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Difference in expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 in patients with persistent ovarian cysts
Difference in expression of matrix metalloproteinase2 and matrix metalloproteinase-9 in patients with persistent ovarian cysts Matrix metalloproteinase-1 (MMP-1), MMP-2, and MMP-9 expression with real-time quantitative polymerase chain reaction were analyzed in endometriotic and nonendometriotic ovarian cysts. Although MMP-1 was not detected, MMP-9 and MMP-2 were expressed in all of the cysts. In particular, in five of six nonendometriotic cysts (83.3%) MMP-2 expression was higher than in endometriotic cysts. These data may represent new molecular elements helpful in differential diagnosis of endometriotic lesions. (Fertil Steril威 2005;84:1049 –52. ©2005 by American Society for Reproductive Medicine.)
In women of reproductive age, ovarian cysts are more frequently observed by gynecologists compared to those encountered by pathologists, because, in most cases, they involute spontaneously or after a course of hormonal therapy (HT). Patients with persistent ovarian cysts for more than 6 months, which do not respond to HT, usually should undergo surgical treatment and subsequent histopathological examination. The differential diagnosis of endometriotic lesions compared to nonendometriotic lesions is important for the management of these patients to establish an adequate pharmacologic therapy. Sometimes, the evaluation, during surgery, of the presence of a cyst that contains dark brown fluid or the presence of pelvic synechia may indicate an endometriotic lesion, but it should be emphasized that hemorrhaging occurs in many ovarian cysts and also in neoplastic lesions. The diagnosis of endometriotic disease may be clinically suspected by the patient’s history and by the surgeon’s evaluation, but it can only be established by histopathological evaluation. Yet, sometimes the histological diagnosis of endometriosis is very difficult or even impossible because of the changes occurring in the lesions after bleeding and fibrosis. Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopeptidases secreted by various cells that are involved in the extracellular matrix and basement membrane degradation. They play an important role in many physiological processes, such as cell migration and neural growth, and also in pathological processes (1). In addition, MMPs have been implicated in the cancer progression and imetastatic diffusion (2, 3). The MMPs are regulated by hormones, growth factors, and cytokines, and are involved in ovarian function (4, 5).
Received November 8, 2004; revised and accepted February 4, 2005. Reprint requests: Maria Rosaria Raspollini, M.D., Department of Human Pathology and Oncology, University of Florence, School of Medicine, viale G.B. Morgagni, 85, 50134 Florence, Italy (FAX: 39-055-4379868; E-mail: [email protected]).
0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2005.02.058
The role of MMP-1, MMP-2, and MMP-9 in the pathogenesis of polycystic ovary syndrome (PCOS) has recently been investigated by Lahav-Baratz et al. (6) and by BenShlomo et al. (7). Some studies were also focusing on the expression of MMPs in endometriosis (8 –10). However, to the best of our knowledge, the expression of MMPs has never been compared in endometriotic and nonendometriotic cysts. The aim of the study was to analyze the expression levels of MMP-1, MMP-2, and MMP-9 in endometriotic and nonendometriotic ovarian cysts with real-time quantitative polymerase chain reaction (PCR) in fresh tissue to identify a possible molecular marker in cases with difficult differential diagnosis of endometriotic lesions. Ovarian cysts were removed from 15 patients who underwent laparoscopy for persistent ovarian cysts (⬎6 months) at the Department of Gynecology, Perinatology and Reproductive Medicine of the University of Florence. Histopathologic examination and quantification for expression levels of MMP-1, MMP-2, and MMP-9 were done at the Department of Human Pathology and Oncology of the University of Florence. Patient and family medical histories were obtained. Patients had undergone pelvic ultrasonography every 3 months since the diagnosis. After completion of surgical treatment, the follow-up consisted of a pelvic examination and ultrasonogram every 6 months. The patients with a clinical and histologic diagnosis of endometriosis subsequently underwent therapy with GnRH analogues (GnRH-a). Fifteen ovarian samples were harvested in the operating room and were immediately immersed in RNAlater (Qiagen, Hilden, Germany) and stored at ⫺80°C in our tissue bank. Additional tissue samples were used for histopathologic examination. All samples were collected for therapeutic or diagnostic purposes, with the informed consent of each patient.
