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We have previously reported that endometrial mRNA expression of both tissue inhibitors of metalloproteinase-4 (TIMP-4) and matrix metalloproteinase-26 (MMP-26) peaks in the early secretory phase, which implies a role in implantation. The objective of this study was to compare the distribution of TIMP-4 and MMP-26 in endometrial tissue and uterine fluid over the menstrual cycle. Endometrial tissue was analysed with in situ hybridization and immunohistochemistry to localize mRNA and protein for TIMP-4 and MMP-26 in the same set of samples. TIMP-4 mRNA was quantified in separated stromal and epithelial cells using real-time PCR. Uterine fluid was analysed with western blotting. TIMP-4 mRNA was exclusively localized to the stroma, whereas MMP-26 mRNA was expressed by epithelial cells. TIMP-4 protein was only occasionally found in the stroma but was consistently present in granules of the apical part of luminal and glandular epithelial cells. TIMP-4, but not MMP-26, was demonstrated in uterine fluid. Thus, TIMP-4 is produced in the stroma only, secreted by stromal cells, taken up by epithelial cells, accumulated in apical granules and finally secreted to the uterine fluid. Maximal expression of MMP-26, and its strongest inhibitor TIMP-4, in the early and mid-secretory phase suggests a role during implantation. MMP-26 is stored in epithelial cells in its active form, is not released spontaneously and is controlled by TIMP-4 in both stroma and uterine fluid.

Objective: To examine the expression pattern of matrix metalloproteinase-26 (MMP-26) mRNA and estrogen receptor-alpha (ER alpha) in normal, hyperplastic, premalignant and malignant endometrial tissue., Design: Experimental study., Setting: Department of Obstetrics and Gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden, Atherosclerosis Research Unit, King Gustav V Research Institute, Karolinska Hospital, Stockholm, Sweden., Methods: We studied MMP-26 mRNA and ER alpha in 36 normal, 7 hyperplastic, 6 premalignant and 19 malignant endometrial samples. Based on histological examination, all normal specimens were classified according to an ideal 28 day menstrual cycle as early, mid, and late proliferative phase, early, mid and late secretory phase and menstrual phase. Samples with hyperplasia were classified as simple or complex. Premalignant samples were represented by complex hyperplasia with atypia. Malignant samples were histologically classified as well, intermediately and poorly differentiated, respectively. Specimens were analyzed using in situ hybridization and real time PCR. ER alpha was localized by immunohistochemistry., Results: Epithelial MMP-26 mRNA expression was highest in the early secretory phase and in endometrial hyperplasia. Expression levels were low in the late secretory and menstrual phase and in malignant samples decreased gradually with dedifferentiation. Expression pattern of MMP-26 mRNA in normal, hyperplastic, premalignant and malignant endometrial tissue strongly co-variated with that of ER alpha., Conclusion: Co-expression of MMP-26 and ER alpha in normal and pathological endometrial tissue suggests possible regulation of MMP-26 gene by estrogen.

Objective: To examine possible estrogen dependent endometrial expression of MMP-26 in vitro., Design: Experimental study., Setting: Department of Obstetrics and Gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden., Methods: We studied MMP-26 mRNA in 14 normal endometrial samples obtained from the proliferative phase of the menstrual cycle. Samples were cultured for five days either with estradiol alone or in combination with progesterone. Samples cultured with ethanol represented control groups. MMP-26 mRNA expression was examined in frozen samples using in situ hybridization. Immunohistochemistry was used to study the presence of estrogen and progesterone receptors in endometrial explants., Results: MMP-26 mRNA expression was highest in fresh (non cultured) samples. Signal intensity decreased during the first two days of culture and was negligable in the following days. Nuclear intensity for estrogen and progesterone receptor was high after five days of culture., Conclusion: We did not find MMP-26 mRNA in vitro expression to be directly estrogen dependent.

Objective: To examine the endometrial expression pattern of messenger RNA (mRNA) for selected matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases-1 (TIMP-1)., Design: Experimental study., Setting: Department of Obstetrics and gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obsterics and Gynecology, University Hospital, Luna, Sweden., Methods: We studied MMP-1, -3, -7, -10, -11, -12, -13, -14, -16, -26 and TIMP-1 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Based on histological examination, all specimens were classified according to an ideal 28 day menstrual cycle as early (n=8), mid (n=6) and late (n=7) proliferative phase, early (n=4), mid (n=4) and late (n=8) secretory phase and menstrual (n=3) phase. mRNA extracted from frozen tissue samples was quantitated using real time PCR., Results: Four distinct patterns of MMP mRNA expression were detected in cycling endometrium. MMP-1, -3, -10, and -12 were expressed predominantly in perimenstrual period, MMP-7 and -11 had highest levels in proliferative phase, MMP-13, -14 and -16 were expressed throughout the cycle and MMP-26 was found to be maximal in periovulatory period. Levels of TIMP-1 mRNA remained unchanged during the cycle., Conclusion: The specific endometrial expression profiles of MMPs during menstrual cycle point to their specific biologic roles during the cycle. MMP-26 exhibits a unique expression pattern.

