In a murine model, we have linked early life toxicant exposure to reduced uterine sensitivity to progesterone, a phenotype we had previously associated with inflammation in endometriosis patients. Subsequent studies revealed that developmental toxicant exposure not only reduces fertility in male and female mice but also negatively impacts pregnancy leading to spontaneous preterm birth (PTB). An epigenetic alteration of the progesterone receptor gene correlated with reduced fertility and adverse pregnancy outcomes and persisted in multiple generations of mice in the absence of an additional toxicant exposure. Gene-environment interactions in women may explain why some patients “at risk” for PTB deliver at term while others without known risks deliver early. Our model provides a unique system to unravel the interactive influences of inflammation and reduced progesterone responsiveness on PTB and suggests that therapy needs to begin prior to pregnancy (and involve both partners) rather than once the inflammatory cascade has been initiated.
Progesterone action normally mediates the balance between anti-inflammatory and pro-inflammatory processes throughout the female reproductive tract. However, in women with endometriosis, endometrial progesterone resistance, characterized by alterations in progesterone responsive gene and protein expression, is now considered a central element in disease pathophysiology. Recent studies additionally suggest that the peritoneal microenvironment of endometriosis patients exhibits altered physiological characteristics that may further promote inflammation-driven disease development and progression. Within this review, we summarize our current understanding of the pathogenesis of endometriosis with an emphasis on the role that inflammation plays in generating not only the progesterone-resistant eutopic endometrium but also a peritoneal microenvironment that may contribute significantly to disease establishment. Viewing endometriosis from the emerging perspective that a progesterone resistant endometrium and an immunologically compromised peritoneal microenvironment are biologically linked risk factors for disease development provides a novel mechanistic framework to identify new therapeutic targets for appropriate medical management.
Endometriosis; inflammation; progesterone resistance
To examine whether dietary fish oil supplementation reduces development of spontaneous endometriosis-associated adhesions using an established model.
Medical center research laboratory.
Disease-free women of reproductive age and nude mice.
Women were not provided any intervention. Mice were randomized to receive fish oil supplementation or control diet.
Main Outcome Measure(s)
Experimental endometriosis was established in mice via injection of human endometrial tissue within 16 hours of ovariectomy. Mice were provided standard or menhaden fish oil–supplemented diets for ≥2 weeks before initiation of experimental endometriosis and until killing them 1 week later. At necropsy, mice were examined for the presence and extent of adhesions and endometriotic-like lesions. Tissues were excised and morphologically characterized.
Adhesions/lesions were reduced in mice provided with dietary fish oil compared with control animals. Leukocytes were more numerous within the adhesions/lesions of the mice maintained on the standard diet compared with animals provided with fish oil. As indicated by staining intensity, collagen deposition was greater at adhesion sites within control mice compared with fish oil–supplemented animals.
Wound-healing associated with surgery created an inflammatory peritoneal microenvironment that promoted the development of both experimental endometriosis and adhesions in a murine model. Targeting excessive inflammation with fish oil may be an effective adjuvant therapy to reduce the development of postsurgical adhesions related to endometriosis.
Omega-3 fatty acids; adhesions; endometriosis; mice; inflammation
To examine the differentiation-related expression of CB1-R mRNA and protein in endometrial tissue obtained from women with and without endometriosis and to determine the impact of acute TCDD exposure on CB1-R gene expression in isolated endometrial stromal cells.
University-affiliated medical center
Women with and without endometriosis undergoing volunteer endometrial biopsies after informed consent.
Main Outcome Measures
Analysis of in vivo CB1-R mRNA and protein expression in human endometrial tissues and mRNA expression in isolated stromal cells following exposure to TCDD or a progesterone receptor antagonist (Onapristone).
CB1-R mRNA and protein expression was highest during the progesterone-dominated secretory phase in control women, while expression was minimal in endometrial tissues acquired from women with endometriosis, regardless of the cycle phase. Although progesterone was found to induce CB1-R mRNA expression in endometrial stromal cells from control donors, steroid-induced expression of this gene was inhibited by co-treatment with either TCDD or Onapristone.
