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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 10 4782-4791
Copyright © 2002 by The Endocrine Society

Original Article

Steroid and Cytokine Regulation of Matrix Metalloproteinase Expression in Endometriosis and the Establishment of Experimental Endometriosis in Nude Mice

Kaylon L. Bruner-Tran, Esther Eisenberg, Grant R. Yeaman, Ted A. Anderson, Judith McBean and Kevin G. Osteen

Women’s Reproductive Health Research Center (K.L.B.-T., E.E., G.R.Y., T.A.A., K.G.O.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and University of Vermont (J.M.), Burlington, Vermont 05401

Address all correspondence and requests for reprints to: Kevin G. Osteen, Ph.D., Women’s Reproductive Health Research Center, B-1100 Medical Center North, Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville Tennessee 37232. E-mail: kevin.osteen{at}mcmail.vanderbilt.edu.

Abstract

The cyclic expression of matrix metalloproteinases (MMPs) by human endometrium has been suggested to play a role in the invasive process necessary to establish endometriosis. The ability of progesterone exposure to inhibit endometrial MMP-3 and MMP-7 expression requires the local action of TGFß and may also be linked to the local production of retinoic acid by stromal cells. A continuous expression of several MMPs in endometriotic lesions has been reported, indicating a failure of progesterone or locally produced factors to suppress these enzymes. To address cell-specific MMP regulation associated with endometriosis, we examined expression of MMP-3 and MMP-7 mRNA in eutopic endometrium and endometriotic lesions acquired during the secretory phase of the menstrual cycle. We examined the in vitro regulation of MMP-3 and MMP-7 protein in similar tissues. We also examined the in vitro regulation of MMP secretion by progesterone, retinoic acid, and TGFß in endometriosis tissues relative to the establishment of experimental disease. Our studies indicate that either eutopic or ectopic tissue from women with endometriosis exhibit patterns of altered MMP regulation in vivo. A lack of responsiveness to progesterone was demonstrated in vitro, associated with a failure to suppress MMP expression and an enhanced ability of the tissue to establish experimental endometriosis. However, in vitro treatments with retinoic acid and TGFß restored the ability of progesterone to suppress MMPs in vitro and prevented the establishment of experimental disease.

MATRIX DEGRADATION IS a normal component of reproductive tissue function and is associated with the selective expression and action of members of the matrix metalloproteinase (MMP) family (1). In the normal endometrium, numerous MMPs are cyclically expressed and appear to be regulated by changes in levels of ovarian estradiol and progesterone production that mediate distinct patterns of endometrial growth and differentiation across each menstrual cycle. For example, in an early study of MMP expression, mRNAs for MMP-1, MMP-3, and MMP-7 were found to be focally expressed following menstrual breakdown as the endometrium initiated a new cycle of growth and repair during the estrogen-dominant proliferative phase (2, 3). In contrast, after ovulation and the rise in serum levels of progesterone, expression of these MMPs declined and remained largely undetectable until steroid levels began to fall at the end of the cycle (2, 3). Specific tissue inhibitors of MMPs (TIMPs), including TIMP-1, TIMP-2, and TIMP-3, exhibit a low level of expression during the proliferative phase of the menstrual cycle but are more broadly expressed in response to rising levels of progesterone (3, 4). Additionally, recent studies have shown that progesterone limits the effects of proinflammatory cytokines, including IL-1{alpha}, IL-1ß, and TNF-{alpha} (5, 6, 7), which are potent stimulators of MMP expression during menstruation (4). Although the cellular and molecular mechanisms by which progesterone blocks proinflammatory cytokine action has not been elucidated, this steroid is critical to maintaining the appropriate balance between MMPs and TIMPs within the endometrium at the time of normal pregnancy. A failure to properly regulate endometrial MMPs disrupts normal endometrial function and has been associated not only with pregnancy failure but also with the development of uterine cancer (1, 4). Additionally, the growth of endometrial tissue outside the uterine corpus, associated with the disease endometriosis, has also been linked to the altered expression of members of the MMP family (7).