Fertility and Sterility姞 Vol. 84, No. 4, October 2005 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.
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Ribonucleic acid was isolated from fresh tissue. The samples, which were previously immersed in RNAlater (Qiagen) and stored at ⫺80°C, were thawed, cut in small pieces, and homogenized. The RNA was extracted with 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Then, 20-m sections from formalin-fixed paraffin embedded tissues were deparaffinized with xylene/ethanol and incubated overnight at 37°C with 250 g/mL proteinase K. Total RNA was isolated with 6100 Nucleic Acid PrepStation. Total RNA (500 ng) was reverse transcribed to cDNA using High Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer’s instructions. Realtime quantitative PCR was performed on an ABI PRISM 7000 Sequence Detector System (Applied Biosystems). The PCR products for MMP-1, MMP-2, and MMP-9 were detected using gene-specific primers and probes labeled with reporter day FAM (Assay on Demand, Applied Biosystems). gAPDH was used as endogenous control gene for normalization. The PCR reactions were carried out in 96-well plates with 20 L/well using 1⫻ TaqMan Universal PCR MasterMix. After an incubation for 2 minutes at 50°C and 10 minutes at 95°C, the reaction continued for 50 cycles at 95°C for 15 seconds and 60°C for 1 minute. The 2(⫺⌬ ⌬C(t)) method described by Livak and Schmittgen (11) was used to analyze the results. In brief, the Ct (threshold cycle) value of the MMP gene was subtracted from the Ct value of a housekeeping gene (gAPDH) as a standard for the amount of RNA template and efficiencies of reverse transcription. The relative quantities of the targets (nonendometriotic ovarian cysts) were compared to the relative quantities of the ovarian endometriotic cysts. The resulting change in Ct values was then converted to a linear form using 2(⫺⌬ ⌬Ct) and used in subsequent statistical analysis. The different expression levels of MMP-2 and MMP-9 in endometriotic and nonendometriotic ovarian cysts were calculated according to the Wilcoxon Mann-Whitney test (12). Data analysis was performed using the SPSS version 11.0 (Chicago, IL) statistical package. A P value ⱕ.05 was considered to be statistically significant. The histologic evaluation of the persistent ovarian cysts documented endometriotic tissue in 9 cases and the presence of paraovarian cysts (13) in 6 cases. The RNA extraction was successful in all patients and we evaluated the MMP-1, MMP-2, and MMP-9 expression levels in the ovarian cysts. The MMP expression was measured in comparison to housekeeping gene (gAPDH). 1050
Raspollini et al.
Correspondence
MMP-1 was not expressed in ovarian cyst samples. In paraovarian cysts, the MMP-9 expression was between 0.001 and 0.208. In ovarian endometriotic cysts the MMP-9 expression was between 0.001 and 1.268. In paraovarian cysts, the MMP-2 expression was between 0.119 and 62.987. In ovarian endometriotic cysts the MMP-2 expression was between 0.001 and 79.434. In 3 (50%) paraovarian cysts MMP-9 was expressed higher than the mean MMP-9 expression of ovarian endometriotic cysts. In 5 (83.3%) paraovarian cysts MMP-2 was expressed higher than the mean MMP-2 expression of ovarian endometriotic cysts (Fig. 1). The data did not have statistical significance; however, the MMP-2 expression in paraovarian cysts with regard to the mean MMP-2 expression of ovarian endometriotic cysts showed a trend to correlation (P⫽.4, according to the Wilcoxon Mann-Whitney test). In the present study using real-time PCR analysis we investigated different expression levels of three MMPs in paraovarian cysts compared to the mean expression of ovarian endometriotic cysts. The most consistent finding was the different expression levels of MMP-2 between paraovarian cysts and ovarian endometriotic cysts. In fact, in almost all patients with paraovarian cysts MMP-2 was expressed at a higher level than the mean MMP-2 expression in ovarian endometriotic cysts. We observed that MMP-9 was expressed both in paraovarian cysts and endometriotic cysts, whereas MMP-1 was not expressed in paraovarian cysts nor in ovarian endometriotic cysts. Changes in RNA levels for several proteins have an important role in most pathological processes. In recent years new technologies allowed the study of gene expression. Unlike any other technology in molecular biology, PCR has changed the technological armamentarium of molecular scientists working on tissue, in terms of greatly improving sensitivity and accuracy. The determination of mRNA expression levels by real-time quantitative PCR appears to be the most reliable method for accurate determination of gene expression levels in tissues. This technology offers outstanding sensitivity and accuracy in terms of determination of the amount of cDNA molecules. The MMPs are a family of protein that degrade various components of the extracellular matrix. The MMPs have been implicated in tumor cell invasion and metastases (14) and in the pathogenesis of endometriosis (9, 15–18). Our data on expression of MMP-9 in endometriotic lesions support the concept expressed in recent studies (10, 14), that is, extracellular matrix degradation may be important to establish endometriotic lesions, and possibly for their maintenance. In addition, our finding of a lower mean MMP-2 RNA expression in histologically documented endometriotic Vol. 84, No. 4, October 2005
FIGURE 1 In five of six nonendometriotic cysts (83.3%) MMP-2 expression (y axis) was higher than in endometriotic cysts (x axis).