Our studies reveal a role for the anti-inflammatory actions of progesterone in regulating endometrial cannabinoid signaling, which is disrupted in women with endometriosis. Significantly, our studies demonstrate, for the first time, that acute TCDD exposure disrupts cannabinoid signaling in the human endometrium.
Cannabinoid receptor CB1-R; progesterone; endometriosis; 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD;dioxin)
We recently reported that adult male C57BL/6 mice exposed in utero to the environmental toxicant TCDD confer an increased risk of preterm birth (PTB) to unexposed females. Risk of PTB was coincident with decreased placental progesterone receptor (PR) mRNA expression and increased toll-like receptor-4 (TLR-4) mRNA expression, suggesting toxicant exposure induced a heightened inflammatory response at the maternal-fetal interface. Since omega-3 fatty acids exhibit anti-inflammatory activity, herein, we provided TCDD-exposed males a fish oil-enriched diet prior to mating. Although PTB was common in control females mated to TCDD-exposed males on the standard diet, fish oil supplementation of TCDD-exposed males eliminated PTB in unexposed partners. We also determined the influence of preconception, paternal fish oil supplementation on the placental inflammatory response in late pregnancy (E18.5) by examining expression of PR and TLR-4 mRNA as well as expression of 15-hydroxy prostaglandin dehydrogenase (PGDH). PGDH catabolizes the inflammatory PGE2 to an inactive form; thus, reduced expression of this enzyme would promote tissue inflammation. Compared to control pregnancies, examination of E18.5 placentas arising from TCDD-exposed males on the standard diet revealed a significant increase in TLR-4 mRNA expression corresponding to a reduction in PR mRNA and PGDH protein expression. In contrast, fish oil supplementation of toxicant-exposed males led to normalization of placental expression of both PR and TLR-4 mRNA and a marked increase in PGDH expression. These studies suggest that a paternal preconception diet which includes omega-3 fatty acids prevents the toxicant-associated increase in the placental inflammatory response at late gestation, preventing PTB.
Laboratory and population-based studies suggest that exposure to environmental toxicants may be one of several triggers for the development of endometriosis. We discuss evidence that modulation of the endometrial endocrine-immune interface could mechanistically link toxicant exposure to the development of this disease.
Capsule Summary: Environmental toxicant exposure induces an inflammatory-like endometrial response that may promote the development of endometriosis.
endometriosis; dioxin; progesterone; inflammation; leukocytes
Recently we reported that statins, the competitive inhibitors of the key enzyme regulating the mevalonate pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), decrease proliferation of human endometrial stromal (HES) cells. Furthermore, we found that simvastatin treatment reduces the number and the size of endometrial implants in a nude mouse model of endometriosis. The present study was undertaken to investigate the effect of simvastatin on HES cell invasiveness and on expression of selected genes relevant to invasiveness: matrix metalloproteinase 2 (MMP2), MMP3, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), and CD44. Because statin-induced inhibition of HMGCR reduces the production of substrates for isoprenylation—geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)—the effects of GGPP and FPP were also evaluated. Simvastatin induced a concentration-dependent reduction of invasiveness of HES cells. This effect of simvastatin was abrogated by GGPP but not by FPP. Simvastatin also reduced the mRNA levels of MMP2, MMP3, and CD44, but increased TIMP2 mRNA; all these effects of simvastatin were partly or entirely reversed in the presence of GGPP. The present findings provide a novel mechanism of action of simvastatin on endometrial stroma that may explain reduction of endometriosis in animal models of this disease. Furthermore, the presently described effects of simvastatin are likely mediated, at least in part, by inhibition of geranylgeranylation.
Simvastatin decreases invasiveness of human endometrial stromal cells and decreases expression of matrix metalloproteinases 2 and 3 and CD44, while increasing expression of tissue inhibitor of matrix metalloproteinase 2.