Endometriosis, simply defined as the presence of endometrial glands and stroma at an extrauterine site, remains a poorly understood and complex disease afflicting millions of women worldwide. Although the etiology and basic pathophysiology of endometriosis is controversial (8, 9), most practitioners agree that the disease can occur via the ectopic implantation of refluxed endometrial tissue at the time of menstruation (9, 10). Establishment of ectopic sites of endometrial growth is an invasive event requiring degradation of the extracellular matrix (11). Although the precise role of endometrial MMPs in the pathophysiology of endometriosis is not fully understood, several laboratories have independently implicated these enzymes in the establishment and progression of the disease (12, 13, 14). Although endometrial expression of the MMP family is normally tightly regulated during the menstrual cycle, altered patterns of MMPs and TIMP expression have been reported in eutopic and ectopic endometrial tissues obtained from patients with endometriosis. For example, MMP-1 and MMP-2 protein were found to be highly expressed in endometriotic tissues, compared with normal endometrium (15, 16), but expression of TIMP-1 and TIMP-2 proteins were significantly reduced in the diseased tissues (16). More recently, Chung et al. (17) demonstrated lower levels of TIMP-3 mRNA expression in both eutopic endometrium and endometriotic lesions, compared with disease-free women, but MMP-9 mRNA was increased only in the lesions. In this laboratory, we have recently identified expression of MMP-11 mRNA in the eutopic endometrium from women with endometriosis during the secretory phase (18) when its expression is absent in normal women (3).

The dysregulation of MMP and TIMP family members associated with endometriosis may provide a key mechanistic link to both the initial establishment of disease and its subsequent progression. Estrogen, the steroid most often linked to the development and recurrence of endometriosis (19, 20), is associated with endometrial expression of stromal and epithelial-specific MMPs. In contrast, progestin therapy and the progesterone-rich environment of pregnancy have each been demonstrated to have a beneficial impact on the disease in some women (21, 22). Recently, our laboratory more specifically linked the respective actions of estrogen and progesterone on endometrial MMP expression in human endometrium to the development or prevention of experimental endometriosis in nude mice (23). Nevertheless, a lack of predictable tissue differentiation in endometriotic implants is typically observed, perhaps as a result of defects in progesterone receptor expression and function, local estrogen production, or altered steroid metabolism (24, 25, 26, 27, 28).

Although the suppression of MMPs may be one of the benefits of progesterone, the variability of response to this steroid at endometriotic sites may limit the effectiveness of progestin-based therapy. However, a broad array of cytokines and growth factors are critical for the regulation of cell-cell communication within each functional region of the endometrium, acting in concert with the cyclic changes in tissue exposure to ovarian steroids (29, 30). For example, we have previously identified that endometrial exposure to progesterone induces local, cell-specific retinoic acid (RA) synthesis, which can enhance TGFß expression during the secretory phase of the menstrual cycle (31, 32, 33). These studies suggest that the local action of RA and TGFß is important for endometrial function at the time of pregnancy establishment, including the regulation of members of the MMP family (32, 34, 35). Previous studies from our laboratory indicated that the interactive actions of progesterone, RA, and TGFß on MMP regulation can impact the ability of human endometrial tissue to establish experimental endometriosis in nude mice (18, 35).

Taken together, the above studies suggest that a disruption in the normal regulation of endometrial MMP expression could be a component in the pathophysiology of endometriosis. Therefore, in the current study, we initially analyzed the expression pattern of stromal-specific MMP-3 and epithelial-specific MMP-7 mRNAs in archived samples of eutopic and ectopic endometrial tissues from women with endometriosis. We also investigated the in vitro ability of progesterone to suppress pro-MMP-3 and pro-MMP-7 secretion in eutopic and ectopic endometrium from women with endometriosis. We then determined whether progesterone treatment could prevent the development of experimental endometriosis in nude mice receiving these tissues. Finally, we examined whether RA and TGFß treatments in vitro would enhance the ability of progesterone to suppress MMP secretion in tissues obtained from women with endometriosis.

Materials and Methods

Acquisition of human tissues

Normal endometrial tissues (n = 12) were acquired by biopsy during the proliferative phase (d 9–12) of the menstrual cycle from a donor population (aged 18–45 yr) exhibiting normal menstrual cycles and no history of endometriosis. Endometrial samples (n = 7) from women with laparoscopically confirmed endometriosis were also obtained by biopsy during the proliferative phase from patients seen in the Women’s Reproductive Health Clinic at Vanderbilt University. An endometrial thickness of greater than 9 mm (confirmed by vaginal ultrasound) and a serum progesterone level of less than 1.5 ng/ml were required for inclusion in this study. Individuals with a recent (<=3 months) history of hormone therapy (i.e. oral contraceptives) or other medications that might impact study results (i.e. anti-inflammatory agents) were excluded. Biopsies were obtained from the uterine fundus using a Pipelle endometrial suction curette (Unimar, Inc., Wilton, CT), and the tissue was washed in prewarmed, phenol-red free DME/F-12 (Sigma, St. Louis, MO) to remove residual blood and mucous before culturing.

