- Email: [email protected]
Expression of interstitial collagenase (matrix metalloproteinase-1) is related to the activity of human endometriotic lesions
Vol. 68, No. 2, August 1997
FERTILITY AND STERILIT@ Copyright D 1997 American Society for ReproductiveMedicine
Printed
on acid-free
paper
in U. 5. A
Published by Eleevier Science Inc.
Expression of interstitial collagenase (matrix metalloproteinase-I) is related to the activity of human endometriotic lesions*
Isabelle Kokorine,
M.Sc.l
Yves Eeckhout,
Ph.D.?
Michelle Nisolle, M.D., Ph.D.+
Pierre J. Courtoy, M.D., Ph.D.?
Jacques Donnez, M.D., Ph.D.*
Etienne Marbaix, M.D.,
Znternational Brussels,
Institute
of Cellular
and Molecular
Ph.D.t$[l
Pathology and Saint Luc University
Clinics,
University
of Louvain Medical
School,
Belgium
Objective: To determine whether interstitial collagenase (matrix metalloproteinase-1), known to play a pivotal role in the initiation of menstruation, contributes to the pathogenesis of endometriosis. Design: Serial sections of peritoneal red and black endometriotic lesions, ovarian endometriotic cysts, and rectovaginal adenomyotic nodules were analyzed by in situ hybridization for the expression of matrix metalloproteinase-1 by silver staining for the integrity of the fibrillar extracellular matrix and by immunolabeling for the abundance of sex steroid receptors. Snttinrt: ______~_
Amdnmir --_--_-___
hn.mit.nl ____~_‘ __ am-l ---- rennnrch -___ - -_-- lnhnmtnrv. _-___-___J.
Patient(s): Premenopausal women undergoing laparoscopy for endometriosis. Intervention(s): Biopsy of endometriotic lesions, combined with endometrium
whenever
possible.
Main Outcome Measure(s): Expression
of matrix
metalloproteinase-1
messenger
RNA
(mRNA).
Result(s): Matrix metalloproteinase-1 mRNA was expressed focally in red peritoneal and ovarian endometriosis irrespective of the phase of the menstrual cycle but was not detectable in black peritoneal and rectovaginal lesions. Foci of matrix metalloproteinase-1 expression closely correlated with matrix breakdown and with the absence of P receptors in adjacent epithelial cells. Conclusion(s): Correlation of matrix metalloproteinase-1 expression with activity of endometriotic tissue suggests its involvement in tissue remodeling and bleeding, and possibly in the secondary shedding and reimplantation of endometriotic lesions. (Fertil Steril@ 1997;68:246-51. 0 1997 by American Society for Reproductive Medicine.) Key Words: Endometriosis,
collagenase, MMP-1, extracellular
Endometriosis, defined as the presence of ectopic endometrial glands and stroma, is one of the commonest benign gynecologic conditions, but its pathoReceived January 8, 1997; revised and accepted April 2, 1997. * Supported by the Belgian Fonds de la Recherche Scientifique MBdicale, by the Fonds de DBveloppementScientifique of the University of Louvain, Belgium, and by Ipsen Biotech, Paris, France. t Cell Biology Unit, International Institute of Cellular and MoIncnlnr .__-_-_ -Pnt.hnlnmr -___-_-~J_ $ Department
of Gynaecology, Saint Luc University Clinics. 8 Department of Pathology, Saint Luc University Clinics. 11Reprint requests: Etienne Marbaix, M.D., Ph.D., Cliniques Universitaires Saint Luc, Service d’Anatomie Pathologique ANPS-1712, 10 Avenue Hippocrate, B-1200 Bruxelles, Belgium (FAX: 32-2-764-8924; e-mail: [email protected]).
246
matrix, progesterone receptors
genesis and classification remain controversial (1). Two nonmutually exclusive pathogenetic mechanisms have been invoked: transplantation after retrograde menstruation through the fallopian tubes (2) and metaplasia (3). The most common sites of endometriosis are the peritoneum, the ovaries, and the rectovaginal septum (4). On the peritoneal surface, red endometriotic foci, assumed to be early lesions, show high stromal vascularization and epithelial -au* mitntir ~__~.a..“~~indov z--..vIs in 111 onntrad ClVllVIUY” with IVLYII hlarlr UIU”I% lnainnc ‘V”‘“~I”,
which are thought to result from enclosure below the mesothelium of more advanced lesions containing blood pigment, and with white lesions, which correspond to scars (5). In the ovaries, activity of the endometriotic lesions has been linked to the abundance
0015-0282/97/$17.00 PI1 SOOl5-0282(97)00166-O
of the vascular network in the cyst wall and to the epithelial mitotic index (6, 7). The rectovaginal nodules, considered to be similar to uterine adenomyosis, show low vascularization and/or mitotic activity (8). Recurrent bleeding in the endometriotic foci recently has been proposed to be the cause of disease progression (9). The activity of endometriosis thus is likely to be linked with matrix remodeling. Collagen types I, III, and IV have been immunolocalized in the extracellular matrix of peritoneal endometriotic lesions: with a distribution pattern similar to that of eutopic endometrium (10). Accelerated matrix turnover in the early red lesions is supported by the detection of free aminoterminal propeptide of type III procollagen in peritoneal fluid (11). Interstitial collagenase (matrix metalloproteinase-1) is a key member of the family of matrix metalloproteinases (reviewed by Murphy (121, several of which are expressed in the eutopic human endometrium (13-X). Collagenases are the only enzymes able to cleave at neutral pH the triple helix of native fibrillar collagens, including the types I and III, which are the major components of the matrix argyr1 .. . ophilic network (i2 j. Studies by in situ hybridization (13, 15) and by Northern blot analysis (14, 16) have shown that matrix metalloproteinase-1 expression in the normal endometrium is confined in space to stromal foci of the functionalis and restricted in time to the perimenstrual period of the cycle, suggesting a tight control by sex steroids. Matrix metalloproteinase-1 expression in human endometrial cultured explants can be repressed clearly by physiologic concentrations of P (17). In both cultured and noncultured endometrium, wherever matrix metalloproteinase-1 is expressed, P receptors no longer are detected in epithelial cells? and the argyrophilic fibers are disrupted (15). Furthermore, menstrual breakdown can be mimicked in vitro using organotypic cultures of human endometrium and can be prevented by specific inhibitors of matrix metal loproteinases, providing direct evidence for the involvement of at least one matrix metalloproteinase in the initiation of the menstruation process (18). In ectopic endometrium, expression of stromelysin-1 (matrix metalloproteinase-3) and of tissue inhibitor of metalloproteinase-1 have been reported (19, 201, and P receptors as well as estrogen receptors generally are detected, whatever the type _I--J_-_L~--~__-1^__^- -_l--I---,,..1 LI--L-^w.L o1txluull1ebr1us1ti ar1u eve11 WllellllUrlllullal bI-txllmltxl~
with matrix breakdown and abundance of sex steroid receptors.
