Introduction to Preclinical Evidence from Animal Models of Endometriosis.

Endometriosis, the presence and growth of uterine endometrial glandular epithelial and stroma cells outside the uterine cavity, causes pain and infertility in women and girls of reproductive age. As randomized, double-blinded, controlled studies of endometriosis in women are impractical and at times ethically prohibitive, animal models for endometriosis arose as an important adjunct to gain mechanistic insights into the etiology and pathophysiological mechanisms of this perplexing disorder. A more thorough understanding of endometriosis in women may help develop novel noninvasive diagnostics, classification systems, therapeutic regimes, and even preventative methods for the management of endometriosis. This chapter is intended to introduce a brief historical background, biological and epidemiological aspects, the major symptoms, the effects of endocrine-disrupting chemicals, and an example of an epigenetic factor of endometriosis in women.

Identifying Mechanisms of Endometriosis-Associated Reduced Fecundity in a Rat Model: Novel Insights toward Understanding Human Infertility.

The existence of endometriosis has been known since at least the nineteenth century, yet the lack of understanding of causes of infertility and therefore inadequate treatment approaches in endometriosis creates a significant challenge in reproductive medicine. Women worldwide suffer not only pain and infertility but also economical, societal, and physiological burdens. Studies of reproductive events in women are difficult to conduct due to a host of confounding personal and environmental factors and ethically limited due to the very nature of working with reproductive tissues and cells, especially embryos. Animal models are a viable adjunct to study mechanisms causing human reproductive anomalies and infertility in endometriosis. This chapter discusses reproductive anomalies causing infertility in endometriosis and well-established animal models which help decipher the problems and lead to heretofore unknown nonsurgical, nonhormonal methods to manage endometriosis in women. In addition, studies of effects of developmental exposure to endometriosis are revealing for the first time, in both female and male offspring, transgenerational subfertility in a rat model providing insights into the familial nature of endometriosis and possible epigenetic involvement.

World Endometriosis Society consensus on the classification of endometriosis.

