Rodent Models of Experimental Endometriosis: Identifying Mechanisms of Disease and Therapeutic Targets

In large part, current research efforts in the field of endometriosis are focused on the development of new and better clinical therapies for women with this condition. However, designing more effective therapeutic strategies requires that we develop a deeper understanding of the trigger mechanisms driving the development and progression of endometriosis. Detailed below are examples of mechanistic studies, utilizing the models described above, which attempt to identify the pathogenic mechanisms associated with development of endometriosis. Successful establishment of endometriosis is a multi-step process requiring the rapid occurrence of peritoneal attachment, extracellular matrix degradation and neovascularization. In order to examine early events associated with ectopic attachment of endometrial tissues, our laboratory utilized the chimeric human/mouse model system. Using this model, we explored the potential role of matrix metalloproteinases (MMPs) in the invasive establishment of experimental endometriosis in nude mice [ 36 , 99 ]. The MMPs are highly regulated enzymes that are necessary for normal and pathologic tissue remodeling, including the cellular migration and matrix restructuring associated with wound-healing or tumor metastasis [ 100 ]. Our studies demonstrated that blocking endometrial MMP expression or action prevented the establishment of human endometrial growth at ectopic sites within the peritoneal cavity of nude mice [ 36 ]. Following the physical attachment of endometrial tissues to ectopic sites, survival of lesions requires the rapid establishment of a vascular supply and, in a collaborative study, we demonstrated that blocking angiogenesis is effective in preventing lesion development [ 101 ]. In a follow-up study focused on very early disease, we demonstrated that vascular development is apparent by 24 hrs with extensive vascularization occurring about 5 days post-injection [ 102 ]. Importantly, we noted that peritoneal attachment and acquisition of a vascular supply occurs more rapidly following experimental disease establishment with eutopic endometrial tissues acquired from women with endometriosis, suggesting that the endometrial phenotype associated with endometriosis patients exhibits a greater innate capacity to stimulate peritoneal blood vessel growth in our model [ 79 ]. Furthermore, acute exposure of disease-free control endometrial tissue to TCDD, prior to injection into the peritoneal cavity of recipient mice, leads to a similar host vascular response within the peritoneal cavity as observed with tissue acquired from endometriosis patients [ 79 ]. Thus, in addition to stimulating MMP expression in endometrial fragments [ 103 ], TCDD exposure also promotes a vascular response at the site of endometrial invasion into the murine peritoneal wall, suggesting that this toxicant may promote lesion development via multiple mechanisms. In order to specifically examine the individual cellular responses within the human endometrium to TCDD, we have begun to utilize organ-on-chip models [ 104 ]. These microscaled models enable compartmentalized, heterogeneous cell culture systems that better recapitulate in vivo anatomy and allow the direct evaluation of paracrine and endocrine crosstalk among cell types. Using such a system, in conjunction with the in vivo models described herein, should provide detailed information with regard to the responses of individual cells to TCDD as well as the whole tissue response within the peritoneal cavity. Although the phenotype of endometrial tissue fragments significantly contributes to disease pathogenesis, recent studies provide evidence that the peritoneal phenotype also plays a critical role in endometriosis. Moreover, recent studies suggest that the eutopic endometrial phenotype works in concert with peritoneal inflammation, synergistically promoting ectopic endometrial growth. For example, in response to retrograde menstruation, there is a large influx of immune cells into the peritoneal cavity, which in healthy women would clear the dead and dying menstrual debris. However, in women with endometriosis, both heavier menstrual bleeding and immune cell dysfunction may reduce the capacity of the innate immune system to scavenge refluxed tissues. Equally important, inflammatory mediators produced at inappropriate levels likely impact the invasive and neoangiogenic processes that are necessary for establishment of endometriosis [ 68 , 105 ]. Menstrual debris may also stimulate pro-inflammatory cytokine production as a consequence of inflammasome activation. The inflammasome is a family of multiprotein complexes that are expressed by macrophages and other immune cells. These components act as immune system receptors and sensors and regulate the inflammatory response associated with both infectious microbes and damaged-associated host proteins. To date, four inflammasome complexes have been identified; each having a distinct protein composition which are formed in a stimulus-dependent manner (reviewed [ 106 ]). Among these, the NLRP3 (NLR Family Pyrin Domain Containing 3) complex is the most well-studied and is present in a variety of normal and disease states [ 107 ]. All of the inflammasome complexes