failed (21). We therefore investigated the expression of matrix metalloproteinase-1 messenger RNA (mRNA) in the different types of endometriotic lesions throughout the menstrual cycle and examined its relationship
tion, slides were washed, incubated for 25 minutes at 50°C in a stringent washing buffer, and treated
Vol. 68, No. 2, August 1997
MATERIALS
AND
METHODS
Biopsy specimens of endometriotic tissue (n = 47; 11 ovarian cysts, 13 red peritoneal, 12 black peritoneal, and 11 rectovaginal lesions), whenever possible combined with the corresponding eutopic endometrium (n = 17), were taken throughout the menstrual cycle from 37 patients (age range, 19 to 49 years; mean, 31 years! during laparoscopy for infertility or lower abdominal pain. Twenty-six patients had regular ovulatory cycles: 9 were in the proliferative phase (1 ovarian, 1 red peritoneal, 6 black peritoneal, and 2 rectovaginal lesions), 14 in the secretory phase (5 ovarian, 9 red peritoneal, 4 black peritoneal, and 2 rectovaginal lesions), and 3 in the menstrual phase (2 ovarian lesions and 1 rectovaginal lesion). Two patients had anovulatory cycles (1 ovarian, 1 red peritoneal, 1 black peritoneal, and 1 rectovaginal lesion) and 9 patients were receiving hormonal treatment: 5 were receiving an estrogen-progestin combination (2 red peritoneal and 4 rectovaginal lesionsj, i a progestin alone (1 ovarian lesion), 2 a GnRH agonist (1 ovarian lesion and 1 rectovaginal lesion), and 1 danazol @anofi-Winthrop, Brussels, Belgium) (1 black peritoneal lesion). Two patients who were receiving treatment with estrogen-progestin were bleeding at the time of the biopsies (1 red peritoneal lesion and 1 rectovaginal lesion). These two lesions were considered as having been sampled at the menstrual phase of the cycle. Tissue collection was approved by the Ethical Committee of the University of Louvain. Tissue samples were fixed in 4% paraformaldehyde for 6 to 24 hours and were embedded in paraffin. All morphological techniques used in this study were performed exactly as described (16). Briefly, in situ hybridization was performed on tissue sections digested by proteinase K, acetylated, and air dried. Sections were hybridized with 36S-labeled sense (as control) or antisense riboprobes transcribed from the human matrix metalloproteinase-1 complementary DNA (H. Nagase, Ph.D., Kansas University Medical Center, Kansas City, KS). The hybridization solution contained 30,000 to 50,000 cpm/pL of probes, and hybridization was all,....-.,.l c,. ,,.-.,,..-.,.I ,...,.-:...Lc ,"Wtxl b" ptJ'"c;eeu""tx"'~;"~
.T+ .cAOP Ac+'c,..hA.&,4:".n Llb._J" ".NLcL 1lyLnlulaa-
with RNAses. Sections were dehydrated, dipped into K5 Ilford emulsion (Ilford Ltd, Mobberley, United Kingdom), exposed for 1 week at 4”C, developed
Kokorine
et al.
Collagenase
in endometriosis
247
in Agfa G150 (Agfa Gevaert AG, Leverkusen, Germany), counterstained with hematoxylin, and mounted in Eukitt medium (Kindler GmbH, Freiburg, Germany). Hybridizations with the sense riboprobe were always negative (not shown). Sex steroid receptors were immunolocalized using mouse monoclonal antibodies (Novocastra Laboratories Ltd, Newcastle-upon-Tyne, United Kingdom, for P receptors; Dako A/S, Glastrup, Denmark, for estrogen receptors) after antigen retrieval with a microwave oven. Specific binding was detected with biotinylated sheep anti-mouse antibodies (Boehringer Mannheim GmbH, Mannheim, Germany) for 1 hour at room temperature, followed by 30 minutes of incubation with peroxidase-conjugated streptavidin (Boehringer Mannheim GmbH) and 10 minutes of incubation with HzOz and 3,3’-diaminobenzidine in tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4. Matrix breakdown was defined as fragmentation or disappearance of the argyrophilic fibers, stained by silver impregnation according to the method of Gordon and Sweets (15). RESULTS
Matrix metalloproteinase-1 mRNA was detected in at least one focus of approximately one-third of ovarian (4/11) and of red peritoneal (4/13) endometriotic biopsies but in none of the black peritoneal and the rectovaginal lesions (Table 1; Figs. 1B and 2D). To increase the sensitivity of detection, five semiserial sections, 20 pm apart, were analyzed in a limited number of samples, all of which, with the exception of one ovarian and one red peritoneal lesion, initially were negative. With this procedure, matrix metalloproteinase-1 mRNA was detected in three of three ovarian lesions (l/5, 3/5, and 5/5 sections, respectively; Fig. 1B) and
Table 1 Matrix Metalloproteinase-1 (MMP-1) Expression Correlates with Activity and Focal Matrix Breakdown Matrix Breakdown* Endometriotic Lesions Active Ovarian cysts Red peritoneal Inactive Black peritoneal Rectovaginal
MMP-1 mRNA
MMP-1 mRNApositive foci
MMP-1 mRNAnegative tissue?