STUDY QUESTION: What is the global consensus on the classification of endometriosis that considers the views of women with endometriosis? SUMMARY ANSWER: We have produced an international consensus statement on the classification of endometriosis through systematic appraisal of evidence and a consensus process that included representatives of national and international, medical and non-medical societies, patient organizations, and companies with an interest in endometriosis. WHAT IS KNOWN ALREADY: Classification systems of endometriosis, developed by several professional organizations, traditionally have been based on lesion appearance, pelvic adhesions, and anatomic location of disease. One system predicts fertility outcome and none predicts pelvic pain, response to medications, disease recurrence, risks for associated disorders, quality of life measures, and other endpoints important to women and health care providers for guiding appropriate therapeutic options and prognosis. STUDY DESIGN, SIZE, DURATION: A consensus meeting, in conjunction with pre- and post-meeting processes, was undertaken. PARTICIPANTS/MATERIALS, SETTING, METHODS: A consensus meeting was held on 30 April 2014 in conjunction with the World Endometriosis Society's 12th World Congress on Endometriosis. Rigorous pre- and post-meeting processes, involving 55 representatives of 29 national and international, medical and non-medical organizations from a range of disciplines, led to this consensus statement. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 28 consensus statements were made. Of all, 10 statements had unanimous consensus, however none of the statements was made without expression of a caveat about the strength of the statement or the statement itself. Two statements did not achieve majority consensus. The statements covered women's priorities, aspects of classification, impact of low resources, as well as all the major classification systems for endometriosis. Until better classification systems are developed, we propose a classification toolbox (that includes the revised American Society for Reproductive Medicine and, where appropriate, the Enzian and Endometriosis Fertility Index staging systems), that may be used by all surgeons in each case of surgery undertaken for women with endometriosis. We also propose wider use of the World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonisation Project surgical and clinical data collection tools for research to improve classification of endometriosis in the future, of particular relevance when surgery is not undertaken. LIMITATIONS, REASONS FOR CAUTION: This consensus process differed from that of formal guideline development, although based on the same available evidence. A different group of international experts from those participating in this process may have yielded subtly different consensus statements. WIDER IMPLICATIONS OF THE FINDINGS: This is the first time that a large, global, consortium-representing 29 major stake-holding organizations, from 19 countries - has convened to systematically evaluate the best available evidence on the classification of endometriosis and reach consensus. In addition to 21 international medical organizations and companies, representatives from eight national endometriosis organizations were involved, including lay support groups, thus generating and including input from women who suffer from endometriosis in an endeavour to keep uppermost the goal of optimizing quality of life for women with endometriosis. STUDY FUNDING/COMPETING INTERESTS: The World Endometriosis Society convened and hosted the consensus meeting. Financial support for participants to attend the meeting was provided by the organizations that they represented. There was no other specific funding for this consensus process. Mauricio Abrao is an advisor to Bayer Pharma, and a consultant to AbbVie and AstraZeneca; G David Adamson is the Owner of Advanced Reproductive Care Inc and Ziva and a consultant to Bayer Pharma, Ferring, and AbbVie; Deborah Bush has received travel grants from Fisher & Paykel Healthcare and Bayer Pharmaceuticals; Linda Giudice is a consultant to AbbVie, Juniper Pharmaceutical, and NextGen Jane, holds research grant from the NIH, is site PI on a clinical trial sponsored by Bayer, and is a shareholder in Merck and Pfizer; Lone Hummelshoj is an unpaid consultant to AbbVie; Neil Johnson has received conference expenses from Bayer Pharma, Merck-Serono, and MSD, research funding from AbbVie, and is a consultant to Vifor Pharma and Guerbet; Jörg Keckstein has received a travel grant from AbbVie; Ludwig Kiesel is a consultant to Bayer Pharma, AbbVie, AstraZeneca, Gedeon Richter, and Shionogi, and holds a research grant from Bayer Pharma; Luk Rombauts is an advisor to MSD, Merck Serono, and Ferring, and a shareholder in Monash IVF. The following have declared that they have nothing to disclose: Kathy Sharpe Timms; Rulla Tamimi; Hugh Taylor. TRIAL REGISTRATION NUMBER: N/A.

TIMP1 contributes to ovarian anomalies in both an MMP-dependent and -independent manner in a rat model.

Ovulatory dysfunction occurs in women with endometriosis, yet the mechanisms are unknown. We have shown that endometriotic lesions synthesize and secrete tissue inhibitor of metalloproteinase (TIMP) 1 into the peritoneal cavity in humans and a rat model of endometriosis, where excess TIMP1 localizes in the ovarian theca in endometriosis and modulating peritoneal TIMP1 alters ovarian dynamics. Here, we evaluated whether mechanisms whereby excessive peritoneal fluid TIMP1 negatively impacts ovarian function are matrix metalloproteinase (MMP)-dependent and/or MMP-independent actions. Rats were treated with a mutated TIMP1 without MMP inhibitory function (Ala-TIMP1), wild-type TIMP1 (rTIMP1), or PBS. Rats treated with Ala-TIMP1 or rTIMP1 had fewer antral follicles, fewer new corpora lutea, and the presence of luteinized unruptured follicle syndrome compared with PBS rats. Ala-TIMP1 and rTIMP1 differentially caused downstream changes in gene expression and protein localization related to ovulation, as measured by whole-genome microarray with quantitative real-time PCR validation and immunohistochemistry. More vascular endothelial growth factor and FN were expressed and localized in ovaries of Ala-TIMP1-treated rats compared to rTIMP1- and PBS-treated rats inferring MMP-independent functions. Less caspase 3 localized in ovaries of rTIMP1 compared with the other two groups, and was thus dependent on MMP action. Furthermore, after coimmunoprecipitation, more CD63 was bound to TIMP1 in ovaries of rats treated with Ala-TIMP1 than in rTIMP1-treated rats, providing evidence for another MMP-independent mechanism of ovulatory dysfunction. We predict that MMP-dependent and MMP-independent events are involved in improper fortification of the follicular wall through multiple mechanisms, such as apoptosis inhibition, extracellular matrix components and angiogenesis. Collectively, excessive peritoneal TIMP1 causes changes in ovarian dynamics, both dependently and independently of MMP inhibition.