cleave pro-IL-1β, leading to its activation and promoting an inflammatory cascade. Activation of this system in the context of endometriosis may inadvertently promote tissue repair rather than tissue clearing [ 108 ]. This acute inflammatory response system can also activate the local vasculature, further promoting lesion survival. To this end, using the nude mouse model of experimental endometriosis, we found that vessels present within lesions revealed anastomosis had occurred between human and murine endothelial cells [ 101 ], suggesting that the human tissue produces chemoattractants that promote growth of the murine vasculature. Supporting these findings, chimeric human/murine vessels were also identified by Alvarez-Gonzalez et al . in a SCID mouse model of human experimental endometriosis in [ 109 ]. This latter study confirmed that the chimeric vessels were functional, with a circulating blood supply established by 3 weeks. Clearly, development of a vascular supply is essential for ectopic endometrial survival. Therefore, a large number of studies have utilized murine models to examine the potential therapeutic value of angiogenesis inhibitors [ 101 , 110 - 117 ]. Collectively, these studies indicate that both natural and pharmaceutical agents that inhibit angiogenesis can impede maintenance and growth of experimental endometriosis and support investigation of angiostatic agents as potential therapeutic agents for women with this disease. Normal endometrial function is largely dependent on the sequential action of the ovarian steroids estrogen and progesterone [ 118 ]. Following menstruation, rising levels of estradiol act to induce endometrial regrowth and estrogen levels remain high even after ovulation when progesterone levels increase to induce endometrial maturation. Progesterone not only acts on the endometrium to promote differentiation in preparation for pregnancy, but is also a potent anti-inflammatory steroid. If nidation does not occur, progesterone levels will begin to decline, resulting in a loss of endometrial steroid support and culminating in the acute inflammatory process of menstruation. Discussing the multiple cellular processes within the endometrium that are controlled by ovarian steroids is beyond the scope of this review examining experimental models of endometriosis. Nevertheless, researchers interested in the pathogenesis of endometriosis have long recognized the critical roles of the sex steroids in endometrial function whether within the uterus or at ectopic sites of growth. Exposure to estrogen is one of the principal endocrine risk factors for developing endometriosis [ 19 ], largely as result of this steroid’s ability to promote rapid endometrial proliferation. In contrast to estrogen action, progesterone exposure (ie, pregnancy or combined oral contraceptives) acts to reduce proliferation and induce cellular maturation, thus serving as a negative risk factor for development of endometriosis [ 19 , 119 ]. However, numerous studies have demonstrated that women with endometriosis exhibit altered expression of a variety of genes and proteins that are normally regulated by the sex steroids [ 120 , 121 ] revealing potential therapeutic targets [ 122 , 123 ]. Importantly, both steroids act via their respective receptors, primarily progesterone receptor-B (PR-B) and estrogen receptor-α (ESR1). Additionally, PR-A, a truncated isoform of PR-B, can act as a dominant repressor of PR-B [ 124 , 125 ] and has been found to be overexpressed in endometriosis [ 103 , 126 , 127 ]. Similarly, estrogen receptor-β (ESR2) is overexpressed in women with endometriosis and appears to play a role in disease development [ 119 , 128 , 129 ]. Estrogen’s ability to promote endometrial proliferation in both eutopic [ 118 ] and ectopic [ 19 ] sites of growth has made this steroid an important target of investigation. For example, using a syngeneic mouse model of endometriosis, [ 51 ] the Korach laboratory demonstrated estradiol-mediated signaling was required in the development of endometriosis-like lesions. Specifically, this group utilized estrogen receptor knockout (αERKO and βERKO) mice to explore the influence of expression or absence of each receptor in the ectopic lesions as well as within the recipient animal. Their studies demonstrated that, compared to wild-type donors, the number of successful lesions was dramatically reduced in mice receiving minced uterine tissue from either αERKO or βERKO mice, which they surmise was related to an inability of αERKO uteri to acquire a vasculature in response to estradiol. Interestingly, the ER status of the recipient animal had little impact on lesion establishment. Unlike estrogen, progesterone exposure (ie, pregnancy) is known to reduce the risk of endometriosis. For this reason, experimental models of endometriosis have been useful for preclinical studies examining the potential benefit of various progestins. A potential confounding issue with progestin-based therapy for endometriosis is that many patients with this disease exhibit sensitivity to progesterone [ 97 , 130 ]. As noted earlier, our studies more than a decade ago demonstrated that the reduced response to progesterone action in endometrial tissue acquired from endometriosis patients is associated with increased expression of MMPs and an