4/l 1 4113
313 2l3
117 217
o/12 o/11
-
l/10 l/7
* In a few cases, this parameter could not be analyzed because adjacent sections were not available. t Also includes MMP-l-negative areas of sections containing MMP-l-positive foci.
248
Kokorine
et al.
Collagenase
in endometriosis
in two of three red peritoneal lesions (in 2/5 and 3/ 5 sections) but in none of the three black peritoneal lesions nor in the rectovaginal nodule tested. Matrix metalloproteinase-1 mRNA was expressed in more than one site in several sections, underlining the multifocal nature of the process. Confirming previous reports (13-161, expression of matrix metalloproteinase-1 mRNA in eutopic endometrium was restricted to the perimenstrual phase (3/4 perimenstrual and O/13 nonperimenstrual samples). In contrast, ectopic endometrium expressed matrix metalloproteinase-1 mRNA at nonperimenstrual phases of the cycle in ovarian endometriosis sampled during the early secretory (2/3) or midsecretory (l/2) phase of the spontaneous menstrual cycle and in red peritoneal lesions sampled during the middle (l/l) or late (l/6) secretory phase of the spontaneous cycle and even during treatment with estrogenprogestin combination (l/l). In endometriotic tissues sampled during the menstrual phase, matrix metalloproteinase-1 mRNA was expressed in one of two ovarian lesions and in the red peritoneal lesion from a patient bleeding on estrogen-progestin treatment. Conversely, matrix , 11 metalloproteinase-i mRNA was not detected in the two rectovaginal nodules sampled during the menstrual phase. When or where matrix metalloproteinase-1 mRNA was not detected in eutopic endometrium, estrogen and P receptors were expressed abundantly in stroma1 cells (1607 and 14/17, respectively) and in epithelial cells (16/17 and 12/17, respectively), in agreement with our previous report (15). Similarly, in endometriotic lesions where no matrix metalloproteinase-1 mRNA was detected, estrogen and P receptors also were expressed in both cell types in a majority of the 47 cases tested (37 and 35 cases for stromal cells; 31 and 27 cases for epithelial cells). Conversely, matrix metalloproteinase-1 mRNA expression was restricted to areas where P receptors were not detected in the adjacent epithelial cells (6/7; Fig. 2E). Finally, both in eutopic and ectopic endometrium, focal expression of matrix metalloproteinase-1 mRNA was related inversely to the integrity of the argyrophilic fibrillar network (Figs. 1E and 2F; Table 1). Matrix breakdown was observed in almost all areas expressing matrix metalloproteinase-1 mRNA (5161, whereas it was observed infrequently in lesions not expressing matrix metalloproteinase1 m.~,rte;an I “L “UUUIU.z
.r,wn9n u1.zacJ
nt-elntr;v “I IllaLd1*
mntollmm.~+r\;n~o, Illr;~arr”yL”~=;lllcr~r;-l
1 c.v r;n-
pression (5/31). DISCUSSION
Endometriotic lesions of the peritoneal lining of the pelvis have various macroscopic appearances,
Fertility
and Sterility@
n-&WA and inverse relation with the integrity Figure 1 Focal expression of matrix metalloproteinase-1 of the argyrophilic fibrillar network. This typical ovarian endometriotic biopsy specimen was obtained during the early secretory phase of the menstrual cycle. Two pairs of sections from the same ribbon, 20 pm apart, were hybridized with the antisense matrix metalloproteinase-13% riboprobe (A to C) or were silver stained (D to F). In the first series, no matrix metalloproteinase-1 mRNA was detected (A) and the argyrophilit fibrillar network (D) was fully preserved. In the second series, matrix metalloproteinase-1 mRNA was detected in foci (B, arrows) where the matrix was extensively broken down (E) but not where the matrix was preserved (C and F). Bar, 100 pm.
which have been proposed to reflect their age aiiruor activity. 1ne rea suotie lesions (vesicumr, red flamelike, and glandular excrescences) are more vascularized and have a higher epithelial mitotic index than the typical, puckered black or bluish peritoneal lesions, whereas the vascularization and the mitotic index are lowest in the white lesions (white opacification, yellow-brown patches, and circular peritoneal defects) (5). Thus, red lesions are thought to correspond to the first, active stage of early implantation of endometrial glands and stroma, and would evolve toward the typical black or bluish lesion after enclosure beneath the peritoneal lining. The white lesions are believed to correspond to fibrotic, quiescent lesions. Similarly, foci of increased vascularization and epithelial mitotic index are present in the wall of ovarian endometriotic cysts and have been thought to correspond to foci of activity (6, 7). In contrast, the rectovaginal nodules show low vascularization and/or mitotic activity, and a different pathogenesis has been proposed for this less active form of disease (8). Foci of matrix metalloproteinase-1 mRNA were detected only in red peritoneal lesions and in the wall of ovarian cysts, i.e., the two types of easily bleeding endometriotic lesions (9) that show at least fneallxr ricma nf a&kT;tv I”YU&IJ “16”” “I U’ Y’.IVJ
(!i_?). \Y
Tbae ~nmncar;~rm “““‘p.aI lU”ll ho_ U”
tween these combined active lesions and the inactive lesions (black peritoneal and rectovaginal) is statistically significant (P = 0.004, Fisher’s exact test). It
Vol. 68, No. 2, August 1997
is likely that the proportion of positive cases in the -A!active lesions would increase if all of them had been analyzed by serial sectioning. The lack of matrix metalloproteinase-1 expression in inactive lesions, even when the corresponding eutopic tissue undergoes menstruation, is further evidence of their inactivity. As previously shown for eutopic perimenstrual endometrium (151, expression of matrix metalloproteinase-1 appears to be correlated with bleeding, because ovarian and red peritoneal endometriosis frequently show hemorrhages at laparoscopic examination (5-7, 9). Matrix breakdown essentially was observed in areas of matrix metalloproteinase-1 expression, as is the case in the eutopic endometrium (15). With respect to peritoneal endometriosis, expression of the metalloproteinase was observed in red but not in black lesions. The former are more superficial and show a prominent vascularization and a conspicuous stroma undergoing differentiation, fibrillar remodeling, and eventual breakdown (5). In the enclosed black lesions, the scanty stroma barely shows menstrual remodeling. Thus, matrix metalloproteinase-1 could be involved, probably in concert with other matrix metalloproteinases, in the implantation of retrograde menstrual fragments (22), generating early, active, rnrl nnrllnr“I LaI”” nlen nn..lrl Qnrl I uu lnainna ICIUIVII”, CIIIW x,“uLAtAnrnr~&n pl” * “1-b hlnarlinm UI~c_da111~UllU tearing of the tissue, so that detached viable fragments could reimplant on the peritoneum and propagate the disease (4). The invasive potential of endo-
Kokorine
et al.
