Mais les résultats doivent être attendus longtemps et il n'y a généralement pas de temps metronidazole prix L'autre cas, c'est que l'achat d'un ou d'un autre antibiotique dans une pharmacie classique nécessite des dépenses matérielles considérables et pas toutes les personnes ne peuvent acheter des produits pharmaceutiques aussi coûteux.

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Journal of Reproduction & Contraception (2005) 16 (4):225-234 Multifactor Regulation on Expressions of MMP-9 and
TIMP-1 in Endometrial Stromal Cells
Xue-mei LIU, Gang ZHONG, Feng-li SONG, Li YIN Department of Obstetric and Gynecology, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China Objective To investigate the regulatory effect of mult ifactor on the matrix
metalloproteinases-9 (MMP-9) and the tissue inhibitor of metalloproteinase-1 (TIMP-1) Methods The endometrial stromal cells separated from the proliferative endometrial
ti ssues were i ncubated with medi um al one, 17-β est radiol (E2, 10-8 mol /L ), medroxyprogesterone acetate (MPA, 10-6 mol/L), E2 (10-8 mol/L)+MPA (10-6 mol/L), E2 (10-8 mol/L)+MPA (10-6 mol/L)+RU486 (10-5 mol/L) or HB-EGF (10 ng/ml) for 48 h respectively. The expressions of MMP-9 and TIMP-1 were detected by in situ hybridization, immunocytochemistry, reverse transcriptase-polymerase chain reaction Results Compared with control group [mRNA, 0.729 ± 0.090 (MMP-9) and 1.056 ±
0.154 (TIMP-1); protein, 0.545 ± 0.086 (MMP-9) and 0.745 ± 0.154 (TIMP-1)], expressions of MMP-9 and TIMP-1 in E2 alone, progestin alone or E2 combined with progestin group were respectively: mRNA, 0.413 ± 0.069, 0.402 ± 0.073 and 0.407± 0.039; 0.487 ± 0.093, 0.503 ± 0.093 and 0.468 ± 0.075; protein, 0.294 ± 0.076,0.331 ± 0.064 and 0.265 ± 0.049; 0.425 ± 0.085, 0.397 ± 0.065 and 0.435 ± 0.099. RU486 weakened the expression level of down-regulation, while HB-EGF elevated the level of MMP-9 and TIMP-1 after 48 h treatment (mRNA, 0.955 ± 0.068 and 1.396 ± 0.238; protein, 0.780 ± 0.109 and 0.985 ± 0.165). Conclusions 1) Both E2 and progestin can down-regulate the expressions of MMP-9
and TIMP-1 in endometrial stromal cells, but RU486 can inhibit the effect. 2) HB-EGF can elevate the level of MMP-9 and TIMP-1. 3) E2, progestin and HB-EGF have effect Key words: matrix metalloproteinases-9 (MMP-9); tissue inhibitor of metalloproteinase-1
(TIMP-1); endometrial stromal cell; regulation Corresponding author: Xue-mei LIU; E-mail: [email protected] Recent evidence suggests that preparation of the endometrium for implantation is not merely a question of adequate hormonal stimulation but that implantation also depends on theinteraction between the blastocyst and the endometrium and is mediated by cytokines, growthfactors, and adhesion molecules, which are produced and secreted by the endometrium andthe blastocyst[1]. Implantation is a complex process that involves embryo apposition andattachment to the maternal endometrial epithelium, the extracellular matrix proteolysis ofendomtrium, and invasion into the endometrial stroma. The extracellular matrix proteolysisof endomtrium is the most important in the process of implantation. Biological extracellularmatrix proteolysis of endometrium depends on the balance of the levels between activatedmatrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Recentresearches suggest that MMP-9 and TIMP-1 play an important role in the process [2, 3]. However,there are few researches on the regulations of MMP-9 and TIMP-1. To discuss the effectsof MMP-9 and TIMP-1 in implantation, we investigate the regulatory effect of multifactoron MMP-9 and TIMP-1 in proliferative endometrial stromal cells.
Materials & Methods
Materials
Endometrial samples (15 cases) from regularly cycling women were collected at prolif- erative phase (d 0-14) of the menstrual cycle after hysterectomy. DMEM/F12 (1:1)
(Invitrogen, USA). Phenol red-free DMEM/F12 (1:1) (Hyclone, USA). FBS (Hyclone, USA).