Cellular and molecular basis for endometriosis-associated infertility.

Endometriosis is a gynecological disease characterized by the presence of endometrial glandular epithelial and stromal cells growing in the extra-uterine environment. The disease afflicts 10%-15% of menstruating women causing debilitating pain and infertility. Endometriosis appears to affect every part of a woman's reproductive system including ovarian function, oocyte quality, embryo development and implantation, uterine function and the endocrine system choreographing the reproductive process and results in infertility or spontaneous pregnancy loss. Current treatments are laden with menopausal-like side effects and many cause cessation or chemical alteration of the reproductive cycle, neither of which is conducive to achieving a pregnancy. However, despite the prevalence, physical and psychological tolls and health care costs, a cure for endometriosis has not yet been found. We hypothesize that endometriosis causes infertility via multifaceted mechanisms that are intricately interwoven thereby contributing to our lack of understanding of this disease process. Identifying and understanding the cellular and molecular mechanisms responsible for endometriosis-associated infertility might help unravel the confounding multiplicities of infertility and provide insights into novel therapeutic approaches and potentially curative treatments for endometriosis.

Neutralizing TIMP1 restores fecundity in a rat model of endometriosis and treating control rats with TIMP1 causes anomalies in ovarian function and embryo development.

Human and rat endometriotic lesions synthesize and secrete tissue inhibitor of metalloproteinase 1 (TIMP1). More TIMP1 localizes in the ovarian theca in an established rat model for endometriosis (Endo) when compared to surgical controls (Sham). We hypothesized that endometriotic TIMP1 secreted into peritoneal fluid (PF) negatively affects ovarian function and embryogenesis by altering the balance of matrix metalloproteinases (MMPs) and TIMPs. Three experiments were performed modulating TIMP1 in vitro and in vivo to investigate ovarian and embryonic anomalies. The first experiment demonstrated control embryos treated in vitro with endometriotic PF concentrations of TIMP1 developed abnormally. In the second experiment where TIMP1 was modulated in vivo, TIMP1-treated Sham rats had fewer zygotes, ovarian follicles, and corpora lutea (CLs) and poorer embryo quality and development, which is analogous to the findings in Endo rats. Importantly, Endo rats treated with a TIMP1 function-blocking antibody had zygote, follicle, and CL numbers and embryo quality similar to Sham rats. In addition, more TIMP1 inhibitory activity was found in ovaries from Endo and TIMP1-treated Sham rats than in ovaries from Sham or TIMP1 antibody-treated Endo rats. In experiment three, control rats (no surgery) treated with Endo PF had fewer follicles and CLs and increased TIMP1 localization in the ovarian theca whereas treatment with Endo PF stripped of TIMP1 or with Sham PF had no effect, providing further evidence that endometriotic TIMP1 sequesters in the ovary and inhibits MMPs necessary for ovulation. Collectively, these results showed that excessive TIMP1 was deleterious to ovulation and embryo development. Thus, novel TIMP1-modulating therapies may be developed to alleviate infertility in women with endometriosis.

Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media.

OBJECTIVE: Validate single versus sequential culture media for murine embryo development. DESIGN: Prospective laboratory experiment. SETTING: Assisted Reproduction Laboratory. ANIMALS: Murine embryos. INTERVENTIONS: Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. MAIN OUTCOME MEASURES: On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. RESULTS: Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts. CONCLUSIONS: Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed.

Using rats as a research model for the study of endometriosis.