Collagenase
in endometriosis
249
that they correspond to a distinct disease, perhaps one that is related more closely to uterine adenomyosis (8). Finally, despite the lack of consistent response of endometriotic tissue to hormonal treatment and the fact that eutopic and ectopic endometrium are not always in phase (251, both sex steroid receptors generally were detected in both cell types of endometriotic tissue not expressing matrix metalloproteinase1, whereas P receptors generally were absent in epithelial cells surrounding stromal foci of matrix metalloproteinase-1. Thus, P still exerts some regulation on ectopic lesions, which challenges the commonly held view that endometriotic lesions are purely autonomous and hormone independent (22). However, the failure of hormonal therapy in the treatment of endometriosis easily could be accounted for by the absence of sex steroid receptors at the critical sites of matrix breakdown, suggesting that a direct pharmacologic inhibition of the proteinase activity could be more efficient for controlling the disease.
Acknowledgments. The authors thank H. Nagase, Ph.D., from the Kansas University Medical Center, Kansas City, Kansas, for the gift of MMP-1 cDNA. The staBof the Department of Pathology is acknowledged for its technical help. This article represents research results of the Belgian Programme on Interuniversity Poles of Attraction and of Concerted Research Actions of the Communaute Francaise de Belgique.
Typical relation between matrix metalloproteinase1 mRNA, P receptors, and argyrophilic fibrillar network. Three biopsies were performed simultaneously in the same patient during the midsecretory phase of the menstrual cycle: one of eutopic endometrium (A to C), one of a red peritoneal endometriotic tissue (D to F), and one of a black peritoneal endometriotic tissue (G to I). Serial sections were hybridized with the antisense matrix metalloproteinase-1 35Sriboprobes (A, D, and Gl, incubated with monoclonal anti-P receptor antibodies (B, E, and I-I), or silver stained (C, F, and I). Matrix metalloproteinase-1 mRNA was detected only in stromal foci of the red peritoneal lesion (D, arrows) that showed no detectable P receptors (E) but did show extensive matrix breakdown (F). When and where no matrix metalloproteinase-1 mRNA was detected (A and G), P receptors were abundantly expressed (B and H) and the argyrophilic fibrillar network was well preserved (C and I). Bar, 100 pm. Figure 2
metriotic peritoneal lesions has been demonstrated by collagen gel invasion assays (23). Expression of matrix metalloproteinase-1 during remodeling of newly metaplastic areas is another pathogenetic possibility. The same considerations apply to ovarian cysts, which have been considered as free or mucosal lesions (24) where the stroma is more developed focally. In contrast, the argyrophilic fibrillar network was well-preserved in rectovaginal nodules and showed no cyclic change, supporting the view
260
Kokorine
et al.
Collagenase in endometriosis
REFERENCES
1. Rock JA, Markham SM. Pathogenesis of endometriosis. Lancet 1992;340:1264-7. 2. Sampson JA. Peritoneal endometriosis due to menstlval dissemination of endometrial tissue into the pelvic cavity. Am J Obstet Gynecol 1927; 14:422-67. 3. Meyer R. Ueber den Stand der Frage der Adenomyositis und Adenomyome in Allgemeinen und Insbesondere ueber Adenomyositis seroepithelialis und Adenomyometritis sarcomatosa. Zentralbl Gynakol 1919;36:743-59. 4. Brosens IA. Is mild endometriosis a progressive disease? Hum Reprod 1994;9:2209-11. 5. Nisolle M, Casanas-Roux F, Anaf V, Mine J-M, Donnez J. Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil Steril 1993;59:681-4. 6. Donnez J, Nisolle M, Gillet N, Smets M, Bassil S, CasanasRoux F. Large ovarian endometriomas. Hum Reprod 1996; 11:641-6. 7. Brosens IA, Puttemans PJ, Deprest J. The endoscopic localization of endometrial implants in the ovarian chocolate cyst. Fertil Steril 1994;61:1034-8. 8. Donnez J, Nisolle M, Smoes P, Gillet N, Beguin S, CasanasRoux F. Peritoneal endometriosis and “endometriotic” nodules of the rectovaginal septum are two different entities. Fertil Steril 1996;66:362-8. 9. Brosens IA. Endometriosis: a disease because it is characterized by bleeding. Am J Obstet Gynecol 1997; 176:263-7. 10. Stovall DW, Anners JA, Halme J. Immunohistochemical de-
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tection of type I, III, IV collagen in endometriosis implants. Fertil Steril 1992;57:984-9. Spuijbroek MDEH, Dunselman GAJ, Menheere PPCA, Evers JLH. Early endometriosis invades the extracellular matrix. Fertil Steril 1992;58:929-33. Murphy G. Matrix metalloproteinases and their inhibitors. Acta Orthop &and 1995;66(Suppl266):55-60. Rodgers WI-I, Matrisian LM, Guidice LC, Dsupin B, Cannon P, Svitek C, et al. Patterns of matrix metalloproteinase’ expression in cycling endometrium imply differential functions and regulation by steroid hormones. J Clin Invest 1994; 94:946-53. Hampton AL, Salamonsen LA. Expression of messenger ribonucleic acid encoding matrix metalloproteinases and their tissue inhibitors is related to menstruation. J Endocrinol 1994;141:Rl-R3. Kokorine I, Marbaix E, Henriet P, Okada Y, Donnez J, Eeckhout Y, et al. Focal cellular origin and regulation of interstitial collagenase (matrix metalloproteinase-1) are related to menstrual breakdown in the human endometrium. J Cell Sci 1996;109:2151-60. Marbaix E, Kokorine I, Henriet P, Donnez J, Courtoy PJ, Eeckhout Y. The expression of interstitial collagenase in human endometrium is controlled by progesterone and by oestradiol and is related to menstruation. Biochem J 1995; 305:1027-30. Marbaix E, Donnez J, Courtoy PJ, Eeckhout Y. Progesterone regulates the activity of collagenase and related gelatinases A and B in human endometrial explants. Proc Nat1 Acad Sci USA 1992;89:11789-93. Marbaix E, Kokorine I, Moulin P, Donnez J, Eeckhout Y,
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19.