Charcoal-stripped FBS (Invitrogen). 17-β estradiol (E2), MPA, RU486, HB-EGF (Sigma,
USA). MMP-9, TIMP-1 in situ hybridization assay (Boster Biological Technology Co., Ltd,
Wuhan). Goat polyclonal anti-MMP-9 antibody, goat polyclonal anti-TIMP-1 antibody (Santa
Cruz Biotechnology). Mouse monoclonal anti-vimentin antibody, Goat SP Kit, DAB Kit
(Zhongshan Biotechnology). Trizol (Invitrogen). RT-PCR system (Promega, USA); ECL
Western blotting detection reagents (Pierce Biotechnology, USA).
Isolation, cell culture and grouping of human endometrial stromal cells
Hysteretomy was performed for uterine myomas. The stromal cells were separated from endometrial tissue by mechanical dissociation and collagenase digestion, followed byfiltration through a 40 µm sieve. The stromal cells were found in the filtrate and were pelleted,washed, and suspended in DMEM/F12 (1:1) containing antibiotics and 10% FBS[4]. Thecells were incubated at 37℃ in a 5% CO incubator. The purity (98%) of the isolated cells was assessed by immunocytochemistry.
The stromal cells were obtained from each endometrial sample. The cells (near to 70%-80% density, about 72 h) were exposed in phenol red-free DMEM/F12 (1:1) supple-mented with 0.5% charcoal-stripped FBS and 1% penicillin-streptomycin for 24 h. Then thecells were exposed to medium alone (control) or medium containing 17-β estradiol (10-8 mol/L) alone or in combination with MPA (10-6 mol/L), or with 10 ng/ml heparin-bindingepidermal growth factor (HB-EGF) [5]. The hormones were added from 1 000-fold concentratedstocks prepared in pure ethanol. The appropriate vehicle (ethanol) was added to controlcultures in each experiment. In certain experiments, the antagonist of antiprogestin (RU486,10-5 mol/L)[5] was also added to endometrial stromal cells along with E2 and MPA. Thetreatment media consisted of phenol red-free DMEM/F12 (1:1) supplemented with 0.5%charcoal-stripped FBS and 1% penicillin-streptomycin. The time of exposure was 48 h.
Every experimental group and control group all included three wells.
In situ hybridization
After termination of treatment, cells were rinsed in PBS thrice for 5 min, and then fixed in 4% paraformaldehyde in PBS (0.1 mol/L) for 30 min at room temperature. Hybridizationwas carried out in a humidified chamber using DIG-labeled sense and antisense complemen-tary RNA probes specific for MMP-9 and TIMP-1 at 42℃ overnight. For negative control,prehybridization solution was used in hybridization instead of hybridization solution. After aseries of washes, nonspecific binding sites were blocked with blocking solution at 37℃ for30 min. The cells were stained with diaminobenzadine (DAB) for 5 min.
Immunocytochemistry
After termination of treatment, cells were washed in PBS thrice for 5 min, and then fixed in ice-cold acetone/methanol (1:1) for 15 min. Immunocytochemistry staining was per-formed as SP kit instructions. Cells were incubated with monoclonal mouse anti-vimentinand polyclonal goat anti-MMP-9 and anti-TIMP-1 at a concentration of 1:50. Control experi-ments included staining without the primary antibody and substitution of PBS.
Semi-quantitative RT-PCR
Total RNA was prepared from cells using GIBCO reagent according to the manufacturer’s instructions. The RT-PCR protocol was performed to ascertain that thelinear amplification range, where the density of the bands correlated with the amount of geneexpression. Four micrograms total RNA from each samples were denatured at 70℃ for 5min to melt secondary structure and cooled immediately on ice to prevent secondary struc-ture from reforming. The RNA was then reverse transcribed in a 30 µl reaction mixturecontaining 0.5 µg/µl oligo (dT)15 1 µl, 10 mmol/L dNTP1 µl, 50 u/µl RNAsin 0.5 µl, 200 U/µlM-MLV 1 µl and 5×M-MLV-RT buffer 6 µl (reaction conditions: 37℃ for 1 h, 94℃ for 5min in a sterile RNase-free microcentrifuge tube). After reverse transcription, the PCRreaction was conducted using 2 µl cDNA in a total volume of 50 µl containing 10 × PCRbuffer 5 µl, 25 mmol/L MgCL 3 µl, 10 mmol/L dNTP 1 µl, 10 µmol/L each of 5' and 3' primers1 µl, 5 u/µl Taq DNA polymerase 0.5 µl and sterile water 36.5 µl (reaction conditions: 94℃for 5 min, 94℃ for 30 s→50℃(MMP-9) or 60℃ (TIMP-1) for 1 min →72℃ for 1 min ×25 cycles, 72℃ for 10 min). All prmers were synthesized by the Bioasia Biologic Techonology(Co. Ltd). The primers used for amplification of the control G3PDH (a 788 bp product) had the following sequence[6]: sense: 5'-GGTCGGAGTCAACGGATTTGGTCG-3' and antisense:5'-CCTCCGACGCCTGCTTCACCAC-3'. MMP-9 and TIMP-1 primers were selected usingthe primer selection program Primer3. MMP-9: sense: 5'-GGGACGGCAATGCTGAT-3'and antisense: 5'-CGCCACGAGGAACAAACT-3'; TIMP-1: sense: 5'-TTCCGACC-TCGTCATCAG-3' and antisense: 5'-GCATTCCTCACAGCCAAC-3'.The PCR productsand molecular weight markers were separated on 2% agarose gels containing ethidium biomide(10 mg/ml) and visualized by ultraviolet (UV) light. The intensity of each band was normal-ized to its corresponding G3PDH band to compare values between samples semiquantitatively.