Although there are disadvantages of extrapolating data across species, the rat model may be used to study events involved in the pathogenesis and pathophysiologies of endometriosis or novel therapeutic approaches for this disorder that are not accessible in humans. Rat endometriotic tissues are similar to human lesions in vivo, and rat endometriotic tissues and cells perform in a similar manner as human endometriotic tissues and cells in organ explant culture and isolated cell culture. The rat model permits studies of mechanisms and regulators in a controlled manner free from confounding influences such as individual patient variation and environmental influences. The primary method used for induction of endometriosis in rats has been autotransplantation of uterine squares (implants) into the peritoneal cavity. Beyond mere growth of endometrium in ectopic locations, rats with endometriosis display similar symptoms, including a reduction in fertility and fecundity, and the endometriotic implants react similarly to therapeutics as those of humans with the disease. Similar alterations in gene expression and protein production have been observed in endometriotic tissues from rats and humans that may, in part, be causative agents involved in the pathogenesis or pathophysiologies of endometriosis.

Paracrine regulation of matrix metalloproteinase expression in endometriosis.

Following retrograde menstruation, shed endometrial tissue fragments attach to and invade the peritoneal surface to form established endometriotic lesions. With disease progression, the biochemically active lesions undergo remodeling and become fibrotic. Matrix metalloproteinase enzymes (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) play a significant role in normal endometrial remodeling during menses. Anomalous expression of MMPs and TIMPs has been identified in endometriotic lesions as compared to their highly regulated expression in eutopic endometrium. The paracrine mechanisms regulating misexpression of MMPs and TIMPs by endometriotic lesions are, however, not well defined. Misexpression of the MMPs and TIMPs may be due to innate anomalies in the eutopic endometrium from women with endometriosis, in the resident immune cells and peritoneal cells that juxtapose the ectopic endometrium, and/or numerous substances present in peritoneal fluid of women with endometriosis. The majority of MMPs are under strict transcriptional regulation. Steroid hormones and cytokines appear to act on the MMP promoter, either independently or in consort, to provide both positive and negative regulation of these genes. Misregulated expression of MMPs and TIMPs is associated with a more aggressive phenotype and a cascade of events facilitating peritoneal extracellular matrix degradation and establishment or remodeling of endometriotic lesions. The mechanisms by which MMP and TIMP expression are misregulated warrant further investigation as such information may provide insight into novel therapeutic modalities for endometriosis.

Understanding endometriosis is the key to successful therapeutic management.

Understanding endometriosis on a molecular, biochemical, and genetic level will lead to better methods for the diagnosis and therapeutic management of this enigmatic disease.

Endometriotic haptoglobin binds to peritoneal macrophages and alters their function in women with endometriosis.

OBJECTIVE: To evaluate the effects of endometriotic haptoglobin on peritoneal macrophage function. DESIGN: Prospective laboratory study. SETTING: School of medicine. PATIENT(S): Twenty-three women with and without endometriosis. INTERVENTION(S): Peritoneal macrophages cultured without or with haptoglobin. MAIN OUTCOME MEASURE(S): Peritoneal macrophage haptoglobin immunoreactivity, adhesion, and interleukin-6 (IL-6) production. RESULT(S): In vivo, significantly more peritoneal macrophages from women with endometriosis bound haptoglobin and exhibited reduced adhesion compared to women without endometriosis. In vitro, haptoglobin treatment significantly decreased peritoneal macrophage adherence only in women without endometriosis; this effect was not seen in women with endometriosis, probably owing to in vivo haptoglobin saturation. Conversely, haptoglobin treatment robustly increased IL-6 production only by macrophages from women with endometriosis, suggesting differential immune response in these women. CONCLUSION(S): Endometriotic lesions synthesize and secrete a unique form of haptoglobin (endometriosis protein-I) that is up-regulated by IL-6. This study shows that haptoglobin adheres to peritoneal macrophages; decreases adhesion, which may influence phagocytic function; and up-regulates IL-6 production. Hence, a feed-forward loop is proposed whereby endometriotic lesion haptoglobin decreases macrophage phagocytic function while increasing IL-6 production, which in turn increases endometriotic haptoglobin and promotes establishment of endometriosis.

Expression of soluble adhesion molecules in sera of women with stage III and IV endometriosis.