In addition, ectopic tissues (n = 10) were acquired at the time of surgery from patients undergoing laparoscopic resection of ovarian endometriosis (endometrioma). All tissues were acquired during the proliferative interval from patients who were not receiving medication for the treatment of endometriosis for at least the preceding 3 months. As above, tissues were immediately placed in prewarmed, phenol-red free DME/F-12 to remove residual blood and mucous. Informed consent was obtained before endometrial biopsy or surgical removal of endometriotic tissue. A portion of each sample was formalin fixed for histological confirmation of cycle stage. Archived samples of normal endometrium (proliferative phase, n = 10; secretory phase, n = 8) and eutopic (proliferative phase, n = 4; secretory phase, n = 5) and ectopic (proliferative phase, n = 4; secretory phase, n = 6) endometrial tissues from women with endometriosis were obtained from the Histopathology Department at Vanderbilt University Hospital and the Department of Pathology at Thomas Jefferson University Hospital (Philadelphia, PA). Although names were kept confidential, each patient’s age, cycle stage, and medication history were made available. The use of human tissues for this study was approved by Vanderbilt University’s Institutional Review Board and Committee for the Protection of Human Subjects.

Organ cultures of human tissues

Endometrial biopsies or endometriomas were dissected into small cubes (1 x 1 mm3), and 8–10 pieces per treatment group were suspended in tissue culture inserts (Millipore Corp., Bedford, MA) as described previously (36). Each organ culture was maintained under serum-free conditions in DME/F-12 supplemented with 1% insulin-transferrin-selenium (ITS+; Collaborative Biomedical, Bedford, MA) and 0.1% Excyte (Miles Scientific, Kankakee, IL) and incubated at 37 C in a humidified chamber with 5% CO2. Treatments included estradiol (1 nM) and estradiol plus progesterone (1 nM and 500 nM, respectively) or estradiol, progesterone, RA, and TGFß (1 nM, 500 nM, and 10 nM, 2 ng/ml, respectively). Cultures were maintained for 24 h before injection into mice or 48 h before analysis of protein expression. Estradiol, progesterone, and RA were purchased from Sigma, and TGFß was obtained from R&D Systems (Minneapolis, MN).

In situ hybridization analysis

The in situ hybridization method using 35S-labeled probes was derived from Cox et al. (37) and adapted for application with formalin-fixed, paraffin-embedded tissues (3). Hybridization probes were prepared from cloned human MMP cDNAs as previously described (2, 3). Briefly, full-length RNA transcripts from the sense and antisense strands were synthesized from the linearized plasmid by in vitro transcription using random primers and 35S -labeled CTP. Following hybridization, slides were dehydrated and dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) and exposed for 2 wk at 4 C. Slides were developed using D-19 (Eastman Kodak Co.) and counterstained with Gill’s hematoxylin 3 (Sigma).

Western analysis

Proteins present in medium collected from tissue cultures were quantified using the Coomassie Plus protein assay (Pierce Chemical Co., Rockford, IL) and 20 µg total protein subjected to 10% SDS-PAGE. Proteins were transferred to polyvinyl difluoride membrane (Immobilon-P, Amersham Life Sciences, Piscataway, NJ) and blocked in PBS with 10% nonfat milk and 0.05% Tween 20. Blots were incubated overnight at 4 C in PBS/milk/Tween with primary antibody, washed in PBS/Tween, and incubated with secondary antibody for 1 h. Proteins were visualized by chemiluminescence (Amersham) and autoradiography. As a negative control, identical blots were not incubated in primary antibody but remained in blocking solution until addition of the secondary antibody. Antibodies for pro-MMP-3, pro-MMP-7, and the secondary antibodies were commercially obtained and have been described previously (35).