enhanced capacity to establish experimental endometriosis [ 130 ]. A later study revealed that although endometrial tissues from women with endometriosis established as experimental disease in nude mice were resistant to regression by progesterone, treatment with tanaproget, a selective PR-B agonist, effectively reduced disease burden [ 131 ]. Additionally, experimental endometriosis using transgenic mice in which one of the major steroid receptors has been deleted further underscores the important and complex relationship between estrogen and progesterone action and endometriosis. Using a syngeneic endometriosis model with wild-type and progesterone receptor knockout (PRKO) mice, Bulun and colleagues demonstrated that the presence of PR in ectopic uterine tissue was essential to prevent estrogen-mediated proliferation and survival of lesions. The authors concluded that resistance to progesterone of human endometriosis may be a consequence of reduced PR expression or function [ 53 ]. Taken together, these data suggest that normalizing progesterone action within the endometrium would effectively reduce a woman’s risk for developing endometriosis. The presence of menstrual debris within the peritoneal cavity is generally accepted as a mechanical factor in a woman’s risk for the development of endometriosis. Nevertheless, an important, but, unanswered question is why only certain women develop this disease despite retrograde menstruation being a common phenomenon among reproductive age women [ 9 ] One explanation may be the intrinsic differences in progesterone responses noted between endometrial tissues of patients compared to women without endometriosis. However, as reviewed by Brosens et al . [ 132 ], alterations within the eutopic endometrium of women with endometriosis are not limited to reduced progesterone responsiveness. Indeed, alterations in expression of angiogenic factors, identification of nerve fibers and expression of inflammatory cytokines have been demonstrated to be altered in patients compared to disease-free women. Clearly, these differences likely promote either the development of endometriosis or play a role in co-morbidities associated with this disease. In our laboratory, we identified elevated expression of IL-1α and IL-1β in endometrial tissues acquired from women with endometriosis which was associated with an enhanced ability to establish experimental disease in our chimeric nude mouse model [ 129 ]. Indeed, an important advantage of immunocompromised mice is the ability to compare endometrial tissues from women with and without endometriosis in an in vivo system. Our published data using this model, in which human endometrial fragments are introduced into the peritoneal cavity of immunocompromised mice, demonstrated an invasive advantage of eutopic endometrium obtained from women with endometriosis [ 37 , 79 ]. Specifically, although endometrial tissues from women with and without endometriosis are equally capable of establishing ectopic disease, tissues from women with endometriosis establish larger lesions that are more quickly able to establish a vasculature [ 102 ]. Significantly, we found that peripheral immune cells obtained from control women limited the growth of experimental endometriosis [ 46 ]; however, in contrast, a similar immune cell preparation obtained from women with endometriosis enhanced the development of experimental disease (Fig. 3 and Table 2 ). These data suggest an important phenotypic difference in immune cells , in addition to endometrial tissue, obtained from women with and without endometriosis. The hyperinflammatory peritoneal environment observed in association with endometriosis in women has prompted a number of studies in rodents examining the efficacy of anti-inflammatory agents. Many of these studies, targeting specific inflammatory cytokines, were recently reviewed by Nothnick and Alali [ 105 ] and will not be detailed here. However, in contrast to targeting a specific inflammatory cytokine, a number of recent studies have explored the utility of dietary modulators of inflammation for the treatment of endometriosis. For example, using the rat surgical model of endometriosis, Akyol et al . [ 133 ] demonstrated that omega-3 fatty acids (found in fish oil) reduced experimental disease burden in association with a reduction in peritoneal levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and vascular endothelial growth factor (VEGF). Using a similar approach, İlhan et al . [ 134 ] treated rats with surgically induced endometriosis with a mixture of sea buckthorn and St. John's wort oils. These compounds have been used for many years in folk medicine for the treatment of a wide array of inflammatory disorders [ 135 - 138 ], but had not previously been examined with regard to endometriosis. In their study, İlhan et al . (2016) found that following 4 weeks of treatment with the oil mixture, rats in the treated group exhibited significantly fewer endometriotic lesions and adhesions compared to vehicle treated rats. Reduction in disease burden was associated with decreased peritoneal levels of IL-6, VEGF and TNF-α. Given the side effects of traditional pharmacologic approaches for the treatment of endometriosis, these studies support the exploration of anti-inflammatory diets for women with this disease.