Raspollini. MMP-2 and MMP-9 in ovarian cysts. Fertil Steril 2005.
cysts, compared to MMP-2 RNA expression in nonendometriotic lesions, suggests that different genes are expressed in the development and maintenance of these lesions. These data may represent new molecular elements helpful in the differential diagnosis of endometriotic lesions. Further studies, focusing on the genome expression, may support this concept and give pathologists additional molecular elements to combine with morphological and immunohistochemical data to achieve this difficult differential diagnosis. Moreover, these data, when confirmed by other analyses, may provide useful information for finding candidate genes whose products could serve as molecular targets for the management of endometriotic lesions (19). Maria Rosaria Raspollini, M.D.a Francesca Castiglione, M.D.a Duccio Rossi Degl’Innocenti, B.Sc.a Francesca Garbini, M.D.a Maria Elisabetta Coccia, M.D.b Gian Luigi Taddei, M.D.a Departments of aHuman Pathology and Oncology and b Gynecology, Perinatology and Reproductive Medicine, University of Florence, School of Medicine, Florence, Italy REFERENCES 1. Salamonsen LA. Matrix metalloproteinase and their tissue inhibitors in endocrinology. Trend Endocrinol Metab 1996;7:28 –34. 2. Shiozawa J, Ito M, Nakayama T, Nakashima M, Kohno S, Semine I. Expression of matrix metalloproteinase-1 in human colorectal carcinoma. Mod Pathol 2000;13:925–33.
Fertility and Sterility姞
3. Manetti L, Paganoni P, Floriani I, Bandoni F, Torri V, Buda A, et al. Expression levels of vascular endothelial growth factor, matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases 1 and 2 in the plasma of patients with ovarian carcinoma. Eur J Cancer 2003;39:1948 –56. 4. Hulboy DL, Rudolph LA, Matrisian LM. Matrix metalloproteinase as mediators of reproduction function. Mol Hum Reprod 1997;3:27– 45. 5. McIntush EW, Smith MF. Matrix metalloproteinase and tissue inhibitors of metalloproteinase in ovarian function. Rev Reprod 1998;3:23–30. 6. Lahav-Baratz S, Kraiem Z, Shiloh H, Koifman M, Ishai D, Dirnfeld M. Decreased expression of tissue inhibitor of matix metalloproteinases in follicular fluid from women with polycystic ovaries compared with normally ovulating patients undergoing in vitro fertilization. Fertil Steril 2003;79:567–71. 7. Ben-Shlomo I, Goldman S, Shalev E. Regulation of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of MMP, and progesterone secretion in luteinized granulosa cells from normally ovulating women with polycystic ovary disease. Fertil Steril 2003;79(Suppl 1): 694 –701. 8. Huang HF, Hong LH, Tan Y, Sheng JZ. Matrix metalloproteinase 2 is associated with changes in steroid hormones in the sera and peritoneal fluid of patients with endometriosis. Fertil Steril 2004;81:1235–9. 9. Wolber EM, Kressin P, Meyhofer-Malik A, Diedrich K, Malik E. Differential induction of matrix metalloproteinase 1 and 2 in ectopic endometrium. Reprod Biomed Online 2003;6:238 – 43. 10. Collette T, Bellehumeur C, Kats R, Maheux R, Mailloux J, Villeneuve M, et al. Evidence for an increased release of proteolytic activity by the eutopic endometrial tissue in women with endometriosis and for involvement of matrix metalloproteinase-9. Hum Reprod 2004;19:1257– 64. 11. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-⌬⌬C(T)) method. Methods 2001;25:402– 8. 12. Fisher LD, Van Belle G. Biostatistics: a methodology for the health sciences. 2nd ed., 3th serie. New York: Wiley Interscience Publication, 1993.