Objective: Matrix metalloproteinases (MMPs) are key players in the degradation of extracellular matrix and basement membranes, and are thus important in tumor invasion. Recently, MMP-26 (endometase), a novel matrilysin-type member of the MMP family, was cloned from an endometrial tumor. This study examines the expression of MMP-26 mRNA in hyperplastic, premalignant and malignant endometrial samples, and compares with normal endometrial tissue., Methods: Endometrial carcinoma samples (19) were histologically classified as well, intermediately and poorly differentiated. Samples with hyperplasia (n = 15) were classified as simple, complex, or complex with atypia. Normal endometrial specimens (n = 39) were classified according to an ideal 28-day menstrual cycle and subsequently grouped in the early, middle, and late parts of the cycle. All samples were analyzed using in situ hybridization and real time PCR. The probes used for in situ hybridization and real time PCR recognized non-overlapping sequences. MMP-26 protein was localized by immunohistochemistry., Results: MMP-26 mRNA was exclusively localized in the epithelial component of normal, hyperplastic, premalignant, as well as malignant samples. It was not found in the stroma of any tissue category. Quantifications with real time PCR as well as semi-quantifications of the in situ hybridization signal revealed maximal levels in normal tissue at midcycle and in endometrial hyperplasia both with and without atypia. The amount of MMP-26 mRNA decreased progressively with loss of histological differentiation in malignant samples. Immunostaining localized MMP-26 in epithelial glandular and luminal cells, in vessel walls, and in tumor cells. Since the pattern of MMP-26 expression mimicked that of ER-alpha, we searched the MMP-26 promoter region for a potential estrogen response element (ERE). A sequence at position -130 to -116 had high homology to the consensus sequence of an ERE. Based on these observations, we suggest that ER-alpha is involved in regulation of the MMP-26 gene., Conclusions: MMP-26 mRNA is selectively localized in the epithelial compartment of normal, hyperplastic, and malignant endometrial tissue. Expression is high in normal and hyperplastic endometria, but is downregulated in the late part of the cycle and in malignant tumors. The expression pattern of MMP-26 mRNA mimics that of ER-alpha, and the promoter region of the MMP-26 gene has a potential ERE.

We have previously reported that endometrial expression of matrix metalloproteinase (MMP)-26 mRNA comes to a maximum in the early secretory phase. Since tissue inhibitor of metalloproteinase (TIMP)-4 is a potent inhibitor of MMP-26, the objective of this study was to identify the pattern of TIMP-4 mRNA expression in the normal endometrial cycle. We also evaluated hyperplastic, pre-malignant (atypical hyperplasia) and malignant endometrial tissue. Endometrial TIMP-4 mRNA was localized in tissue sections using in situ hybridization, and quantified in tissue extracts using real-time PCR. Estrogen receptor alpha (ERalpha) was assayed in the same set of samples using immunohistochemistry. In situ hybridization demonstrated TIMP-4 mRNA in the stroma of both normal and pathological tissues. TIMP-4 mRNA increased in the proliferative phase to a maximum in the early secretory phase, and then decreased in the late part of the cycle. Expression was comparable in normal and hyperplastic (including atypical) endometrial samples, whereas lower levels were detected in malignant tumours. Since this general pattern of expression suggests estrogen dependence, we evaluated ERalpha in our samples. Tissue sections from the normal proliferative phase, hyperplasia and pre-malignant atypical hyperplasia tissue stained strongly for ERalpha, whereas weak staining was seen in the secretory phase and in malignant tumours. Thus, low level of ERalpha was accompanied by down-regulated TIMP-4 mRNA, supporting the hypothesis that ERalpha contributes to regulation of the TIMP-4 gene. In addition, we identified a putative estrogen response element (ERE) in the promoter region of the TIMP-4 gene at position -930 to -916. Similarities in the cyclic patterns of TIMP-4 mRNA and MMP-26 mRNA, together with the fact that TIMP-4 is a potent inhibitor of MMP-26, suggest a functional relationship, and furthermore a role in human implantation.