OBJECTIVE: To determine the effect of stage III and IV endometriosis on serum concentration of soluble intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), and E-selectin. METHODS: Sera from women with laparoscopically proven stage III and IV endometriosis were analyzed for the presence of soluble ICAM-1, VCAM-1, and E-selectin using enzyme-linked immunosorbent assays. Concentrations of these molecules were compared with those of laparoscopically proven disease-free controls. RESULTS: When compared with controls, women with stage III and IV endometriosis had higher serum concentrations of soluble VCAM-1, lower serum concentration of soluble ICAM-1, and no difference in serum concentration of soluble E-selectin. CONCLUSION: Significant aberrations in levels of circulating adhesion molecules were found in women with stage III and IV endometriosis. These findings might shed light on the pathogenesis of endometriosis and be useful in the development of biochemical markers for disease stage.

Developmental exposure of fetal ovaries and fetal germ cells to endometriosis in an endometriosis model causes differential gene expression in the preimplantation embryos of the first-generation and second-generation embryos.

OBJECTIVE: To characterize multigenerational gene expression anomalies in eight-cell stage embryos associated with developmental exposure to endometriosis. DESIGN: Using an endometriosis model in rats (F0 founder generation) to evaluate gene expression in F1 (fetal exposure) and F2 (fetal germ cell exposure) generation eight-cell stage embryos. SETTING: Laboratory. ANIMAL(S): Endometriosis model in rats (Endo) and controls (Sham). INTERVENTION(S): F0 Endo and Sham rats were bred; half the pregnant rats were killed on gestational day 3 to collect F1 eight-cell stage embryos and the others gestated to term (F1 females). Adult F1 females bred; F2 eight-cell embryos collected. MAIN OUTCOME MEASURE(S): Maintenance of differential gene expression in F1 and F2 generation eight-cell embryos in endometriosis. RESULT(S): Developmental exposure to endometriosis altered the gene signaling pathways, with changes found in apoptosis, the cell cycle process, the response to oxidative stress, negative regulation of molecular function, and RNA processing. The apoptotic genes Diablo, Casp3, Parp1, Cad, and Dnaja3 were increased and the Nfkbia transcripts were decreased in F1 Endo versus F1 Sham embryos. In F2 Endo versus Sham embryos, Casp3 and Cad were statistically significantly increased, and Parp1 and Nfkbia tended to be elevated. CONCLUSION(S): Fetal and germ cell exposure to endometriosis alters apoptotic gene expression in first- and second-generation eight-cell stage embryos, supporting the hypothesis of multigenerational inheritance resulting from exposure to endometriosis in utero.

Elevated peritoneal fluid TNF-α incites ovarian early growth response factor 1 expression and downstream protease mediators: a correlation with ovulatory dysfunction in endometriosis.

Endometriosis-associated infertility manifests itself via multiple, poorly understood mechanisms. Our goal was to characterize signaling pathways, between peritoneal endometriotic lesions and the ovary, leading to failed ovulation. Genome-wide microarray analysis comparing ovarian tissue from an in vivo endometriosis model in the rat (Endo) with controls (Sham) identified 22 differentially expressed genes, including transiently expressed early growth response factor 1 (Egr1). The Egr1 regulates gene requisites for ovulation. The Egr1 promoter is responsive to tumor necrosis factor-alpha (TNF-α) signaling. We hypothesized that altered expression of ovarian EGR1 is induced by elevated peritoneal fluid TNF-α which is upregulated by the presence of peritoneal endometriosis. Endo rats, compared to controls, had more peritoneal fluid TNF-α and quantitative, spatial differences in Egr1 mRNA and EGR1 protein localization in follicular compartments. Interactions between elevated peritoneal fluid TNF-α and overexpression of follicular Egr1/EGR1 expression may affect downstream protease pathways impeding ovulation in endometriosis. Preliminary studies identified similar patterns of EGR1 protein localization in human ovaries from women with endometriosis and compared to those without endometriosis.

Mouse model of surgically-induced endometriosis by auto-transplantation of uterine tissue.

