that are disrupted in adenomyosis. 35,36 The propensity of endometrial cells to form endometriosis lesions in smooth muscle of the intestinal tract and adenomyosis lesions in the myometrium, and to similarly be associated with hyperplastic smooth muscle (or myofibroblasts) in other ectopic contexts such as the bladder, ureter, rectocervical space, and relatively absent from the mesentery and omentum, suggests an interesting tropism for microenvironments that may offer mechanical stimulation. Although the ability of drugs and hormones to alter contractile phenotype of myometrial cells has been used as a screen in explant and 3D culture assays, 81,113,129 and to compare general contractile abilities of normal and adenomyotic myometrium, 36 extension to more complex questions of etiology and interaction with endometrium under dynamic conditions is only nascent. Dynamic me- chanical stimulation of human uterine cells in 2D has shown that endometrial stromal cells can acquire a con- tractile phenotype 163 and that human myometrial cells exhibit dramatic shifts in the phosphoproteome under stretch. 164 A study designed to illuminate the possible effects of myometrial contractions on endometrial pheno- type used a novel micro fluidic reactor to coculture an endometrial cell line on a layer of myometrial cells, in a manner that exposed the coculture to dynamic peristalsis that generated shear flow in the fluid impinging on the endometrial epithelial cell layer. 165 This proof-of-principle study performed with cell lines showed morphological changes in the epithelial barrier in response to the mechan- ical forces,165 and provides a foundation for next-generation experiments with primary cells and a more complex tissue architecture, building on the well-developed observations about tissue engineering of vascular smooth muscle under pulsatile mechanical stimulation. 166 Innervation In addition to the well-known animal dorsal root ganglia sources of neurons, the ef ficient derivation of genetically diverse peripheral sensory neurons from human cells has provided novel avenues for investigating and culturing hu- man nerve fibers in vitro 167,168 and for building models for neuromotor action and pain. 167 While models of the endo- metrium have not yet been explored, micro fluidic models of enteric nerve –epithelial interactions in the intestine are exemplary of the approaches. 169 Similarly, a micro fluidic model of neuronal activation of vascularized skeletal muscle contraction, which allows quantitative analysis of axonal growth, muscle maturations, and contraction, provides a template for design of a similar device for the myome- trium. 170 Ultimately, these tools might be combined to build models of innervated, vascularized adenomyosis lesions, using patient-specific samples to understand the manifesta- tion of debilitating menorrhea. Systemic and Organ –Organ interactions Finally, adenomyosis and other chronic inflammatory diseases both exert and respond to systemic effects, potentially includ- ing those emanating from the gut microbiome. A growing number of micro fluidic and meso fluidic models are being developed to connect MPSs representing multiple different organ systems (e.g., gut, liver, heart, brain) in a continuously communicating fluidic network for extended (weeks) culture periods, using fabrication materials that avoid the problems of PDMS.171–173 A new pumping technology driven by integrat- ing microfluidic pumps that are safe for immune cells onto the platform has been applied to examine the interplay between tissue-resident cells in the gut and liver and circulating im- mune cells in response to short-chain fatty acid metabolites produced in the colon, revealing paradoxical responses. 173 Such technologies might be used in the future to examine chronic cell trafficking between the vasculature and lesions in a single-MPS model of adenomyosis as a function of hormone cycles, or, more ambitiously, the interplay between the bone marrow–adenomyosis lesion axis with respect to circulating cells and factors, given the interplay between bone marrow – derived cells and wound healing/lesion phenomena. 174 Data-Driven Analysis and Predictive Modeling of Human Responses A premise of the physiomimetics approach ( ►Fig. 1 )i st h a t patients can first be stratified on the basis of a combination of clinical phenotypes and molecular network analysis, to gener- ate hypotheses for mechanistically distinguishable subgroups. In vitro tissue-engineered models of these subgroups can then be constructed using well-characterized patient-derived specimens and perturbed. Finally, multi-omic measurements combined with phenotypic metrics can be interpreted to drive identification and validation of therapies for patient sub- groups, thus providing a foundation for stratified clinical trials. Molecular stratification of endometrium-derived diseases is still in early stages. A meta-analysis of genome-wide associa- tion studies for endometriosis showed stronger reproducibili- ty across eight of nine disease-associated loci for patients with Stage III/IV disease