Average densitometry was calculated and MMP-9 (or TIMP-1)-to-G3PDH ratios werecompared by F test.
Western blot analysis
For Western blot analysis, cells were washed with ice-cold PBS for thrice, scraped into lysis buffer [50 mmol/L Tris-HCL (pH8.0), 150 mmol/L NaCl, 0.1% sodioum dodecyl sul-phate (SDS), 0.5% sodium deoxycholate, 1% NP-40, 0.02% NaN3, and freshly added proteininhibitors 10 µg/µl phenylmethylsulphonyl fluoride and 1 µg/ml aprotinin], and cooled on icefor 20 min. Lyastes were transferred to a microcentrifuge at 12 000 × g for 5 min at 4℃,and supernatants were collected as the total cell lysates. Protein concentrations were mea-sured using coomassie brilliant blue G-250 assay. Aliquots of each sample containing 100 µgof total protein were fractionated on a 12% polyacrylamide-SDS gel and transferred to anitrocellulose membrane. Membranes were blocked with 5% nonfat dry milk in Tris-buff-ered saline containing 0.2% Tween-20 for 1 h at room temperature, and then incubated withprimary antibody (MMP-9 or TIMP-1 diluted in 1:500) at 4℃ overnight. After a TBS-Twash for 30 min, the blots were incubated with a 1:3 000 rabbit peroxidase-conjugated anti-goat IgG secondary antibody for 1 h at 37℃. After being washed with TBS-T for 30 min, theimmunoreative protein complexes were detected using an enhanced chemiluminescencedetection kit and exposed to Kodak Biomax ML film for 1-5 min to achieve satisfactoryexposure. Average densitometry was calculated and MMP-9 (or TIMP-1)-to-β-actin ratioswere compared using F test.
Statistical analysis
All experiments were performed at least six times yielding similar results. The results were expressed as x- ± s. Data were statistically tested by F test for treated and untreatedsamples. Significance was based on a 95% confidence interval and a P<0.05.
Regulatory effect of multifactor on expression of MMP-9 and TIMP-1 mRNA in
endometrial stromal cells
Immunocytochemistry revealed 92%-96% purity of stromal cell fractions (Figure 1).
The expressions of MMP-9 and TIMP-1 mRNA in endometrial stromal cell were examinedby RT-PCR (Figure 2). In all experiments, MMP-9 and TIMP-1 mRNA could be detected,and their expressions dramatically decreased after treatment with either estrogen or progesterone (P<0.05). To determine that the progesterone effect on MMP-9 and TIMP-1 expressions wasmediated through the progesterone receptor in endometrial stromal cells, the cells werepretreated with the antiprogesterone RU486. RU486 blocked the response to progesterone(P<0.05). On the contrary, MMP-9 and TIMP-1 mRNA expressions dramatically increasedafter treatment with HB-EGF (P<0.05) (Figure 3, Tables 1, 2).
To further determine the subcellular localization of MMP-9 and TIMP-1 mRNA, we performed in situ hybridization. MMP-9 and TIMP-1 mRNA transcripts were observed incytoplasm of all endometrial stromal cells (Figure 4).
Table 1 The regulation of MMP-9 in endometrial stromal cells (x ± s)
Item Control E2
*: P<0.05, compared with the control**: P<0.05, compared with the control, E2 and MPA group Table 2 The regulation of TIMP-1 in endometrial stromal cells (x ± s)
Item Control E2
*: P<0.05, compared with the control**: P<0.05, compared with the control, E2 and MPA group Regulatory effect of multifactor on expressions of MMP-9 and TIMP-1 protein in
endometrial stromal cells
The expressions of MMP-9 and TIMP-1 protein in endometrial stromal cell were examined by Western blotting (Figure 5). In all experiments, MMP-9 and TIMP-1 proteinscould be detected in endometrial stromal cells, and their expressions obviously decreased after treatment with either estrogen or progesterone (P<0.05). To determine thatthe progesterone effect on MMP-9 and TIMP-1 expressions was mediated through theprogesterone receptor in endometrial stromal cells, the cells were pretreated with theantiprogesterone RU486. RU486 blocked the response to progesterone (P<0.05). On the contrary,MMP-9 and TIMP-1 protein expressions dramatically increased after treatment withHB-EGF (P<0.05) (Figure 6, Tables 1, 2).