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25.
Courtoy PJ. Menstrual breakdown of human endometrium can be mimicked in vitro and is selectively and reversibly blocked by inhibitors of matrix metalloproteinases. Proc Nat1 Acad Sci USA 1996;93:9120-5. Saito T, Mizumoto H, Kuroki K, Fujii M, Mori S, Kudo R. Expression of MMP-3 and TIMP-1 in the endometriosis and the influence of danazol. Acta Obstetrica Gynaecologica Japonica 1995;47:495-6. Sharpe Timms KL, Penney LL, Zimmer RL, Wright JA, Zhang Y, Surewicz K. Partial purification and aminoacid sequence analysis of endometriosis protein-II (ENDO-II) reveals homology with tissue inhibitor of metalloproteinases-1 (TIMP-1). J Clin Endocrinol Metab 1995;80:3784-7. Nisolle M, Casanas-Roux F, Wyns C, de Menten Y, Mathieu PE, Donnez J. Immunohistochemical analysis of estrogen and progesterone receptors in endometrium and peritoneal endometriosis: a new quantitative method. Fertil Steril 1994; 62:751-g. Sillem M, Hahn U, Coddington CC, Gordon K, Runnebaum B, Hodgen GD. Ectopic growth of endometrium depends on its structural integrity and proteolytic activity in the cynomolgus monkey (Mucacu fuscicularis) model of endometriosis. Fertil Steril 1996;66:468-73. Gaetje R, Kotzian S, Herrmann G, Baumann R, StarzinskiPowitz A. Invasiveness of endometriotic cells in vitro. Lancet 1995;346:1463-4. Nieminen U. Studies on the vascular pattern of ectopic endometrium with special reference to cyclic changes. Acta Obstet Gynecol Stand 1962;41(Suppl 3):1-81. Metzger DA, Szpak CA, Haney AI?. Histologic features associated with hormonal responsiveness of ectopic endometrium. Fertil Steril 1993;59:83-8.
Kokorine
et al.
Collagenase in endometriosis
261
FERTILITY AND STERILIT@ Copyright D 1997 American Society for ReproductiveMedicine
Printed
on acid-free
paper
in U. 5. A
Published by Eleevier Science Inc.
Expression of interstitial collagenase (matrix metalloproteinase-I) is related to the activity of human endometriotic lesions*
Isabelle Kokorine,
M.Sc.l
Yves Eeckhout,
Ph.D.?
Michelle Nisolle, M.D., Ph.D.+
Pierre J. Courtoy, M.D., Ph.D.?
Jacques Donnez, M.D., Ph.D.*
Etienne Marbaix, M.D.,
Znternational Brussels,
Institute
of Cellular
and Molecular
Ph.D.t$[l
Pathology and Saint Luc University
Clinics,
University
of Louvain Medical
School,
Belgium
Objective: To determine whether interstitial collagenase (matrix metalloproteinase-1), known to play a pivotal role in the initiation of menstruation, contributes to the pathogenesis of endometriosis. Design: Serial sections of peritoneal red and black endometriotic lesions, ovarian endometriotic cysts, and rectovaginal adenomyotic nodules were analyzed by in situ hybridization for the expression of matrix metalloproteinase-1 by silver staining for the integrity of the fibrillar extracellular matrix and by immunolabeling for the abundance of sex steroid receptors. Snttinrt: ______~_
Amdnmir --_--_-___
hn.mit.nl ____~_‘ __ am-l ---- rennnrch -___ - -_-- lnhnmtnrv. _-___-___J.
Patient(s): Premenopausal women undergoing laparoscopy for endometriosis. Intervention(s): Biopsy of endometriotic lesions, combined with endometrium
whenever
possible.
Main Outcome Measure(s): Expression
of matrix
metalloproteinase-1
messenger
RNA
(mRNA).
Result(s): Matrix metalloproteinase-1 mRNA was expressed focally in red peritoneal and ovarian endometriosis irrespective of the phase of the menstrual cycle but was not detectable in black peritoneal and rectovaginal lesions. Foci of matrix metalloproteinase-1 expression closely correlated with matrix breakdown and with the absence of P receptors in adjacent epithelial cells. Conclusion(s): Correlation of matrix metalloproteinase-1 expression with activity of endometriotic tissue suggests its involvement in tissue remodeling and bleeding, and possibly in the secondary shedding and reimplantation of endometriotic lesions. (Fertil Steril@ 1997;68:246-51. 0 1997 by American Society for Reproductive Medicine.) Key Words: Endometriosis,
collagenase, MMP-1, extracellular
Endometriosis, defined as the presence of ectopic endometrial glands and stroma, is one of the commonest benign gynecologic conditions, but its pathoReceived January 8, 1997; revised and accepted April 2, 1997. * Supported by the Belgian Fonds de la Recherche Scientifique MBdicale, by the Fonds de DBveloppementScientifique of the University of Louvain, Belgium, and by Ipsen Biotech, Paris, France. t Cell Biology Unit, International Institute of Cellular and MoIncnlnr .__-_-_ -Pnt.hnlnmr -___-_-~J_ $ Department
of Gynaecology, Saint Luc University Clinics. 8 Department of Pathology, Saint Luc University Clinics. 11Reprint requests: Etienne Marbaix, M.D., Ph.D., Cliniques Universitaires Saint Luc, Service d’Anatomie Pathologique ANPS-1712, 10 Avenue Hippocrate, B-1200 Bruxelles, Belgium (FAX: 32-2-764-8924; e-mail: [email protected]).