compared with Stage I, 175 suggesting there may be clues to processes that amplify lesion characteristics. However, the loci implicate genes widely expressed through- out the body; thus, one could reasonably construct hypotheses around gut permeability in fluencing systemic immune func - tion, for example, as a possible contributing mechanism. While systems-level physiomimetic modeling of complex systemic immune reactions involving gut permeability are emerging, 173 these are not the most likely place to begin to parse adeno- myosis. Similarly, transcriptomic changes in endometrial bi- opsies are more pronounced in severe versus mild endometriosis,176 suggesting several possible pathways for intervention. However, without other measurements of lesion tissue context and associated symptom characteristics, these Seminars in Reproductive Medicine Vol. 38 No. 2-3/2020 Physiomimetic Models of Adenomyosis Gnecco et al.190 datasets provide incomplete directions on designing physio- mimetic models to test mechanisms. These types of analyses might be paired, though, with phenotypic assays representing dynamic lesion properties. For example, the fine-grained in vitro analysis of pancreatic tumor organoid invasion characteristics, used for parsing mechanistic relationships between pancreatic cancer muta- tion subtypes and survival,177 might provide phenotypic dis- crimination among patient subgroups, which could in turn lead to additional molecular phenotyping based on hypotheses regarding mechanisms governing the phenotypes. Even the assay used for the pancreatic tumors, however, revealed paradoxical relationships with survival 177—suggesting an as- say incorporating more complex tumor –stroma interactions could be more revealing. A piece of a physiomimetic puzzle for endometriosis, involving a bioinformatics prediction that JNK would govern cytokine release from peritoneal macrophages, which was then confirmed in vitro with patient samples, 14 may also have implications for adenomyosis, as macrophages are also involved. A physiomimetic model incorporating patient macrophages along with lesions may reveal disease-related phenotypic characteristics that could be modulated with a JNK inhibitor. At a systems level, the effects of potential new therapeutics can also be assessed using multi-MPS systems including liver and the immune system, incorporating features of liver metabolism and other metabolic and systemic trans- formations, using multi-omics to parse complex system responses. 172,173 These technologies are developing quickly and may greatly enhance physiomimetic modeling of gynecol- ogy diseases in general once resources to clinically phenotype patients and build corresponding tissue banks are identified.

and Future Directions The enormous spectrum of symptoms, histological/morpho- logical appearances, and associated comorbidities that occur in patients with adenomyosis suggest that there may be distinct subsets of patients who could be targeted with personalized therapies—if the rules for defining these patient populations and approaches for developing the targeted therapies are established and implemented in an integrated fashion. The complexity of adenomyosis requires a physio- mimetic approach: parsing the phenomena that may con- tribute using computational modeling approaches and then building a physiological model that provides information that translates back into the clinic. The fundamental in vitro tissue engineering approaches necessary to create physio- mimetic models have largely been established: methods to conceptually and computationally characterize the disease to generate hypotheses about patient strati fication approaches; methods to create tissue banks of relevant cell types derived from carefully phenotyped patients; 3D syn- thetic matrices to engineer microenvironments in a repro- ducible way; micro fluidic platforms to control vascular and nerve interactions with 3D tissues; and analytical outputs using functional and molecular assays. The future of model- ing adenomyosis lesion complexity arguably depends on how well these tools become democratized, whether large enough patient populations can be pooled to de fine the bedside-to-bench-to-bedside paradigm of physiomimetic modeling, and whether standardized methods can be de- fined and implemented for access to patient myometrial/ adenomyotic tissue in younger patients who undergo fertili- ty-sparing procedures. We are optimistic that the roadmap described here will spur multidisciplinary teams to hasten development and implementation of better treatments for patients. Funding National Institutes of Health http://dx.doi.org/10.13039/ 100000002 EB029132 National Science Foundation http://dx.doi.org/10.13039/100000001 Conflict of Interest None declared. Acknowledgments We thank Hilary Critchley, Stacey Missmer, and Doug Lauffenburger for critical reading of the manuscript. This work was supported by the John and Karine Begg Foundation, the Manton Foundation, the National Science Foundation, and NIH U01 EB029132.

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