CD44; endometriosis; human endometrial stromal cells; invasion assay; metalloproteinases; simvastatin; tissue inhibitor of metalloproteinases
Statins are potent inhibitors of the endogenous mevalonate pathway. Besides inhibiting
cholesterol biosynthesis, statins may also demonstrate anti-inflammatory properties.
Inflammation is implicated in the attachment and invasion of endometrial cells to the
peritoneal surface and growth of ectopic endometrium by inducing proliferation and
angiogenesis. In this study, the effect of statins on monocyte chemotactic protein 1
(MCP-1) expression in endometriotic implants in nude mouse model and in cultured
endometriotic cells was evaluated. In mouse model, simvastatin decreased MCP-1 expression
in a dose-dependent manner in endometriotic implants (P < .05).
Similarly, both simvastatin and mevastatin revealed a dose-dependent inhibition of MCP-1
production in cultured endometriotic cells (P < .01). This inhibitory
effect of the statins on MCP-1 production was reversed by the downstream substrates of the
mevalonate pathway. Moreover, statins decreased MCP-1 messenger RNA expression in cultured
endometriotic cells (P < .05). In conclusion, statins exert
anti-inflammatory effect in endometriotic cells and could provide a potential treatment of
endometriosis in the future.
endometriosis; statin; MCP-1; inflammation
Endometriosis is an often painful disorder in which the endometrial glands and stroma grow outside the uterus. The disease affects women’s quality of life and is a common cause of infertility. In this review, we describe promising new developments in the field based on in vitro assays and rodent models, each of which has the potential to be beneficial in the treatment of this disease. We will specifically describe the role of anti-inflammatory drugs, selective estrogen, or progesterone modulators, statins, antiangiogenic agents, and the potential for targeting stem cells as likely methods to hone in and eliminate endometriosis. The most promising of these potential therapies are currently slated for further testing in both rodent and nonhuman primate trials.
Endometriosis; ICON; tissue factor; statins; stem cells; SERM; SPRM
Endometriosis is a common, complex gynecologic disorder characterized by the presence of endometrial glands and stroma at extrauterine (ectopic) sites. In women who develop this disease, alterations in specific biological processes involving both the endocrine and immune systems have been observed, which may explain the survival and growth of displaced endometrial tissue in affected women. In the past decade, a considerable amount of research has implicated a role for alterations in progesterone action at both eutopic and ectopic sites of endometrial growth which may contribute to the excessive inflammation associated with progression of endometriosis; however, it remains unclear whether these anomalies induce the condition or are simply a consequence of the disease process. In this article, we summarize current knowledge of alterations within the immune system of endometriosis patients and discuss how endometrial cells from women with this disease not only have the capacity to escape immunosurveillance, but also use inflammatory mechanisms to promote their growth within the peritoneal cavity. Finally, we discuss evidence that exposure to an environmental endocrine disruptor, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, can mediate the development of an endometrial phenotype that exhibits both reduced progesterone responsiveness and hypersensitivity to proinflammatory stimuli mimicking the endometriosis phenotype. Future studies in women with endometriosis should consider whether a heightened inflammatory response within the peritoneal microenvironment contributes to the development and persistence of this disease.
chemokines; cytokines; dioxin; endocrine-disrupting chemicals; endometriosis; estrogen; immune–endocrine interactions; inflammation; peritoneum; progesterone; TCDD
TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a ubiquitous environmental contaminant and known endocrine disruptor. Since humans and animals are most sensitive to toxicant exposure during development, we previously developed a mouse model of in utero TCDD exposure in order to examine the impact of this toxicant on adult reproductive function. Our initial in utero toxicant-exposure study revealed a dose-dependent reduction in uterine sensitivity to progesterone; however, we did not previously explore establishment or maintenance of pregnancy. Thus, in the current study, we examined pregnancy outcomes in adult C57BL/6 mice with a history of developmental TCDD exposure. Herein we demonstrate reduced fertility and an increased incidence of premature birth (PTB) in F1 mice exposed in utero to TCDD as well as in three subsequent generations. Finally, our studies revealed that mice with a history of developmental TCDD exposure exhibit an increased sensitivity to inflammation which further negatively impacted gestation length in all generations examined.