246
matrix, progesterone receptors
genesis and classification remain controversial (1). Two nonmutually exclusive pathogenetic mechanisms have been invoked: transplantation after retrograde menstruation through the fallopian tubes (2) and metaplasia (3). The most common sites of endometriosis are the peritoneum, the ovaries, and the rectovaginal septum (4). On the peritoneal surface, red endometriotic foci, assumed to be early lesions, show high stromal vascularization and epithelial -au* mitntir ~__~.a..“~~indov z--..vIs in 111 onntrad ClVllVIUY” with IVLYII hlarlr UIU”I% lnainnc ‘V”‘“~I”,
which are thought to result from enclosure below the mesothelium of more advanced lesions containing blood pigment, and with white lesions, which correspond to scars (5). In the ovaries, activity of the endometriotic lesions has been linked to the abundance
0015-0282/97/$17.00 PI1 SOOl5-0282(97)00166-O
of the vascular network in the cyst wall and to the epithelial mitotic index (6, 7). The rectovaginal nodules, considered to be similar to uterine adenomyosis, show low vascularization and/or mitotic activity (8). Recurrent bleeding in the endometriotic foci recently has been proposed to be the cause of disease progression (9). The activity of endometriosis thus is likely to be linked with matrix remodeling. Collagen types I, III, and IV have been immunolocalized in the extracellular matrix of peritoneal endometriotic lesions: with a distribution pattern similar to that of eutopic endometrium (10). Accelerated matrix turnover in the early red lesions is supported by the detection of free aminoterminal propeptide of type III procollagen in peritoneal fluid (11). Interstitial collagenase (matrix metalloproteinase-1) is a key member of the family of matrix metalloproteinases (reviewed by Murphy (121, several of which are expressed in the eutopic human endometrium (13-X). Collagenases are the only enzymes able to cleave at neutral pH the triple helix of native fibrillar collagens, including the types I and III, which are the major components of the matrix argyr1 .. . ophilic network (i2 j. Studies by in situ hybridization (13, 15) and by Northern blot analysis (14, 16) have shown that matrix metalloproteinase-1 expression in the normal endometrium is confined in space to stromal foci of the functionalis and restricted in time to the perimenstrual period of the cycle, suggesting a tight control by sex steroids. Matrix metalloproteinase-1 expression in human endometrial cultured explants can be repressed clearly by physiologic concentrations of P (17). In both cultured and noncultured endometrium, wherever matrix metalloproteinase-1 is expressed, P receptors no longer are detected in epithelial cells? and the argyrophilic fibers are disrupted (15). Furthermore, menstrual breakdown can be mimicked in vitro using organotypic cultures of human endometrium and can be prevented by specific inhibitors of matrix metal loproteinases, providing direct evidence for the involvement of at least one matrix metalloproteinase in the initiation of the menstruation process (18). In ectopic endometrium, expression of stromelysin-1 (matrix metalloproteinase-3) and of tissue inhibitor of metalloproteinase-1 have been reported (19, 201, and P receptors as well as estrogen receptors generally are detected, whatever the type _I--J_-_L~--~__-1^__^- -_l--I---,,..1 LI--L-^w.L o1txluull1ebr1us1ti ar1u eve11 WllellllUrlllullal bI-txllmltxl~
with matrix breakdown and abundance of sex steroid receptors.
failed (21). We therefore investigated the expression of matrix metalloproteinase-1 messenger RNA (mRNA) in the different types of endometriotic lesions throughout the menstrual cycle and examined its relationship
tion, slides were washed, incubated for 25 minutes at 50°C in a stringent washing buffer, and treated
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MATERIALS
AND
METHODS
Biopsy specimens of endometriotic tissue (n = 47; 11 ovarian cysts, 13 red peritoneal, 12 black peritoneal, and 11 rectovaginal lesions), whenever possible combined with the corresponding eutopic endometrium (n = 17), were taken throughout the menstrual cycle from 37 patients (age range, 19 to 49 years; mean, 31 years! during laparoscopy for infertility or lower abdominal pain. Twenty-six patients had regular ovulatory cycles: 9 were in the proliferative phase (1 ovarian, 1 red peritoneal, 6 black peritoneal, and 2 rectovaginal lesions), 14 in the secretory phase (5 ovarian, 9 red peritoneal, 4 black peritoneal, and 2 rectovaginal lesions), and 3 in the menstrual phase (2 ovarian lesions and 1 rectovaginal lesion). Two patients had anovulatory cycles (1 ovarian, 1 red peritoneal, 1 black peritoneal, and 1 rectovaginal lesion) and 9 patients were receiving hormonal treatment: 5 were receiving an estrogen-progestin combination (2 red peritoneal and 4 rectovaginal lesionsj, i a progestin alone (1 ovarian lesion), 2 a GnRH agonist (1 ovarian lesion and 1 rectovaginal lesion), and 1 danazol @anofi-Winthrop, Brussels, Belgium) (1 black peritoneal lesion). Two patients who were receiving treatment with estrogen-progestin were bleeding at the time of the biopsies (1 red peritoneal lesion and 1 rectovaginal lesion). These two lesions were considered as having been sampled at the menstrual phase of the cycle. Tissue collection was approved by the Ethical Committee of the University of Louvain. Tissue samples were fixed in 4% paraformaldehyde for 6 to 24 hours and were embedded in paraffin. All morphological techniques used in this study were performed exactly as described (16). Briefly, in situ hybridization was performed on tissue sections digested by proteinase K, acetylated, and air dried. Sections were hybridized with 36S-labeled sense (as control) or antisense riboprobes transcribed from the human matrix metalloproteinase-1 complementary DNA (H. Nagase, Ph.D., Kansas University Medical Center, Kansas City, KS). The hybridization solution contained 30,000 to 50,000 cpm/pL of probes, and hybridization was all,....-.,.l c,. ,,.-.,,..-.,.I ,...,.-:...Lc ,"Wtxl b" ptJ'"c;eeu""tx"'~;"~