Descriptions of endometriosis, defined as the presence of endometrial glands and stroma outside the uterus, can be found in medical literature at least as early as the mid-1800’s [ 1 , 2 ]; however, it would be several decades later before the name “endometriosis” was coined. In his landmark paper, Dr. John Sampson [ 3 ] suggested development of endometriosis was due to ectopic implantation of refluxed menstrual tissue [ 3 ]. Although research studies continue to support the “retrograde menstruation” theory as one mechanism by which endometriosis can develop [ 4 - 6 ], deposition of menstrual tissue within the peritoneal cavity cannot account for all incidences of disease. Additionally, retrograde menstruation is common among reproductive age women [ 7 - 9 ]; however, only about 10% of women develop endometriosis, suggesting other mechanisms are also involved. Alternative explanations for the occurrence of endometriosis include coelomic metaplasia [ 10 - 12 ] and peritoneal activation of embryonic cell rests [ 13 , 14 ]. The latter theory likely explains the unusual and rare occurrence of endometriosis in men [ 15 , 16 ]. Recently, ectopic endometrial tissue has been described in human fetuses which has been theorized to develop as a consequence of ectopic localization of primitive endometrial tissue during organogenesis [ 17 , 18 ]. Other factors likely affecting an individual’s risk for development of endometriosis include: genetic predisposition, immune dysregulation and/or a history of environmental toxicant exposure (reviewed by [ 19 ]). More recently, Brosens and colleagues have postulated a role for neonatal menstruation, as a consequence of early onset steroid responsiveness, in developing endometriosis as an adolescent [ 20 ]. While generally considered a benign condition, endometriosis exhibits cancer-like features and can spread throughout the peritoneal cavity and to distal sites. Endometriosis is frequently physically debilitating, as patients often suffer from chronic pelvic pain, dyspareunia, dysmenorrhea and subfertility. Unfortunately, most women with this disease also exhibit one or more co-morbidities including adenomyosis, adhesive disease and inflammatory conditions such as interstitial cystitis and inflammatory bowel disease [ 21 - 23 ]. However, understanding the natural history of endometriosis, as well as the myriad of equally poorly understood co-morbidities, has proven elusive, despite extensive research in each disease area. Given our lack of understanding of the etiology of endometriosis, treatment options for women with this disease remain limited and generally involve a combination of hormonal manipulation and surgery to remove diseased tissue. Unfortunately, the side effects of hormonal therapy for endometriosis cause many women to abandon this treatment; nevertheless, surgical treatment alone is frequently non-curative and many women suffer recurrence [ 24 - 32 ]. Thus, identifying better diagnostic and treatment strategies for this disease is a major focus of many endometriosis research laboratories. Endometriosis is rare in non-menstruating species, suggesting that shedding of endometrial tissue may be a causative factor as originally proposed by Sampson [ 3 ]. Indeed, numerous studies have demonstrated that the transfer of endometrial tissue to the peritoneal cavity can lead to the development of ectopic endometrial lesions. For example, in the mid-1950s, Sampson’s retrograde menstruation theory was experimentally examined in women via intraperitoneal injection of their own menstrual tissue several months prior to a scheduled surgery for fibroids. This study identified ectopic peritoneal lesions in 2 of 13 women (15%), providing support for the retrograde menstruation theory [ 4 ]. Given the obvious limitations and ethical considerations of human experimentation, current endometriosis researchers rely heavily on non-human primate or rodent models in order to investigate elements of disease pathophysiology.

Not applicable.