1051
13. Robboy SJ, Anderson MA, Russell P. Pathology of the female reproductive tract. Toronto: Churchill Livingstone, 2002. 14. Huang LW, Garrett AP, Bell DA, Welch WR, Berkowitz RS, Mok SC. Differential expression of matrix metalloproteinase-9 and tissue inhibition of metalloproteinase-1 protein in mRNA in epithelial ovarian tumors. Gynecol Oncol 2000;77:369 –76. 15. Witz C. Current concepts in the pathogenesis of endometriosis. Clin Obstet Gynecol 1999;42:566 – 85. 16. Zhou HE, Nothnick WB. The relevancy of the matrix metalloproteinase system to the pathophysiology of endometriosis. Front Biosci 2005;10:569 –75.
1052
Raspollini et al.
Correspondence
17. Ramon L, Gilabert-Estelles J, Castello R, Gilabert J, Espana F, Romeu A, et al. mRNA analysis of several components of the plasminogen activator and matrix metalloproteinase systems in endometriosis using a real-time quantitative RT-PCR assay. Hum Reprod 2005;20:272– 8. 18. Mulayim N, Savlu A, Guzeloglu-Kayisli O, Kayisli UA, Arici A. Regulation of endometrial stromal cell matrix metalloproteinase activity and invasiveness by interleukin-8. Fertil Steril 2004;81 (Suppl 1):904 –11. 19. Nezhat FR, Kalir T. Comparative immunohistochemical studies of endometriosis lesion and endometriotic cysts. Fertil Steril 2002;78:820 – 4.
Vol. 84, No. 4, October 2005
In women of reproductive age, ovarian cysts are more frequently observed by gynecologists compared to those encountered by pathologists, because, in most cases, they involute spontaneously or after a course of hormonal therapy (HT). Patients with persistent ovarian cysts for more than 6 months, which do not respond to HT, usually should undergo surgical treatment and subsequent histopathological examination. The differential diagnosis of endometriotic lesions compared to nonendometriotic lesions is important for the management of these patients to establish an adequate pharmacologic therapy. Sometimes, the evaluation, during surgery, of the presence of a cyst that contains dark brown fluid or the presence of pelvic synechia may indicate an endometriotic lesion, but it should be emphasized that hemorrhaging occurs in many ovarian cysts and also in neoplastic lesions. The diagnosis of endometriotic disease may be clinically suspected by the patient’s history and by the surgeon’s evaluation, but it can only be established by histopathological evaluation. Yet, sometimes the histological diagnosis of endometriosis is very difficult or even impossible because of the changes occurring in the lesions after bleeding and fibrosis. Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopeptidases secreted by various cells that are involved in the extracellular matrix and basement membrane degradation. They play an important role in many physiological processes, such as cell migration and neural growth, and also in pathological processes (1). In addition, MMPs have been implicated in the cancer progression and imetastatic diffusion (2, 3). The MMPs are regulated by hormones, growth factors, and cytokines, and are involved in ovarian function (4, 5).
Received November 8, 2004; revised and accepted February 4, 2005. Reprint requests: Maria Rosaria Raspollini, M.D., Department of Human Pathology and Oncology, University of Florence, School of Medicine, viale G.B. Morgagni, 85, 50134 Florence, Italy (FAX: 39-055-4379868; E-mail: [email protected]).