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat and later transferred to the mouse. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system, hormones and environmental factors affect endometriosis as well as the effects of endometriosis on fertility and pain.

Haptoglobin expression in endometrioid adenocarcinoma of the uterus.

OBJECTIVE: Elevated serum haptoglobin (Hp) concentrations have been reported in patients with malignant diseases. We have shown that Hp is produced by and localizes only in the stroma and not the epithelium of endometriotic lesions, which share many characteristics of carcinoma. Furthermore, Hp mRNA and protein are found exclusively in the stroma of eutopic endometrium from women with endometriosis and not those without endometriosis. We hypothesized that characteristic patterns of Hp gene expression and protein localization in endometrioid adenocarcinoma of the uterus may provide insight into the clinical utility of Hp as a tumor marker or alternative therapeutic approach. METHODS: Biopsies of endometrioid adenocarcinoma tumors of the uterus and their adjacent nonaffected endometrium were collected. Normal endometrium was collected from healthy women. Haptoglobin messenger RNA (mRNA) levels were quantified by quantitative polymerase chain reaction (Q-PCR). Haptoglobin protein cell-specific localization was identified by immunohistochemistry. RESULTS: Haptoglobin mRNA levels were significantly greater (P < .005) in endometrioid adenocarcinoma and adjacent nonaffected endometrial tissues than normal endometrium. No correlation was found between Hp levels and cancer stage (P = .673) or grade (P = .739). Haptoglobin protein localized in both stromal and glandular epithelial cells of endometrioid adenocarcinoma and their adjacent nonaffected tissue but not in control endometrium. CONCLUSIONS: Our results have identified, for the first time, unique patterns of Hp mRNA expression and protein localization in the stromal and glandular epithelial cells of endometrioid adenocarcinoma of the uterus. We propose that this unique pattern of endometrioid adenocarcinoma Hp expression may be developed as a novel diagnostic marker. Modulation of Hp, with its immunomodulatory and angiogenic properties, may generate novel methods of prevention or treatment for endometrial cancer.

Aberrant gene expression profile in a mouse model of endometriosis mirrors that observed in women.

OBJECTIVE: To define the altered gene expression profile of endometriotic lesions in a mouse model of surgically induced endometriosis. DESIGN: Autologous experimental mouse model. SETTING: Medical school department. ANIMAL(S): Adult C57Bl6 mice. INTERVENTION(S): Endometriosis was surgically induced by autotransplantation of uterine tissue to the intestinal mesentery. Endometriotic lesions and eutopic uteri were recovered at 3 or 29 days after induction. MAIN OUTCOME MEASURE(S): Altered gene expression was measured in the endometriotic lesion relative to the eutopic uterus by genome-wide complementary DNA microarray analysis and was confirmed by real-time reverse transcriptase-polymerase chain reaction for six genes. Relevant categories of altered genes were identified using gene ontology analysis to determine groups of genes enriched for altered expression. RESULT(S): The expression of 479 and 114 genes was altered in the endometriotic lesion compared with the eutopic uterus at 3 or 29 days after induction, respectively. Gene ontology enrichment analysis revealed that genes associated with the extracellular matrix, cell adhesions, immune function, cell growth, and angiogenesis were altered in the endometriotic lesion compared with the eutopic uterus. CONCLUSION(S): According to gene expression analysis, the mouse model of surgically induced endometriosis is a good model for studying the pathophysiology and treatment of endometriosis.

Reduced fecundity in female rats with surgically induced endometriosis and in their daughters: a potential role for tissue inhibitors of metalloproteinase 1.