As stated above, women with endometriosis frequently exhibit serious co-morbidities, but the drivers of these disease associations remain unclear. However, experimental rodent models of endometriosis have begun to reveal that inflammation may be contributing to epigenetic changes that play an important role in understanding the relationship between endometriosis and other diseases/conditions. Epigenetic marking of DNA, unlike genetic mutations, alters gene expression without changing the DNA sequence. Epigenetic modification is one mechanism by which DNA accessibility is controlled, altering the ability of the cellular machinery to activate transcription. The most well-studied epigenetic mechanisms are methylation and acetylation of DNA and histones [ 139 ]. Importantly, although these marks can be reversible, they are stable and, when occurring within the germline, these changes can be inherited [ 140 - 142 ]. During development, epigenetic reprogramming of individual cells results in cellular differentiation specific for each organ system, despite each cell having the same DNA. Thus, it is the epigenetic marks that ultimately determine each cell’s fate and allows the formation of a complex, multi-organ animal from the same genetic blueprint. Additionally, epigenetic modifications accumulate as we age, largely in response to our own choices (diet, activity level) or where we live (environmental exposures). These accumulated epigenetic marks are primarily why we become susceptible to age-related diseases. Additionally, environmental factors that affect epigenetic modification of the germ cells have the potential to contribute to an offspring’s phenotype [ 141 ]. Significantly, the presence of aberrant epigenetic patterns can cause developmental abnormalities and are associated with the etiology of certain human diseases [ 143 ]. As will be discussed below, recent research suggests that epigenetic changes, perhaps driven by inflammation related to environmental toxicant exposure, may promote the development of endometriosis and/or associated comorbidities. Infertility impacts up to 40% of women with endometriosis [ 19 ], but whether infertility is a function of inflammation that is associated with the disease or a consequence of intrinsic endometrial defects or both remains unclear. In an effort to address this question, Stilley et al . [ 58 ] used the rat surgical model of endometriosis to examine the impact of ectopic disease on fertility. Perhaps not surprisingly, fertility in animals with surgically induced endometriosis was significantly compromised, while fertility was not compromised in sham operated rats (hemi-hysterectomy, but no ectopic lesions). Furthermore, this study identified a number of ovarian alterations (fewer ovarian follicles and corpora lutea with luteinized unruptured follicles), poor preimplantation embryo development and spontaneous abortion in rats bearing endometriosis-like lesions compared the sham rats. Finally, this study also identified the same reproductive abnormalities in the daughters of rats with endometriosis-like lesions, suggesting that exposure to the inflammatory environment of this disease during in utero development may lead to permanent epigenetic changes in the offspring. Using our developmental toxicant exposure model [ 54 , 98 ], we initially reported that exposure of pregnant C57bl/6 mice to TCDD led to adult daughters (F1 females) with a uterine phenotype similar to that of women with endometriosis [ 98 ]. More specifically, we found that F1 females exhibit reduced uterine PR expression and loss of progesterone-sensitive TGF-β2 (transforming growth factor β) expression, proteins essential for establishment and maintenance of pregnancy. Thus, we were not surprised to find that subfertility was also common among F1 females mated to control breeder males. Approximately 50% of female F1 mice failed to exhibit signs of pregnancy (weight gain/nipple prominence) despite multiple matings and observation of vaginal plugs (4+). In addition to subfertility, F1 females which achieved pregnancy also exhibited a high rate of spontaneous preterm birth [ 54 ], a pregnancy outcome that has recently been linked to endometriosis [ 132 ]. Importantly, F2-F4 female mice continued to exhibit an endometrial phenotype similar to women with endometriosis, even though these animals were not subjected to additional toxicant exposure. Specifically, although the animals in our study did not have experimental endometriosis, we identified both multi-generational (F1-F2) and transgenerational (F3-F4) occurrence of reproductive disorders that are similar to those encountered by endometriosis patients [ 54 ]. Finally, we have also examined whether the male offspring of mice exposed to TCDD during pregnancy could transfer the endometriosis phenotype to his female progeny. Similar to our findings in F1 females, F1 males also exhibit a hyper-inflammatory phenotype and reduced fertility [ 144 ]. Importantly, the daughters of F1 males (F2P females) exhibited the same reproductive abnormalities identified in F1 females and their offspring, including reduced PR expression, subfertility and increased risk of spontaneous PTB (Table 3 ). Our animal studies, taken with the studies by other groups described