Experimental model of endometriosis

The model of endometriosis was performed as previously described (23) with minor modifications. Briefly, 5-wk-old athymic (ncr/nude), ovariectomized mice (Taconics Laboratory Animals) were sc implanted with a 60-d time-release pellet containing 1.5 mg 17ß-estradiol (Innovative Research, Sarasota, FL). Twenty-four hours after the pellets were placed, mice were injected with a PBS suspension of 8–10 tissue fragments/mouse. These fragments had been incubated as described above for the preceding 24 h. Subcutaneous tissue injections were given on the ventral midline just below the umbilicus. Subcutaneous injection of tissues allows for more accurate quantification of both size and number of lesions (18). Ten to twelve days after tissue injection, mice were killed by cervical dislocation and examined for endometriosis. Before any invasive procedure or killing, mice were anesthetized with Metafane (Schering Plough, Union, NJ).

Statistical analysis

Significance of differences in ability of tissues to establish lesions was assessed with two-sided Fisher’s exact test using Prism software (GraphPad Software, Inc., San Diego, CA) (38).

Results

MMP expression in human endometriosis

To assess the expression pattern of MMPs in endometriosis, we acquired a panel of archived eutopic and ectopic endometrial tissues obtained during each phase of the menstrual cycle. Using in situ hybridization localization, the expression pattern of mRNAs for MMP-3 and MMP-7 was examined and compared with the endometrial expression pattern in normal women. Representative results of the stromal cell-specific MMP-3 and epithelial cell-specific MMP-7 mRNAs are shown in Fig. 1Go. Expression of MMP mRNA in normal tissues was similar to results previously described (2, 3), but altered expression was found in secretory-phase samples from women with endometriosis.



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  		Figure 1. In situ hybridization analysis of MMP-3 (A–F) and MMP-7 (G–L) mRNA expression in formalin-fixed, paraffin-embedded normal proliferative endometrium (A and G), normal secretory endometrium (B and H), and eutopic endometrium removed during the proliferative (B and H) or secretory phase (E and K) from patients with endometriosis and endometriotic lesions removed during the proliferative (C and I) or secretory phase (F and L). Insets in A and G depict hybridization using the sense probe of MMP-3 or MMP-7, respectively. Original magnification, x150. Results are representative of at least four samples of each tissue type per MMP.

 
Specifically, in normal proliferative endometrium we found that MMP-3 mRNA exhibited focal expression that was typically adjacent to glandular epithelium (Fig. 1AGo). A similar pattern of MMP-3 mRNA expression was observed in both eutopic (Fig. 1BGo) and ectopic (Fig. 1CGo) endometrium removed during the proliferative phase from patients with endometriosis. In contrast, MMP-3 mRNA was absent in normal secretory-phase endometrium (Fig. 1DGo), but a variable level of MMP-3 mRNA expression was observed in secretory endometrium removed from a women with endometriosis (Fig. 1EGo). Endometriotic lesions removed during the secretory phase also demonstrated expression of MMP-3 mRNA (Fig. 1FGo), which could be observed primarily in stromal cells adjacent to glandular epithelium. As previously reported, epithelial cell-specific MMP-7 mRNA demonstrated a broad expression pattern in normal proliferative endometrium (Fig. 1GGo). Abundant expression of MMP-7 mRNA could also be detected in samples of eutopic (Fig. 1HGo) or ectopic (Fig. 1IGo) endometrium obtained during the proliferative phase of the cycle from patients with endometriosis. MMP-7 mRNA could not be detected in samples of normal, secretory-phase endometrium (Fig. 1JGo), but strong expression of this enzyme was observed in eutopic (Fig. 1KGo) or ectopic (Fig. 1LGo) endometrium removed during the secretory phase from a patient with endometriosis. Control slides were probed concurrently using RNA synthesized from the sense strand of the linearized plasmid; representative slides using the sense strand of MMP-3 and MMP-7 are shown in Fig. 1Go, A and G, respectively (inset).