.T+ .cAOP Ac+'c,..hA.&,4:".n Llb._J" ".NLcL 1lyLnlulaa-
with RNAses. Sections were dehydrated, dipped into K5 Ilford emulsion (Ilford Ltd, Mobberley, United Kingdom), exposed for 1 week at 4”C, developed
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in Agfa G150 (Agfa Gevaert AG, Leverkusen, Germany), counterstained with hematoxylin, and mounted in Eukitt medium (Kindler GmbH, Freiburg, Germany). Hybridizations with the sense riboprobe were always negative (not shown). Sex steroid receptors were immunolocalized using mouse monoclonal antibodies (Novocastra Laboratories Ltd, Newcastle-upon-Tyne, United Kingdom, for P receptors; Dako A/S, Glastrup, Denmark, for estrogen receptors) after antigen retrieval with a microwave oven. Specific binding was detected with biotinylated sheep anti-mouse antibodies (Boehringer Mannheim GmbH, Mannheim, Germany) for 1 hour at room temperature, followed by 30 minutes of incubation with peroxidase-conjugated streptavidin (Boehringer Mannheim GmbH) and 10 minutes of incubation with HzOz and 3,3’-diaminobenzidine in tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4. Matrix breakdown was defined as fragmentation or disappearance of the argyrophilic fibers, stained by silver impregnation according to the method of Gordon and Sweets (15). RESULTS
Matrix metalloproteinase-1 mRNA was detected in at least one focus of approximately one-third of ovarian (4/11) and of red peritoneal (4/13) endometriotic biopsies but in none of the black peritoneal and the rectovaginal lesions (Table 1; Figs. 1B and 2D). To increase the sensitivity of detection, five semiserial sections, 20 pm apart, were analyzed in a limited number of samples, all of which, with the exception of one ovarian and one red peritoneal lesion, initially were negative. With this procedure, matrix metalloproteinase-1 mRNA was detected in three of three ovarian lesions (l/5, 3/5, and 5/5 sections, respectively; Fig. 1B) and
Table 1 Matrix Metalloproteinase-1 (MMP-1) Expression Correlates with Activity and Focal Matrix Breakdown Matrix Breakdown* Endometriotic Lesions Active Ovarian cysts Red peritoneal Inactive Black peritoneal Rectovaginal
MMP-1 mRNA
MMP-1 mRNApositive foci
MMP-1 mRNAnegative tissue?
4/l 1 4113
313 2l3
117 217
o/12 o/11
-
l/10 l/7
* In a few cases, this parameter could not be analyzed because adjacent sections were not available. t Also includes MMP-l-negative areas of sections containing MMP-l-positive foci.
248
Kokorine
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in endometriosis
in two of three red peritoneal lesions (in 2/5 and 3/ 5 sections) but in none of the three black peritoneal lesions nor in the rectovaginal nodule tested. Matrix metalloproteinase-1 mRNA was expressed in more than one site in several sections, underlining the multifocal nature of the process. Confirming previous reports (13-161, expression of matrix metalloproteinase-1 mRNA in eutopic endometrium was restricted to the perimenstrual phase (3/4 perimenstrual and O/13 nonperimenstrual samples). In contrast, ectopic endometrium expressed matrix metalloproteinase-1 mRNA at nonperimenstrual phases of the cycle in ovarian endometriosis sampled during the early secretory (2/3) or midsecretory (l/2) phase of the spontaneous menstrual cycle and in red peritoneal lesions sampled during the middle (l/l) or late (l/6) secretory phase of the spontaneous cycle and even during treatment with estrogenprogestin combination (l/l). In endometriotic tissues sampled during the menstrual phase, matrix metalloproteinase-1 mRNA was expressed in one of two ovarian lesions and in the red peritoneal lesion from a patient bleeding on estrogen-progestin treatment. Conversely, matrix , 11 metalloproteinase-i mRNA was not detected in the two rectovaginal nodules sampled during the menstrual phase. When or where matrix metalloproteinase-1 mRNA was not detected in eutopic endometrium, estrogen and P receptors were expressed abundantly in stroma1 cells (1607 and 14/17, respectively) and in epithelial cells (16/17 and 12/17, respectively), in agreement with our previous report (15). Similarly, in endometriotic lesions where no matrix metalloproteinase-1 mRNA was detected, estrogen and P receptors also were expressed in both cell types in a majority of the 47 cases tested (37 and 35 cases for stromal cells; 31 and 27 cases for epithelial cells). Conversely, matrix metalloproteinase-1 mRNA expression was restricted to areas where P receptors were not detected in the adjacent epithelial cells (6/7; Fig. 2E). Finally, both in eutopic and ectopic endometrium, focal expression of matrix metalloproteinase-1 mRNA was related inversely to the integrity of the argyrophilic fibrillar network (Figs. 1E and 2F; Table 1). Matrix breakdown was observed in almost all areas expressing matrix metalloproteinase-1 mRNA (5161, whereas it was observed infrequently in lesions not expressing matrix metalloproteinase1 m.~,rte;an I “L “UUUIU.z
.r,wn9n u1.zacJ
nt-elntr;v “I IllaLd1*
mntollmm.~+r\;n~o, Illr;~arr”yL”~=;lllcr~r;-l
1 c.v r;n-
pression (5/31). DISCUSSION
Endometriotic lesions of the peritoneal lining of the pelvis have various macroscopic appearances,
Fertility
and Sterility@
n-&WA and inverse relation with the integrity Figure 1 Focal expression of matrix metalloproteinase-1 of the argyrophilic fibrillar network. This typical ovarian endometriotic biopsy specimen was obtained during the early secretory phase of the menstrual cycle. Two pairs of sections from the same ribbon, 20 pm apart, were hybridized with the antisense matrix metalloproteinase-13% riboprobe (A to C) or were silver stained (D to F). In the first series, no matrix metalloproteinase-1 mRNA was detected (A) and the argyrophilit fibrillar network (D) was fully preserved. In the second series, matrix metalloproteinase-1 mRNA was detected in foci (B, arrows) where the matrix was extensively broken down (E) but not where the matrix was preserved (C and F). Bar, 100 pm.