Figure 1 Immunohistochemical staining of stromal M: DNA Marker; 1: control; 2: E2; 3: MPA; 4: E2+MPA; 5: E2+MPA+RU486; 6: HB-EGF Figure 2 Expressions of MMP-9 and TIMP-1 mRNA in endometrial stromal cells *: P <0.05, compared with group 1 P <0.05, compared with groups 2,3,4 1: control; 2: E2; 3: MPA; 4: E2+MPA; 5: E2+MPA+RU486; 6: HB-EGF Figure 3 Regulation of MMP-9 and TIMP-1 mRNA in endometrial stromal cells Figure 4 Subcellular localization of MMP-9(A) and TIMP-1(B) mRNA To further determine the subcellular localization of MMP-9 and TIMP-1 protein, we performed immunocytochemistry. MMP-9 and TIMP-1 proteins were observed in cyto-plasm of all endometrial stromal cells (Figure 7).
Estrogen, progesterone and HB-EGF all had no effect on the ratio of MMP-9/TIMP-1 Table 3 The regulation of the ratio of MMP-9/TIMP-1 in endometrial stromal cells (x ± s)
Item Control E2
1: control; 2: E2; 3: MPA; 4: E2+MPA; 5: E2+MPA+RU486; 6: HB-EGF Figure 5 Expressions of MMP-9 and TIMP-1 proteins in endometrial stromal cells *: P <0.05, compared with group 1 #: P <0.05, compared witn groups 2,3,4 1: control; 2: E2; 3: MPA; 4: E2+MPA; 5: E2+MPA+RU486; 6: HB-EGF Figure 6 Regulation of MMP-9 and TIMP-1 proteins in endometrial stromal cells Figure 7 Subcellular localization of MMP-9(A) and TIMP-1(B) protein Discussion
Regulatory effect of sex steroids on expressions of MMP-9 and TIMP-1 in
endometrial stromal cells
MMP-9 (92 kD), the greatest molecular weight in matrix metalloproteinase family, is present in many major tissues and organs in human, including endometrium. The best knownTIMP is TIMP-1, a ubiquitous 28.5 kD secreted glycoprotein that forms tight stoichiometricnoncovalent complexes with the active forms of all known MMPs and in addition bindsproMMP-2. TIMP-1 is present in all major tissues and organs in human, includingendometrium. TIMP-1 was present in all cellular compartments of the endometrium (luminalepithelium, glands, stroma, endothelial cells, vascular smooth muscle) throughout the menstrualcycle and there was observable cyclical change. The most expression was in luminalepithelium during the early proliferative phase (d 5-7) and throughout the mid-late secretoryand menstrual phase. Epithelial staining intensity was lowest in the mid-to-late proliferativephase. Stromal staining was also lower in the mid-late proliferative phase[7]. Expression ofTIMP-1 mRNA of which was higher in endometrium of ovariectomized lamb, wassubstantially reduced by estrogen and to a less extent by progesterone[8]. In this study, wealso found that the expressions of TIMP-1 and MMP-9 dramatically decreased aftertreatment with either estrogen or progesterone in endometrial stromal cells, but RU486 caninhibit the effect. So we conclude that both estrogen and progestin can down-regulate theexpressions of TIMP-1 and MMP-9 in endometrial cells.
The window of implantation in human (between cycle d 20 and d 24) is the period of increased circulating estrogen and progesterone. The increased circulating estrogen andprogesterone can down-regulate the expression of TIMP-1 to improve the extracellular matrixproteolytic of endometrium. At the same time, the increased circulating estrogen andprogesterone can down-regulate the expression of MMP-9 to prevent the extracellular matrixover proteolytic of endometrium to ensure successful implantation. In contrast, some researches[9,10] find that the expressions of TIMP-1 and MMP-9 increase in the window ofimplantation. It suggests that the expressions of TIMP-1 and MMP-9 are regulated bymultifactor, including estrogen and progesterone.