above, strongly support a developmental origin of endometriosis. Equally important, our findings suggest that the environmental exposure history of either parent can lead to a transgenerational risk for the development of endometriosis. Understanding the potential role of toxicant-mediated inflammation may provide important insight enabling targeted therapies that reduce disease risk. Adenomyosis, the presence of endometrial glands and stroma embedded within the uterine muscle, is frequently identified in women undergoing hysterectomy as a surgical treatment for endometriosis [ 145 ]. Adenomyosis, like endometriosis, has been associated with reduced fertility, pelvic pain, heavy menstrual bleeding and dysmenorrhea [ 22 , 146 ]. The causes of adenomyosis are currently unknown, although both human and animal studies have suggested a role of inflammatory processes in the development of this disease [ 59 , 147 - 149 ]. Furthermore, the occurrence of adenomyosis as a co-morbidity in women with endometriosis, fibroids and menorrhagia, supports a potential role of estrogen action and inflammation in the pathogenesis of this disease as well. In our TCDD exposure model, we recently reported the transgenerational occurrence of adenomyosis in mice exhibiting the endometriosis-like uterine phenotype as a consequence of developmental toxicant exposure of F1 animals [ 147 ]. Within this recent study, we conducted a retrospective analysis of uteri from TCDD exposed F1 female mice and two generations of their offspring to determine whether histological evidence of adenomyosis was present. Although none of the control mice examined exhibited adenomyosis, we identified deep, adenomyotic lesions in the majority of mice with a history of direct (F1-F2) or indirect (F3) TCDD exposure. Since we have demonstrated that F1-F4 mice exhibit a “hyperinflammatory” systemic phenotype, the occurrence of adenomyosis in these animals provides additional support for inflammatory mechanisms in the promotion of this disease. As stated above, in addition to inflammation, several studies have reported the promotion of adenomyosis by estrogen. For example, Koike et al . [ 150 ] exposed pregnant mice (ICR/Jcl, CLEA) to 0.01 mg ethinyl estradiol (EE2)/kg per day or vehicle (olive oil) by gavage from day 11 to 17 of gestation. Adult female offspring (F1 mice) were either treated with the same dose EE2 or to vehicle twice a week until 20 weeks of age. The control female offspring were also exposed to either vehicle of 0.01 mg EE2. All mice were euthanized at 7 months. Adenomyosis was common in in the EE2 -exposed uteri, and incidence of ectopic glands and serous cysts were significantly increased in the prenatally EE2 -exposed ovaries as compared with respective controls. This study suggests that continuous EE2 exposure can promote the development of adenomyosis. Using a very different model, Otto et al ., [ 151 ] also demonstrated an important role of estrogen in the development of adenomyosis. This study examined the influence of the estrogen modulating compounds Faslodex and cetrorelix in SHN mice. SHN mice have been found to develop adenomyosis after pituitary grafting, which leads to an altered endocrine profile. The GnRH antagonist cetrorelix and the estrogen receptor antagonist Faslodex, which negatively interfered with estrogen-mediated signaling, completely inhibited development of adenoymosis, whereas danazol, was slightly less effective in inhibiting disease in SHN mice [ 151 ]. Abdominal adhesions are fibrous bands of scar tissue that fuse two or more abdominal organs to each other and/or the peritoneum. Although adhesions most commonly occur as a complication of abdominal surgery; de novo formation may also occur in the absence of surgery as a consequence of inflammatory conditions within the abdomen [ 152 ]. For this reason, it is perhaps not surprising that compared to the general population; women with endometriosis are at a higher risk of both surgery-associated adhesions and spontaneous development of adhesive disease [ 153 ]. The basic pathophysiology of postsurgical adhesion development is known to involve inflammatory processes that occur during normal wound healing. Certainly, macrophages and neutrophils play key roles in the initiation of inflammation related to wound healing by releasing both proinflammatory cytokines and proangiogenic factors. Women with endometriosis are known to exhibit dysregulated immune cell function, which likely contributes to an enhanced inflammatory response and increased risk of developing adhesive disease [ 154 ]. In our chimeric nude mouse model, we examined the development of adhesive disease in association with experimental endometriosis [ 154 , 155 ]. As reported in our published studies, we noted a cooperative effect of the presence of endometrial tissue fragments on the development of post-surgical adhesions when tissue fragments were introduced into the peritoneum within 16 hours of ovariectomy. Sham surgery (removal of the fat pad surrounding ovary, but not the ovary) or saline injection in the absence of human tissue was not associated with increased adhesive disease [ 154 , 155 ]. Thus, within the peritoneal cavity, immune cell responses to injury, similar to infection or radiation therapy, may