TCDD; fertility; preterm birth; inflammation; progesterone receptor; development
We have previously described diminished uterine progesterone response and increased uterine sensitivity to inflammation in adult female mice with a history of developmental exposure to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin). Since parturition in mammals is an inflammatory process mediated in part by a decline in progesterone action, toxicant-mediated disruption of progesterone receptor (PR) expression at the maternal-fetal interface would likely impact the timing of birth. Therefore, in the current study, we examined pregnancy outcomes in adult female mice with a similar in utero exposure to TCDD. We also examined the impact of in utero TCDD exposure of male mice on pregnancy outcomes in unexposed females since the placenta, a largely paternally derived organ, plays a major role in the timing of normal parturition via inflammatory signaling. Our studies indicate that developmental exposure of either parent to TCDD is associated with preterm birth in a subsequent adult pregnancy due to altered PR expression and placental inflammation.
placenta; decidua; progesterone receptor; preterm birth; TCDD; mice; inflammation
To examine the impact of a recent surgery on development of endometriosis-related adhesions in a chimeric model and to determine the therapeutic efficacy of pioglitazone (PIO).
Human endometrial biopsies were maintained in estradiol (E) with or without PIO for 24 hrs prior to intraperitoneal injection into immunocompromised mice at multiple timepoints following peritoneal surgery. The presence and extent of adhesions was examined in animals relative to the initial establishment of experimental endometriosis.
Medical School Research Center
Endometrial biopsies for experimental studies described herein were provided by normally cycling women without a medical history indicative of endometriosis or adhesions.
Main Outcome Measure
Examination of the development of endometriosis-related adhesions in an experimental model.
Without therapeutic intervention, injection of E-treated human endometrial tissue into mice near the time of peritoneal surgery resulted in multiple adhesions and extensive endometriotic-like disease. In contrast, PIO treatment reduced adhesive disease and experimental endometriosis related to surgical injury.
The presence of human endometrial tissue fragments in the peritoneal cavity of mice with a recent surgical injury promoted development of both adhesive disease and experimental endometriosis. Targeting inflammation and angiogenesis with PIO therapy limited the development of postsurgical adhesions associated with ectopic endometrial growth.
Adhesions; Endometriosis; Surgery; Mice
Endometriosis is a common gynecologic disorder characterized by ectopic attachment and growth of endometrial tissues. Resveratrol is a natural polyphenol with antiproliferative and anti-inflammatory properties. Our objective was to study the effects of resveratrol on human endometriotic implants in a nude mouse model and to examine its impact on human endometrial stromal (HES) cell invasiveness in vitro. Human endometrial tissues were obtained from healthy donors. Endometriosis was established in oophorectomized nude mice by intraperitoneal injection of endometrial tissues. Mice were treated with 17β-estradiol (8 mg, silastic capsule implants) alone (n = 16) or with resveratrol (6 mg/mouse; n = 20) for 10–12 and 18–20 days beginning 1 day after tissue injection. Mice were killed and endometrial implants were evaluated. A Matrigel invasion assay was used to examine the effects of resveratrol on HES cells. We assessed number and size of endometriotic implants in vivo and Matrigel invasion in vitro. Resveratrol decreased the number of endometrial implants per mouse by 60% (P < 0.001) and the total volume of lesions per mouse by 80% (P < 0.001). Resveratrol (10–30 μM) also induced a concentration-dependent reduction of invasiveness of HES by up to 78% (P < 0.0001). Resveratrol inhibits development of endometriosis in the nude mouse and reduces invasiveness of HES cells. These observations may aid in the development of novel treatments of endometriosis.