Regulation of MMP expression in organ cultures of endometrium and endometriosis

Results of the above in situ hybridization analysis provide support for the hypothesis that tissues from women with endometriosis (both eutopic and ectopic) are less sensitive to progesterone-mediated MMP suppression, compared with normal endometrium. To address this postulate more directly, samples of normal endometrium and eutopic and ectopic tissues from women with endometriosis were obtained and established as short-term organ cultures for analysis of cell-specific MMP protein expression. Western analysis of steroid-treated, normal proliferative tissues demonstrated that expression of either pro-MMP-3 or pro-MMP-7 protein can be suppressed following 48 h of exposure to progesterone (Fig. 2AGo). Compared with normal endometrium, results obtained with eutopic endometrium from patients with laparoscopically confirmed endometriosis were more variable. Although some tissues were moderately responsive to progesterone suppression of pro-MMP-3 and pro-MMP-7, the majority of these tissues (70%) continue to secrete these proteins following treatment with progesterone (Fig. 2BGo). Endometriotic tissue obtained from endometriomas and cultured identically were resistant to progesterone, continuing to secrete both of these MMPs (Fig. 2CGo). Interestingly, densitometric analysis of pro-MMP expression indicates a much lower level of expression of either enzyme by the ectopic lesions (Fig. 2Go, D and E). Although these results may indicate a decreased ability of endometriotic tissue to produce MMPs, it is also possible that the fibrous nature of endometriosis impedes the secretion of proteins into the media.



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  		Figure 2. Western analysis of expression and regulation of pro-MMP-3 and pro-MMP-7 protein in organ cultures of normal (A; n = 8), eutopic (B; n = 6), or ectopic (C; n = 4) endometrium from women with endometriosis. All samples were removed during the proliferative phase and cultured for 48 h in the presence of 1 nM estradiol (E) or 1 nM estradiol and 500 nM progesterone (EP). Densitometric analysis of pooled sample data is shown in D (pro-MMP-3) and E (pro-MMP-7).

 
Establishment of experimental disease using tissues from patients with endometriosis

Previously we have demonstrated that normal proliferative-phase endometrial tissue fragments exposed to estradiol in vitro will readily form lesions following intraperitoneal or sc injection into nude mice and that lesions are markedly similar in appearance to human endometriosis (23, 35). We have now ascertained the ability of eutopic and ectopic tissues from women with endometriosis to form lesions in nude mice (Table 1Go) and have assessed both gross and microscopic appearance (Fig. 3Go). Lesions established from either eutopic (Fig. 3Go, A–D) or ectopic (Fig. 3Go, E–H) endometrium from women with endometriosis are vascularized with enlarged glands and disorganized stroma, which greatly resembles the naturally occurring human disease. Although the morphologic and histologic appearance of experimental endometriosis is similar regardless of tissue origin or in vitro steroid treatment, the ability to establish lesions was found to vary considerably between normal endometrium and either endometrium or endometriotic tissues obtained from women with endometriosis. Similar to our studies using normal endometrium, 100% of mice receiving eutopic or ectopic endometrium from women with endometriosis treated in vitro with estradiol developed lesions following sc tissue injection (Table 1Go). However, in contrast to normal tissue, the majority of mice receiving eutopic or ectopic tissues from women with endometriosis exhibited ectopic lesion growth following in vitro treatment of the tissues with progesterone (Table 1Go). In comparison, less than 10% of mice receiving progesterone-treated endometrium from disease-free women developed experimental endometriosis (Table 1Go).


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  		Table 1. Establishment of experimental endometriosis by normal endometrium or tissues from women with endometriosis

 


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  		Figure 3. Experimental endometriosis established from eutopic (A–D) or ectopic (E–H) endometrium from patients with endometriosis. All tissues were obtained during the proliferative phase and treated in vitro for 24 h with 1 nM estradiol (A, B, E, and F) or with 1 nM estradiol plus 500 nM progesterone (C, D, G, and H). Gross and microscopic appearance eutopic endometrium treated with estradiol (A and B) or estradiol plus progesterone (C and D) and established as lesions on the peritoneum of nude mice is markedly similar to lesions established from human endometriomas treated in vitro with estradiol (E and F) or estradiol plus progesterone (G and H). Magnification of photomicrographs: gross, x15; microscopic, x200.

 
Cytokine regulation of MMPs in endometriosis tissues

We have previously demonstrated that in vitro treatments with progesterone or RA induces expression of TGFß, but an antibody to this growth factor blocks the ability of progesterone or RA to suppress MMP expression and prevent experimental disease (18, 31, 32). Intriguingly, TGFß, in the absence or either progesterone or RA, also fails to prevent experimental endometriosis, indicating a critical interrelationship between these agents (18). Therefore, in the next series of experiments, we attempted to correct the defective steroid response of ovarian endometriomas by supplementing progesterone treatment with RA and TGFß. As shown in Fig. 4Go, the treatment with estradiol and progesterone did not suppress expression of pro-MMP-3 or pro-MMP-7 protein, similar to the findings reported in Fig. 2Go. However, culturing ectopic endometrial tissue with a combination of estradiol, progesterone, RA, and TGFß resulted in a marked suppression of pro-MMP-3 and pro-MMP-7 protein (Fig. 4Go). We additionally examined whether this combination of regulatory factors could inhibit the establishment of ectopic lesions by endometrioma-derived tissue in our experimental model of endometriosis. Indeed, as shown in Table 2Go, although 100% of mice receiving tissue treated with either estradiol and progesterone or estradiol and RA developed lesions, only 37% of mice receiving tissues treated with the full combination of factors developed endometriosislike disease. Notably, those mice in this treatment group that developed disease had fewer and smaller lesions, compared with animals in all other treatment groups (Table 2Go).