0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2005.02.058
The role of MMP-1, MMP-2, and MMP-9 in the pathogenesis of polycystic ovary syndrome (PCOS) has recently been investigated by Lahav-Baratz et al. (6) and by BenShlomo et al. (7). Some studies were also focusing on the expression of MMPs in endometriosis (8 –10). However, to the best of our knowledge, the expression of MMPs has never been compared in endometriotic and nonendometriotic cysts. The aim of the study was to analyze the expression levels of MMP-1, MMP-2, and MMP-9 in endometriotic and nonendometriotic ovarian cysts with real-time quantitative polymerase chain reaction (PCR) in fresh tissue to identify a possible molecular marker in cases with difficult differential diagnosis of endometriotic lesions. Ovarian cysts were removed from 15 patients who underwent laparoscopy for persistent ovarian cysts (⬎6 months) at the Department of Gynecology, Perinatology and Reproductive Medicine of the University of Florence. Histopathologic examination and quantification for expression levels of MMP-1, MMP-2, and MMP-9 were done at the Department of Human Pathology and Oncology of the University of Florence. Patient and family medical histories were obtained. Patients had undergone pelvic ultrasonography every 3 months since the diagnosis. After completion of surgical treatment, the follow-up consisted of a pelvic examination and ultrasonogram every 6 months. The patients with a clinical and histologic diagnosis of endometriosis subsequently underwent therapy with GnRH analogues (GnRH-a). Fifteen ovarian samples were harvested in the operating room and were immediately immersed in RNAlater (Qiagen, Hilden, Germany) and stored at ⫺80°C in our tissue bank. Additional tissue samples were used for histopathologic examination. All samples were collected for therapeutic or diagnostic purposes, with the informed consent of each patient.
Fertility and Sterility姞 Vol. 84, No. 4, October 2005 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.
1049
Ribonucleic acid was isolated from fresh tissue. The samples, which were previously immersed in RNAlater (Qiagen) and stored at ⫺80°C, were thawed, cut in small pieces, and homogenized. The RNA was extracted with 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Then, 20-m sections from formalin-fixed paraffin embedded tissues were deparaffinized with xylene/ethanol and incubated overnight at 37°C with 250 g/mL proteinase K. Total RNA was isolated with 6100 Nucleic Acid PrepStation. Total RNA (500 ng) was reverse transcribed to cDNA using High Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer’s instructions. Realtime quantitative PCR was performed on an ABI PRISM 7000 Sequence Detector System (Applied Biosystems). The PCR products for MMP-1, MMP-2, and MMP-9 were detected using gene-specific primers and probes labeled with reporter day FAM (Assay on Demand, Applied Biosystems). gAPDH was used as endogenous control gene for normalization. The PCR reactions were carried out in 96-well plates with 20 L/well using 1⫻ TaqMan Universal PCR MasterMix. After an incubation for 2 minutes at 50°C and 10 minutes at 95°C, the reaction continued for 50 cycles at 95°C for 15 seconds and 60°C for 1 minute. The 2(⫺⌬ ⌬C(t)) method described by Livak and Schmittgen (11) was used to analyze the results. In brief, the Ct (threshold cycle) value of the MMP gene was subtracted from the Ct value of a housekeeping gene (gAPDH) as a standard for the amount of RNA template and efficiencies of reverse transcription. The relative quantities of the targets (nonendometriotic ovarian cysts) were compared to the relative quantities of the ovarian endometriotic cysts. The resulting change in Ct values was then converted to a linear form using 2(⫺⌬ ⌬Ct) and used in subsequent statistical analysis. The different expression levels of MMP-2 and MMP-9 in endometriotic and nonendometriotic ovarian cysts were calculated according to the Wilcoxon Mann-Whitney test (12). Data analysis was performed using the SPSS version 11.0 (Chicago, IL) statistical package. A P value ⱕ.05 was considered to be statistically significant. The histologic evaluation of the persistent ovarian cysts documented endometriotic tissue in 9 cases and the presence of paraovarian cysts (13) in 6 cases. The RNA extraction was successful in all patients and we evaluated the MMP-1, MMP-2, and MMP-9 expression levels in the ovarian cysts. The MMP expression was measured in comparison to housekeeping gene (gAPDH). 1050
Raspollini et al.