promote formation of adhesions [ 156 ]. Interestingly, we found that adhesions did not form when an identical experiment was conducted in rag2γ(c) mice (Table 4 ), animals which are more severely immunocompromised compared to nude mice. Nude mice exhibit loss of T cell function, but other immune cells are functional. In contrast, rag2γ(c) mice lack functional receptors for multiple interleukins, including IL-2, IL-4, IL-7, IL-9, and IL-15. As a consequence, B and T cell maturation are both compromised. Further, rag2γ(c) mice lack functional NK cells. An absence of adhesions in these animals suggests an additional role of one or more of these cytokines and/or immune cell types in post-surgical adhesion development. Interestingly, enhanced expression of IL-4 and IL-15 has been observed in patients with endometriosis [ 157 , 158 ], raising the possibility that these cytokines might contribute to de novo adhesions in these women. In studies using our developmental TCDD exposure model, we made the incidental observation that adhesions were rare in control mice at necropsy, but commonly observed F1-F3 mice. Therefore, in order to prospectively determine whether developmental TCDD exposure promotes the development of endometriosis-associated adhesive disease, we established a syngeneic endometriosis model in control and F1 mice within 16 hours of a surgical ovariectomy similar to the approach used with the chimeric adhesion model described above [ 154 , 155 ]. These studies revealed that 100% of F1 mice developed adhesive disease following ovariectomy and injection of uterine fragments, regardless of the origin of the tissue (control or F1 uterus). In contrast, only 30% of control mice subjected to ovariectomy and uterine tissue injection (control or F1 tissue) developed adhesions [ 59 ]. These data further suggest that alterations in inflammatory responses within the peritoneal environment may be the most important driving force in the development of post-surgical adhesive disease. Endometriosis-associated pain is a significant contributor to morbidity; however, studies correlating pain to extent or location of disease has not yielded advances in treatment (reviewed by [ 159 ]). Significantly, recent studies have illustrated that women with endometriosis exhibit “central sensitization”, a phenomenon by which pain itself promotes sensitivity to subsequent painful stimuli. Over time, a patient develops significant sensitivity to any stimuli, feeling more pain with less stimulation. Ultimately, even stimuli which should not be painful ( i.e . a light touch) can trigger pain as a consequence of central sensitization [ 160 , 161 ]. Central sensitization is particularly refractory to treatment; thus, animal models which can advance our understanding of this condition are desperately needed. Using the rat surgical model described above, Berkley and colleagues have demonstrated that ectopic endometrial tissues become innervated and lead to vaginal hyperalgesia [ 48 ]. Significantly, inflammation appears to be a key player in the development of innervated lesions [ 160 ]; thus several groups are actively using various rodent models to test whether blocking the actions of the proinflammatory agent prostaglandin E2 can reduce experimental disease as well as associated pain [ 34 , 162 ]. For example, using the chimeric rag2γ(c) mouse model, Arosh et al . [ 162 ] demonstrated that selective inhibition of EP2/EP4 not only inhibited survival of ectopic lesions, but also inhibited innervation of lesions and suppressed inflammation of dorsal root ganglia neurons. Significantly, following treatment with the EP2/EP4 inhibitors, animals in this study exhibited a reduction in pelvic pain, as assessed by stimulating the pelvic floor with von-Frey filaments (a standard nociception assay [ 163 ]).

Despite a century of research, the pathogenesis of endometriosis remains poorly understood. Nevertheless, the use of a variety of rodent models of experimental endometriosis has enabled significant advancement in our understanding of disease processes necessary for development of this disease. Although these models have limitations and cannot completely recapitulate the human disease process, studies described herein and in other recent reviews [ 33 , 34 , 164 , 165 ] demonstrate the significant contribution of rodent models in providing a broader understanding of the complex and interactive roles of the endometrial phenotype, the peritoneal microenvironment, host angiogenic responses and the competency of the immune system which collectively determine an individual’s risk of developing endometriosis. Examination of inflammatory events within the peritoneal cavity in concert with identifying genetic and epigenetic alterations associated with endometriosis is expected to further enhance our understanding of why only certain women develop this condition. Clearly, developing new and more effective therapies will require a more complete understanding of the mechanisms which promote this disease. Furthermore, identifying specific factors (phenotypic, genetic and/or epigenetic) which predispose an at risk individual to the development of endometriosis is critical to ultimately preventing the development of this disease as well as its many comorbidities.