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  		Figure 4. Western analysis of expression and regulation of pro-MMP-3 (A) and pro-MMP-7 (B) protein in organ cultures of an endometrioma removed during the proliferative phase. Samples were cultured for 48 h in the presence of 1 nM estradiol (E), 1 nM estradiol and 500 nM progesterone (EP), or 1 nM estradiol, 500 nM progesterone, 10 nM RA, and 2 ng/mL TGFß (EP, RA, TGF-ß). Results are representative of five separate experiments. Densitometric analysis of pooled data from all samples is shown in C (pro-MMP-3) and D (pro-MMP-7).

 

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  		Table 2. Role of cytokines in preventing establishment of experimental endometriosis by human endometrioma

 
Discussion

Extensive clinical and experimental evidence indicates that a woman’s estrogen exposure contributes to her risk for both the development and recurrence of endometriosis. Moreover, ectopic lesions can synthesize estradiol (27) and exhibit decreased estrogen metabolism (28), thus creating a sustained environment of elevated estrogen exposure. In contrast, progesterone exposure, either from synthetic agents or from the normal endocrine environment of pregnancy, decreases a woman’s risk of developing endometriosis and can lessen the impact of the disease in some women (19, 21, 22). Nevertheless, a significant number of women with endometriosis fails to respond to progestin therapy (39, 40), and pregnancy has recently been shown to have little impact on disease progression in a nonhuman primate study (41). Defects in progesterone receptor expression patterns, as well as altered progesterone receptor isotype expression, may play a role in the diminished ability of this steroid to inhibit growth at ectopic sites (24, 25). In addition, the proinflammatory environment present in endometriotic implants may further promote alterations in steroid-directed cell growth and differentiation, compared with normal endometrium. In either case, because endometrial function requires the production of numerous locally active factors, altered steroid sensitivity significantly impacts the expression and action of key steroid-regulated growth factors and cytokines in both the endometrium and the endometriotic implants (31, 42).

In the normal endometrium, investigators have found that both direct and indirect progesterone-mediated pathways are necessary for normal patterns of cell-specific pro-MMP-3 and pro-MMP-7 regulation (31, 43, 44). Recent studies by our group further indicate that progesterone acts not only to suppress pro-MMP-3 expression by stromal cells during the secretory phase of the menstrual cycle but also to limit the stimulation of this MMP by locally produced proinflammatory cytokines (6, 7). Interestingly, we found that prior progesterone treatment of isolated stromal cells blocks subsequent induction of pro-MMP-3 by IL-1{alpha}, TNF{alpha}, or epithelial growth factor (6, 7). In vivo exposure to progesterone appears to have a similar effect because secretory phase organ cultures are also resistant to IL-1{alpha} stimulation of pro-MMP-3 (6, 7). The potent ability of progesterone to suppress MMP expression as well as limit proinflammatory cytokine action seems in conflict with the necessity of these biological agents during in the invasive process of implantation and pregnancy establishment (45). However, we have speculated that the ability of progesterone to maintain suppression of this key stromal-derived MMP within the proinflammatory environment of implantation and placentation may be necessary to protect the endometrium of early pregnancy from undergoing a menstruationlike breakdown (6, 7).

Such a role for progesterone is supported by the observations of Lessey et al. (46), who demonstrated that the progesterone receptor decreases at the site of implantation, suggesting that progesterone action must be blocked focally to allow for pregnancy establishment. Among women with endometriosis, there is increasing evidence that local production of proinflammatory cytokines, which normally occurs within the environment of early pregnancy, may have a more potent impact. For example, stromal cells isolated from both eutopic and ectopic endometrium from women with endometriosis exhibit an increased expression of IL-6 in response to IL-1{alpha} (47). Furthermore, steroid-regulated expression of paracrine factors necessary for normal regulation of MMPs may also be compromised within the inflammatorylike environment of endometriosis. Therefore, it is not surprising that investigators have found a variety of MMPs to be inappropriately expressed in the endometrium of women with endometriosis as well as in endometriotic lesions (7, 15, 16, 17, 18, 48).