Correspondence
MMP-1 was not expressed in ovarian cyst samples. In paraovarian cysts, the MMP-9 expression was between 0.001 and 0.208. In ovarian endometriotic cysts the MMP-9 expression was between 0.001 and 1.268. In paraovarian cysts, the MMP-2 expression was between 0.119 and 62.987. In ovarian endometriotic cysts the MMP-2 expression was between 0.001 and 79.434. In 3 (50%) paraovarian cysts MMP-9 was expressed higher than the mean MMP-9 expression of ovarian endometriotic cysts. In 5 (83.3%) paraovarian cysts MMP-2 was expressed higher than the mean MMP-2 expression of ovarian endometriotic cysts (Fig. 1). The data did not have statistical significance; however, the MMP-2 expression in paraovarian cysts with regard to the mean MMP-2 expression of ovarian endometriotic cysts showed a trend to correlation (P⫽.4, according to the Wilcoxon Mann-Whitney test). In the present study using real-time PCR analysis we investigated different expression levels of three MMPs in paraovarian cysts compared to the mean expression of ovarian endometriotic cysts. The most consistent finding was the different expression levels of MMP-2 between paraovarian cysts and ovarian endometriotic cysts. In fact, in almost all patients with paraovarian cysts MMP-2 was expressed at a higher level than the mean MMP-2 expression in ovarian endometriotic cysts. We observed that MMP-9 was expressed both in paraovarian cysts and endometriotic cysts, whereas MMP-1 was not expressed in paraovarian cysts nor in ovarian endometriotic cysts. Changes in RNA levels for several proteins have an important role in most pathological processes. In recent years new technologies allowed the study of gene expression. Unlike any other technology in molecular biology, PCR has changed the technological armamentarium of molecular scientists working on tissue, in terms of greatly improving sensitivity and accuracy. The determination of mRNA expression levels by real-time quantitative PCR appears to be the most reliable method for accurate determination of gene expression levels in tissues. This technology offers outstanding sensitivity and accuracy in terms of determination of the amount of cDNA molecules. The MMPs are a family of protein that degrade various components of the extracellular matrix. The MMPs have been implicated in tumor cell invasion and metastases (14) and in the pathogenesis of endometriosis (9, 15–18). Our data on expression of MMP-9 in endometriotic lesions support the concept expressed in recent studies (10, 14), that is, extracellular matrix degradation may be important to establish endometriotic lesions, and possibly for their maintenance. In addition, our finding of a lower mean MMP-2 RNA expression in histologically documented endometriotic Vol. 84, No. 4, October 2005
FIGURE 1 In five of six nonendometriotic cysts (83.3%) MMP-2 expression (y axis) was higher than in endometriotic cysts (x axis).
Raspollini. MMP-2 and MMP-9 in ovarian cysts. Fertil Steril 2005.
cysts, compared to MMP-2 RNA expression in nonendometriotic lesions, suggests that different genes are expressed in the development and maintenance of these lesions. These data may represent new molecular elements helpful in the differential diagnosis of endometriotic lesions. Further studies, focusing on the genome expression, may support this concept and give pathologists additional molecular elements to combine with morphological and immunohistochemical data to achieve this difficult differential diagnosis. Moreover, these data, when confirmed by other analyses, may provide useful information for finding candidate genes whose products could serve as molecular targets for the management of endometriotic lesions (19). Maria Rosaria Raspollini, M.D.a Francesca Castiglione, M.D.a Duccio Rossi Degl’Innocenti, B.Sc.a Francesca Garbini, M.D.a Maria Elisabetta Coccia, M.D.b Gian Luigi Taddei, M.D.a Departments of aHuman Pathology and Oncology and b Gynecology, Perinatology and Reproductive Medicine, University of Florence, School of Medicine, Florence, Italy REFERENCES 1. Salamonsen LA. Matrix metalloproteinase and their tissue inhibitors in endocrinology. Trend Endocrinol Metab 1996;7:28 –34. 2. Shiozawa J, Ito M, Nakayama T, Nakashima M, Kohno S, Semine I. Expression of matrix metalloproteinase-1 in human colorectal carcinoma. Mod Pathol 2000;13:925–33.