Objective: To examine the expression pattern of some novel matrix metalloproteinases (MMPs) in cycling endometrium., Design: Experimental study., Setting: Department of Obstetrics and Gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden., Methods: We studied MMP-12, -16, -17, -19 and -26 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Based on histological examination, all specimens were classified according to an ideal 28 day menstrual cycle as early (n=8), mid (n=6) and late (n=7) proliferative phase, early (n=4), mid (n=4) and late (n=8) secretory phase and menstrual (n=3) phase. Cycle variation was examined in frozen samples using in situ hybridization., Results: Three distinct pattern of MMP mRNA expression were detected in cycling endometrium. MMP-12 was expressed predominantly in perimenstrual period, MMP-16, -17 and 19 were expressed throughout the cycle and MMP-26 was found to be maximal in periovulatory period., Conclusion: Different endometrial expression patterns of novel MMPs during menstrual cycle may indicate their specific roles for menstruation, endometrial growth and remodelling and implantation.

Objective: To summarise which metalloproteinases and specific inhibitors have been already described, their regulatory mechanisms in obstetric complications., Design: A literature review., Setting: Department of Gynecology Obstetric and University Hospital, Palacký University, Olomouc., Abstracts: The invasion of extravillous trophoblast cells into the maternal endometrium is one of the key events in human placentation. The ability of these cells to infiltrate the uterine wall and to anchor the placenta to it as well as their ability to infiltrate and adjust utero-placental vessels to pregnancy depends, among other things, on their ability to secrete enzymes that degrade the extracellular matrix. Most of these enzymes belong to the family of matrix metalloproteinases. In the literature review, we focus on the role of these proteases in normal pregnancy as well as on their importance in the development of some pregnancy complications.

The human endometrium is a dynamic tissue, which undergoes extensive tissue remodelling during the menstrual cycle. Due to their involvement in such processes, several well-characterized matrix metalloproteinases (MMP) have previously been studied in the endometrium. MMP-26 is a newly described matrilysin. We studied MMP-26 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Tissue distribution and cycle variation was examined using in-situ hybridization, Northern blot analyis and real time PCR. The probes for Northern blot analysis and real time PCR recognized non-overlapping sequences. MMP-26 was localized exclusively in epithelial cells of both glands and the luminal surface. Expression increased during the proliferative phase to a maximum at mid-cycle, then decreased to non-detectable levels in the late secretory and menstrual phases. Expression of MMP-26 mRNA in endometrial tissue explants in vitro required stimulation with both estradiol and progesterone. The tissue content of c-jun mRNA was assayed, since c-jun, as part of the enhancer complex AP-1, may be involved in regulation of MMP-26 gene transcription. The pattern of c-jun expression over the menstrual cycle was similar to that of MMP-26. Epithelial expression in the peri- and post-ovulatory stages of the menstrual cycle suggests the involvement of MMP-26 in reproductive processes.

Objective: To summarise which metalloproteinases and specific inhibitors have been already described, their regulatory mechanisms, the connection with implantation and in cases of tumour invasion. In conclusion we would like to point to the possibilities of practical use of this information., Design: A literature review., Setting: Department of Obstetrics and Gynecology, Medical Faculty, Palacký University, Olomouc., Abstracts: Metalloproteinases are important for many biological processes including for instance cell proliferation, differentiation and remodelation of extracellular matrix (ECM) or vascularisation and cell migration. These events are in progress during organogenesis both in the normal development and in tumour progression. Mechanisms of activity of metalloproteinases during these processes include proteolysis of growth factors which in this way become available to cells. The motion of cells through tissues and fission of signal receptors made possible by degradation of the ECM is vital for cell migration. The majority of these processes requires a balanced equilibrium between the activity of matrix metalloproteinases (MMP) and their natural tissue inhibitors (TIMP). It is precisely this equilibrium between metalloproteinases and their tissue inhibitors that is decisive for the site and extent trophoblast invasion during embryo implantation and as well as for the cell invasion during tumour progression. In our literary review we would like to shortly summarize which metalloproteinases and specific inhibitors have been already described and what are their regulatory mechanisms. Which of them were detected in tissues in connection with implantation and which are most often expressed in cases of tumour invasion. In conclusion we would like to point to the possibilities of practical use of this information.

During every menstrual cycle the extracellular matrix in the uterus undergoes dramatic changes associated with release and shedding of the endometrium. Classical investigations made in monkeys assumed that menstruation is caused by vasoconstriction, hypoxia and subsequent necrosis. More recent work indicates the importance of the system of the system of matrix metalloproteinases (MMP) for the extracellular architecture of the endometrium. The submitted paper presents a review of crucial characteristics of the MMP system and its importance for cyclic transformation of the endometrium during the menstrual cycle. From the review it is apparent that impairment of the MMP equilibrium and its tissue inhibitors are an essential prerequisite of tissue degradation and menstruation. MMP activation is regulated by hypoxia, a drop progesterone and paracrine factors released by epithelial, stromal and migratory cells.