which have been proposed to reflect their age aiiruor activity. 1ne rea suotie lesions (vesicumr, red flamelike, and glandular excrescences) are more vascularized and have a higher epithelial mitotic index than the typical, puckered black or bluish peritoneal lesions, whereas the vascularization and the mitotic index are lowest in the white lesions (white opacification, yellow-brown patches, and circular peritoneal defects) (5). Thus, red lesions are thought to correspond to the first, active stage of early implantation of endometrial glands and stroma, and would evolve toward the typical black or bluish lesion after enclosure beneath the peritoneal lining. The white lesions are believed to correspond to fibrotic, quiescent lesions. Similarly, foci of increased vascularization and epithelial mitotic index are present in the wall of ovarian endometriotic cysts and have been thought to correspond to foci of activity (6, 7). In contrast, the rectovaginal nodules show low vascularization and/or mitotic activity, and a different pathogenesis has been proposed for this less active form of disease (8). Foci of matrix metalloproteinase-1 mRNA were detected only in red peritoneal lesions and in the wall of ovarian cysts, i.e., the two types of easily bleeding endometriotic lesions (9) that show at least fneallxr ricma nf a&kT;tv I”YU&IJ “16”” “I U’ Y’.IVJ
(!i_?). \Y
Tbae ~nmncar;~rm “““‘p.aI lU”ll ho_ U”
tween these combined active lesions and the inactive lesions (black peritoneal and rectovaginal) is statistically significant (P = 0.004, Fisher’s exact test). It
Vol. 68, No. 2, August 1997
is likely that the proportion of positive cases in the -A!active lesions would increase if all of them had been analyzed by serial sectioning. The lack of matrix metalloproteinase-1 expression in inactive lesions, even when the corresponding eutopic tissue undergoes menstruation, is further evidence of their inactivity. As previously shown for eutopic perimenstrual endometrium (151, expression of matrix metalloproteinase-1 appears to be correlated with bleeding, because ovarian and red peritoneal endometriosis frequently show hemorrhages at laparoscopic examination (5-7, 9). Matrix breakdown essentially was observed in areas of matrix metalloproteinase-1 expression, as is the case in the eutopic endometrium (15). With respect to peritoneal endometriosis, expression of the metalloproteinase was observed in red but not in black lesions. The former are more superficial and show a prominent vascularization and a conspicuous stroma undergoing differentiation, fibrillar remodeling, and eventual breakdown (5). In the enclosed black lesions, the scanty stroma barely shows menstrual remodeling. Thus, matrix metalloproteinase-1 could be involved, probably in concert with other matrix metalloproteinases, in the implantation of retrograde menstrual fragments (22), generating early, active, rnrl nnrllnr“I LaI”” nlen nn..lrl Qnrl I uu lnainna ICIUIVII”, CIIIW x,“uLAtAnrnr~&n pl” * “1-b hlnarlinm UI~c_da111~UllU tearing of the tissue, so that detached viable fragments could reimplant on the peritoneum and propagate the disease (4). The invasive potential of endo-
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249
that they correspond to a distinct disease, perhaps one that is related more closely to uterine adenomyosis (8). Finally, despite the lack of consistent response of endometriotic tissue to hormonal treatment and the fact that eutopic and ectopic endometrium are not always in phase (251, both sex steroid receptors generally were detected in both cell types of endometriotic tissue not expressing matrix metalloproteinase1, whereas P receptors generally were absent in epithelial cells surrounding stromal foci of matrix metalloproteinase-1. Thus, P still exerts some regulation on ectopic lesions, which challenges the commonly held view that endometriotic lesions are purely autonomous and hormone independent (22). However, the failure of hormonal therapy in the treatment of endometriosis easily could be accounted for by the absence of sex steroid receptors at the critical sites of matrix breakdown, suggesting that a direct pharmacologic inhibition of the proteinase activity could be more efficient for controlling the disease.
Acknowledgments. The authors thank H. Nagase, Ph.D., from the Kansas University Medical Center, Kansas City, Kansas, for the gift of MMP-1 cDNA. The staBof the Department of Pathology is acknowledged for its technical help. This article represents research results of the Belgian Programme on Interuniversity Poles of Attraction and of Concerted Research Actions of the Communaute Francaise de Belgique.
Typical relation between matrix metalloproteinase1 mRNA, P receptors, and argyrophilic fibrillar network. Three biopsies were performed simultaneously in the same patient during the midsecretory phase of the menstrual cycle: one of eutopic endometrium (A to C), one of a red peritoneal endometriotic tissue (D to F), and one of a black peritoneal endometriotic tissue (G to I). Serial sections were hybridized with the antisense matrix metalloproteinase-1 35Sriboprobes (A, D, and Gl, incubated with monoclonal anti-P receptor antibodies (B, E, and I-I), or silver stained (C, F, and I). Matrix metalloproteinase-1 mRNA was detected only in stromal foci of the red peritoneal lesion (D, arrows) that showed no detectable P receptors (E) but did show extensive matrix breakdown (F). When and where no matrix metalloproteinase-1 mRNA was detected (A and G), P receptors were abundantly expressed (B and H) and the argyrophilic fibrillar network was well preserved (C and I). Bar, 100 pm. Figure 2
metriotic peritoneal lesions has been demonstrated by collagen gel invasion assays (23). Expression of matrix metalloproteinase-1 during remodeling of newly metaplastic areas is another pathogenetic possibility. The same considerations apply to ovarian cysts, which have been considered as free or mucosal lesions (24) where the stroma is more developed focally. In contrast, the argyrophilic fibrillar network was well-preserved in rectovaginal nodules and showed no cyclic change, supporting the view
260
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