Fertility and Sterility姞
3. Manetti L, Paganoni P, Floriani I, Bandoni F, Torri V, Buda A, et al. Expression levels of vascular endothelial growth factor, matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases 1 and 2 in the plasma of patients with ovarian carcinoma. Eur J Cancer 2003;39:1948 –56. 4. Hulboy DL, Rudolph LA, Matrisian LM. Matrix metalloproteinase as mediators of reproduction function. Mol Hum Reprod 1997;3:27– 45. 5. McIntush EW, Smith MF. Matrix metalloproteinase and tissue inhibitors of metalloproteinase in ovarian function. Rev Reprod 1998;3:23–30. 6. Lahav-Baratz S, Kraiem Z, Shiloh H, Koifman M, Ishai D, Dirnfeld M. Decreased expression of tissue inhibitor of matix metalloproteinases in follicular fluid from women with polycystic ovaries compared with normally ovulating patients undergoing in vitro fertilization. Fertil Steril 2003;79:567–71. 7. Ben-Shlomo I, Goldman S, Shalev E. Regulation of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of MMP, and progesterone secretion in luteinized granulosa cells from normally ovulating women with polycystic ovary disease. Fertil Steril 2003;79(Suppl 1): 694 –701. 8. Huang HF, Hong LH, Tan Y, Sheng JZ. Matrix metalloproteinase 2 is associated with changes in steroid hormones in the sera and peritoneal fluid of patients with endometriosis. Fertil Steril 2004;81:1235–9. 9. Wolber EM, Kressin P, Meyhofer-Malik A, Diedrich K, Malik E. Differential induction of matrix metalloproteinase 1 and 2 in ectopic endometrium. Reprod Biomed Online 2003;6:238 – 43. 10. Collette T, Bellehumeur C, Kats R, Maheux R, Mailloux J, Villeneuve M, et al. Evidence for an increased release of proteolytic activity by the eutopic endometrial tissue in women with endometriosis and for involvement of matrix metalloproteinase-9. Hum Reprod 2004;19:1257– 64. 11. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-⌬⌬C(T)) method. Methods 2001;25:402– 8. 12. Fisher LD, Van Belle G. Biostatistics: a methodology for the health sciences. 2nd ed., 3th serie. New York: Wiley Interscience Publication, 1993.
1051
13. Robboy SJ, Anderson MA, Russell P. Pathology of the female reproductive tract. Toronto: Churchill Livingstone, 2002. 14. Huang LW, Garrett AP, Bell DA, Welch WR, Berkowitz RS, Mok SC. Differential expression of matrix metalloproteinase-9 and tissue inhibition of metalloproteinase-1 protein in mRNA in epithelial ovarian tumors. Gynecol Oncol 2000;77:369 –76. 15. Witz C. Current concepts in the pathogenesis of endometriosis. Clin Obstet Gynecol 1999;42:566 – 85. 16. Zhou HE, Nothnick WB. The relevancy of the matrix metalloproteinase system to the pathophysiology of endometriosis. Front Biosci 2005;10:569 –75.
1052
Raspollini et al.
Correspondence
17. Ramon L, Gilabert-Estelles J, Castello R, Gilabert J, Espana F, Romeu A, et al. mRNA analysis of several components of the plasminogen activator and matrix metalloproteinase systems in endometriosis using a real-time quantitative RT-PCR assay. Hum Reprod 2005;20:272– 8. 18. Mulayim N, Savlu A, Guzeloglu-Kayisli O, Kayisli UA, Arici A. Regulation of endometrial stromal cell matrix metalloproteinase activity and invasiveness by interleukin-8. Fertil Steril 2004;81 (Suppl 1):904 –11. 19. Nezhat FR, Kalir T. Comparative immunohistochemical studies of endometriosis lesion and endometriotic cysts. Fertil Steril 2002;78:820 – 4.
Vol. 84, No. 4, October 2005