Resveratrol administration decreases development of endometriotic implants in a nude mouse endometriosis model as well as invasiveness of human endometrial stromal cells in culture.
endometrial stroma; endometriosis; nude mouse; resveratrol
To develop a chimeric human/mouse model of experimental endometriosis for the examination of the role of human immune cells in this disease.
University-affiliated medical center.
Healthy women undergoing volunteer endometrial biopsies and blood donation.
Main outcome measure(s):
In vivo analysis of the impact of the adoptive transfer of human immune cells into immunocompromised mice receiving autologous human endometrium.
Similar to our previous data using nude mice, human endometrial tissue fragments injected intraperitoneally in rag2γ(c) mice readily established experimental disease. However, in the current study, we found a significant reduction in the severity of peritoneal disease in ragγ(c) mice readily established experimental disease. However, in the current study, we found a significant reduction in the severity of peritoneal disease in rag2γ(c) mice receiving adoptive transfer of human immune cells compared to mice which did not receive immune cells. Interestingly, human immune cells readily track into the ectopic lesions established in mice.
The ability of immune cells to limit intraperitoneal disease in mice suggests that a robust immune system is protective against the development of endometriosis.
Endometriosis; immune cells; immunocompromised mice
Development of endometriosis likely requires multiple, interactive mechanisms involving both the endocrine and immune systems. Environmental toxicants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are of particular interest as potential contributory agents in the development of this disease because they can disrupt both systems. Nevertheless, defining the potential role that environmental exposure to TCDD plays in the development of endometriosis requires a better understanding of how this toxicant affects the biological processes that promote the disease. Although the disease mechanism(s) responsible for progesterone resistance in the endometrium of endometriosis patients remains speculative, our studies indicate that developmental exposure of mice to TCDD leads to a progesterone-resistant phenotype in adult animals that can persist for several generations. These studies and others underscore the importance of developing a greater understanding of the mechanisms of TCDD action that relate to reproductive disorders such as endometriosis.
Endometriosis; TCDD; developmental exposure
A recent survey in the United States identified 287 different chemicals in human cord blood, demonstrating the significant exposure of women and their children to a wide array of environmental toxicants. While reducing contamination and exposure should be an international priority, it is equally appropriate to develop an understanding of the health consequences of increasing world-wide industrialization. Endometriosis, a disease of the female reproductive tract, has emerged as a disease potentially related to environmental exposures. While a number of population-based studies have suggested that a woman's exposure to dioxin-like polychlorinated biphenyls may affect her risk of developing this disease, other studies have failed to find such evidence. In the current manuscript, we will review the limited data regarding polychlorinated biphenyl congeners and endometriosis with a focus on dioxin-like toxicants. We will also discuss the potential importance of early life exposures to these toxicants on the subsequent development of endometriosis.
TCDD; PCB; endometriosis; epigenetics; developmental exposure
Whether environmental toxicants impact an individual woman’s risk for developing endometriosis remains uncertain. Although the growth of endometrial glands and stroma at extra-uterine sites is associated with retrograde menstruation, our studies suggest that reduced responsiveness to progesterone may increase the invasive capacity of endometrial tissue in women with endometriosis. Interestingly, our recent studies using isolated human endometrial cells in short-term culture suggest that experimental exposure to the environmental contaminant 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) can alter the expression of progesterone receptor isotypes. Compared to adult exposure, toxicant exposure during development can exert a significantly greater biological impact, potentially affecting the incidence of endometriosis in adults. To address this possibility, we exposed mice to TCDD at critical developmental time points and subsequently examined uterine progesterone receptor expression and steroid responsive transforming growth factor-β2 expression in adult animals. We find that the uterine phenotype of toxicant-exposed mice is markedly similarly to the endometrial phenotype of women with endometriosis.
Dioxin; TCDD; Progesterone; Progesterone receptor; TGF-β2; Endometrium; Endometriosis; Development; Fetal origin