A recent study employing differential display found no alteration in MMP gene expression in tissues removed from patients with endometriosis during the proliferative phase of the menstrual cycle (49). Because these enzymes are readily detectable in normal endometrium during the proliferative interval, it is not surprising that no difference was observed in MMP expression in eutopic vs. ectopic tissues examined during this phase of the menstrual cycle (49). In the current study, we examined eutopic and ectopic tissues from women with endometriosis during each phase of the menstrual cycle. We found that MMP-3 and MMP-7 mRNAs are detectable not only during the proliferative phase of the cycle but also during the progesterone-dominated secretory phase (Fig. 1Go). Because MMP mRNA expression is absent in the endometrium of normal tissue donors during the secretory phase, the continued expression of MMPs in women with endometriosis following ovulation may indicate a failure of progesterone action, which may be associated with the disease process because protease expression has been linked to development of endometrioticlike disease in experimental models (23, 50). It is also important to note (Fig. 1Go) that stromal cell-specific MMP-3 mRNA expression appears to be more intense near epithelial cells, reflecting the potential influence of an epithelial-stromal pathway of MMP regulation. We have observed a similar focal expression of MMP-3 and MMP-7 mRNA in association with tissue repair processes occurring in organ cultures of human endometrium (51), and numerous investigators have demonstrated an increase in MMP expression in stromal cells in close proximity to epithelial tumor cells (52, 53, 54). Taken together, these observations support a role for epithelial cell control of stromal MMP expression under conditions of injury or disease. It is likely that a similar epithelial-stromal interaction is occurring in endometriosis, perhaps reflecting a loss of normal progesterone sensitivity within stromal cells or enhanced sensitivity to proinflammatory cytokines as noted above (47).

The local cellular mechanisms by which progesterone normally induces cell-specific loss of sensitivity to proinflammatory cytokines in preparation for pregnancy has not been fully elucidated but appears to involve the action of progesterone-associated cytokines, including RA and TGFß (7). Using an in vitro decidualization model established from isolated human endometrial stromal cells, we have observed that RA synthesis from its retinol precursor is driven by progesterone (32), and expression of TGFß2 increases during the progesterone-dominated secretory phase in women (31) and nonhuman primates (55, 56). Although infertility and pregnancy loss is most often associated with a lack of adequate progesterone support to the endometrium (57), an absence of TGFß2 expression has been associated with recurrent spontaneous abortion in women (58) as well as infertility in nonhuman primates (56) and mice (59).

Recently a natural inhibitor of TGFß, ebaf (also known as lefty) has been identified in the endometrium just before menstruation (60). We have found that in organ cultures, ebaf stimulates the expression of pro-MMP-3 and pro-MMP-7 and that progesterone can block this effect (61). Importantly, this protein has also been identified to be inappropriately expressed during the window of implantation in women with endometriosis who were also infertile (62). Tissue response to members of the TGFß family can be highly dependent on the local environment (63), suggesting that endometrial response to this growth factor may be dictated by individual cell-type responses to progesterone or RA. For example, RA has been demonstrated to regulate cellular sensitivity to TGFß in both fibroblast and epithelial cell types (64, 65, 66, 67), and adequate vitamin A levels are also critical for normal reproductive function (68, 69). Tarrade et al. (70) demonstrated the RA receptors RAR{alpha} and RXR{alpha} are expressed at the implantation site in both maternal and fetal tissues, suggesting that RA plays a key role in the development of normal placenta. RA is known to increase TGFß2 expression in human endometrium (32), and pups born to vitamin A-deficient mice have many of the same developmental defects as those exhibited by TGFß2 knockout mice (71, 72). Similar to reproductive failures associated with deficiencies of progesterone or TGFß2, vitamin A deficiency has been associated with pregnancy failure in women and nonhuman primates (69, 73).