The cause of reduced fecundity in women with endometriosis is unknown. Expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by both ectopic and eutopic endometrium reportedly has a role in the pathogenesis of endometriosis. We hypothesize that anomalous endometriotic TIMP protein synthesis, secretion, and localization also cause reproductive pathologies resulting in reduced fecundity. An established rat model for endometriosis (Endo) compared with nonendometriotic controls (Shams) was used to investigate reduced fecundity in endometriosis. Comparing Endo and Sham rats, Endo rats had altered ovarian dynamics, including fewer ovarian follicles and corpora lutea with luteinized unruptured follicles. Furthermore, in vivo anomalies in postovulatory oocyte structure and preimplantation embryo development, including misaligned chromosomes, nuclear and cytoplasmic fragmentation, and delayed or arrested cleavage, as well as spontaneous abortions, were found only in Endo rats. A causative role for TIMP1 in these phenomena is supported by our findings that Endo rats have more TIMP1 in their peritoneal fluid as detected by ELISA and more TIMP1 immunolocalization in the theca of antral follicles as measured by computer-assisted morphometric analysis. These data suggest that in endometriosis the accumulation of TIMP1 disrupts the normal MMP/TIMP enzymatic milieu in the peritoneal cavity and negatively affects ovarian dynamics, oocyte quality, and preimplantation embryo development, thereby decreasing fecundity. Most intriguingly, daughters of Endo rats that had no experimental interventions exhibited these same reproductive abnormalities. We predict that developmental exposure to endometriosis leads to permanent epigenetic changes in subsequent generations.

Decidualization regulates the expression of the endometrial chorionic gonadotropin receptor in the primate.

Chorionic gonadotropin (CG) plays an important role in establishing a receptive endometrium by directly modulating the function of both endometrial stromal and epithelial cells in the baboon. The focus of this study was to characterize changes in CG receptor (LHCGR, also known as CG-R) expression during the menstrual cycle and early pregnancy, particularly during decidualization. LHCGR was localized by using a peptide-specific antibody generated against the extracellular domain. Immunostaining was absent in any of the cell types during the proliferative phase of the cycle. In contrast, during the secretory phase, both luminal and glandular epithelial cells stained positively. Stromal staining was confined to the cells around spiral arteries (SAs) and in the basalis layer. This stromal staining pattern persisted at the implantation site between Days 18 and 25 of pregnancy and after CG infusion. However, as pregnancy progressed (Days 40 to 60), staining for LHCGR was dramatically decreased in the stromal cells. These data were confirmed by nonisotopic in situ hybridization. To confirm whether the loss of LHCGR was associated with a decidual response, stromal fibroblasts were decidualized in vitro, and cell lysates obtained after 3, 6, and 12 days of culture were analyzed by Western blotting. LHCGR protein decreased with the onset of decidualization in vitro, confirming the in vivo results. Addition of CG to decidualized cells resulted in the reinduction of LHCGR in the absence of dbcAMP. We propose that CG acting via its R on stromal cells modulates SA in preparation for pregnancy and trophoblast invasion. As pregnancy progresses, further modification of SA by migrating endovascular trophoblasts and subsequent decidualization results in the downregulation of LHCGR. This inhibition of LHCGR expression also coincides with the decrease of measurable CG in peripheral circulation.

The interplay of cell-cell and cell-matrix interactions in the invasive properties of brain tumors.

Impairment of tissue cohesion and the reorganization of the extracellular matrix are crucial events during the progression toward invasive cell phenotype. We studied the in vitro invasion patterns of nine brain tumor cell lines in three-dimensional collagen gels. Cell-cell and cell-matrix interactions were quantified and correlated with the expression level of specific molecules: N-cadherin, matrix metalloproteinases, and their inhibitor. Pattern evolution was studied as a function of time and collagen concentration. Cells with low metalloproteinase expression or high tissue cohesion showed limited invasive potential. Higher metalloproteinase expression and intermediate tissue cohesion resulted in configurations with hypercellular zones surrounding regions mostly devoid of cells and with digested collagen, akin to pseudopalisades in surgically removed malignant astrocytoma specimens. In physical terms, these configurations arise as the result of competition between cell-cell and cell-matrix interactions. Our findings suggest specific ways to characterize, control, or engineer cell migratory patterns and hint at the importance of the interplay between biophysical and biomolecular factors in the characterization of invasive cell behavior and, more generally, in epithelial-mesenchymal transitions.