During pregnancy establishment, MMPs are broadly expressed by the invading trophoblasts, and MMP expression within the endometrium is limited (1). Although a focal breakdown of the extracellular matrix must occur for pregnancy to be established, limiting the degree of MMP expression within the maternal endometrium is critical to prevent widespread tissue destruction. It is well established that menstruation follows withdrawal of progesterone support of the endometrium (74), and, as discussed earlier, we have demonstrated that progesterone not only suppresses pro-MMPs but can also prevent induction of these proteins by proinflammatory cytokines (6, 7). It is likely that a loss of progesterone-associated cytokines, which control MMP expression, would also be associated with pregnancy failure. Infertility is a common complaint among women with endometriosis and although the interactive role(s) of progesterone, RA, or TGFß in pregnancy establishment or failure has not been clearly established, our studies provide insight into the important relationship between these agents within the normal endometrium. Using DNA microarray, a recent study has examined the expression of genes within the window of implantation, compared with genes expressed during the late proliferative phase (75). The study found 156 genes to be up-regulated at least 2-fold, and 377 were significantly down-regulated. These results underscore the complexity of understanding the role of any particular gene in disease-related infertility. Nevertheless, these investigators have also examined the endometrium of women with endometriosis and have observed a down-regulation of a number of genes normally up-regulated during the window of implantation (76), further suggesting a failure of normal progesterone action in this tissue.

We have previously shown that in vitro treatments with proinflammatory cytokines or disrupting the local expression of TGFß with an environmental toxin increases the ability of normal endometrium to establish endometrioticlike lesions in our experimental, nude mouse model (35, 51, 77). Results presented here indicate that in tissues acquired from women with endometriosis, the failure of progesterone to suppress pro-MMP-3 or pro-MMP-7 secretion in vitro was linked to an increased ability of these tissues to subsequently establish experimental endometriosis. These results are consistent with a disruption of normal, progesterone-mediated cell-cell communication associated with endometriosis because both stromal-specific MMP-3 and epithelial-specific MMP-7 mRNAs were found to be inappropriately expressed in the eutopic endometrium of women with this disease. We have previously shown that treatments of normal endometrial tissue with TGFß alone, in the absence of progesterone or RA, fails to prevent establishment of the experimental disease (18). Taken together with the current study, these data strongly suggest that the actions of progesterone, RA, and TGFß may each be required to fully suppress cell-specific MMP expression in the human endometrium and a failure of this system may promote the development of endometriosis. Nevertheless, it is unclear whether a defect in progesterone-mediated induction of local factors necessary for MMP suppression precedes the development of endometriosis or is a consequence of the disease.

In summary, our observations indicate an important interrelationship among progesterone, RA, and TGFß in the normal endometrium. In endometriosis, tissue response to progesterone appears to be disrupted, which likely impacts the expression of steroid-associated cytokines. Reduced sensitivity or response to progesterone-mediated MMP suppression appears to contribute to the development of experimental endometriosis in nude mice and may provide insight into the development of the human disease. Moreover, our data provide evidence that locally produced factors such as RA and TGFß may enhance progesterone action in endometriotic tissues with diminished progesterone sensitivity. Our results indicate that these cytokines, which are expressed in response to progesterone in the normal endometrium, coregulate progesterone-mediated MMP expression in tissues from women with endometriosis. Treatment of endometriosis tissues with a combination of progesterone, RA, and TGFß greatly reduces the ability of this tissue to express MMPs and establish ectopic sites of growth in a model of experimental endometriosis. Although we have not investigated the potential therapeutic benefits of our strategy in women with endometriosis, our results suggest that understanding cytokine action in normal endometrium will be important to developing more effective diagnostics as well as medical therapies for this disease.

Acknowledgments

We acknowledge the technical assistance of Christina Svitek. We additionally express our gratitude to the many tissue donors who provided endometrial and endometriosis samples and to the physicians within our department for performing these critical biopsies.

Footnotes

This work was supported by NICHHD/NIH Grants HD28128 and HD30472 and through cooperative agreement (U54-HD37321) as part of the Specialized Cooperative Centers Program in Reproductive Research. It was also supported by Environmental Protection Agency Grant GR-826300 (to K.G.O.) and the Endometriosis Association (to K.G.O., G.R.Y., and K.B.-T.). A portion of this work was presented at the 56th Annual Meeting of the American Society for Reproductive Medicine, San Diego, California, October 2000.

Abbreviations: MMP, Matrix metalloproteinase; RA, retinoic acid; TIMP, tissue inhibitor of MMP.

Received March 18, 2002.

Accepted July 15, 2002.

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