Regulatory effect of HB-EGF on expressions of MMP-9 and TIMP-1 in endometrial
stromal cells
HB-EGF, a member of the EGF family of growth factors, has a high affinity for heparin and heparin sulfate. Experiments using animal models suggested that HB-EGF is importantfor endometrial receptivity[11]. HB-EGF mRNA expression is low in the endometrium duringthe proliferative stage of the menstrual cycle and increase in the secretory stage, with highestexpression immediately prior to the implantation window (d 19-21), after which levelsdecrease[12]. In this study, we found that the expressions of MMP-9 and TIMP-1 dramaticallyincreased after treatment with HB-EGF in endometrial stromal cells, and we assumed thatHB-EGF can up-regulate the expressions of MMP-9 and TIMP-1. In the window ofimplantation, the highest expression of HB-EGF up-regulate the expression of MMP-9. Atthe same time HB-EGF up-regulate the expression of TIMP-1 to inhibit the activity of MMP-9 and extracellular matrix proteolytic of endometrium to ensure successful implantation, tokeep extracellular matrix homeostasis and maintenance of tissue integrity.
In conclusion, human implantation is a complex series of steps that under normal cir- cumstance begins even before the blastocyst reaches the uterine cavity and attaches to theendometrial epithelium. To complete this series of events and to accomplish successfulimplantation, the embryo and the uterine endometrium must be synchronized during thislimited period of uterine receptivity. This enigmatic process is the result of an embryonic-maternal dialogue, in which the embryo and the endometrium induce changes in each otherto promote receptivity. Once the embryo has adhered to the maternal uterine surface, it mustbe able to break rapidly through the endometrial basement membranes (BMs) and gainaccess to the maternal circulation to ensure successful implantation. This process of tropho-blast invasion is associated with tissue remodeling of BMs, a specialized form of extracellu-lar matrix (ECM) that separate cells from the underlying or surrounding connective tissuestroma. The trophoblastic invasion is regulated in part by MMPs and TIMPs. In the windowof implantation, the balance of expressions between MMP-9 and TIMP-1 is kept by thedown-regulation of estrogen and progesterone and the up-regulation of HB-EGF, whichensures a successful implantation. The disbalance of expressions between MMP-9 and TIMP-1 willinhibit or overwhelmingly promote the extracellular matrix proteolysis, which will induceinfertility and pathologic pregnancy. Studies on the mechanisms of MMP-9 and TIMP-1regulation will make for the therapy of infertility and increase the pregnancy rate of IVF-ET.
References
1. Giudice LC. Potential biochemical markers of uterine receptivity. Hum Reprod, 1999, 14(Suppl 2): 3-16.
2. Wang H, Wen Y, Mooney S, et al. Matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase
expression in human preimplantation embryos. Fertil Steril, 2003, 80(Suppl 2): 736-42.
3. Chou CS, Tai CJ, MacCalman CD, et al. Dose-dependent effects of gonadotropin releasing hormone on matrix metalloproteinase (MMP)-2, and MMP-9 and tissue specific inhibitor of metalloproteinases-1 messenger
ribonucleic acid levels in human decidual Stromal cells in vitro. J Clin Endocrinol Metab, 2003, 88(2): 680-8.
4. Casey ML, Smith J, Nagai K, et al. Progesterone-regulated cyclic modulation of membrane metalloendopepeidase (enkephalinase) in human endometrium. J Biol Chem, 1991, 266(34): 23 041-7.
5. Apparao KB, Murray MJ, Fritz MA, et al. Osteopontin and its receptor alphavbeta(3) integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially. J Clin Endocrinol
Metab, 2001, 86(10): 4 991-5 000.
6. Taylor HS, Daftary GS, Selam B, et al. Endometrial HOXA10 expression after controlled ovarian hyper- stimulation with recombinant follicle-stimulating hormone. Fertil Steril, 2003, 80(Suppl 2): 839-43.
7. Zhang J & Salamonsen LA. Tissue inhibitor of metalloproteinases(TIMP)-1, -2 and -3 in human endometrium during the menstrual cycle. Mol Hum Reprod, 1997, 3(9): 735-41.
8. Hampton AL, Butt AR, Riley SC, et al. Tissue inhibitors of metalloproteinases in endometrium of ovariec- tomized steroid-treated ewes and during the estrous cycle and early pregnancy. Biol Reprod, 1995, 53(2):
302-11.
9. Gui WW, Qiu Y, Zeng PH, et al. Expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 protein in the implantation window phase of endometrium in ovulation normal women.
Journal of Chongqing Medical University (in Chinese), 2004, 29(1): 1-7.
10. Li L, Chen SL & Xing FQ. Expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase- 3 protein in the implantation window phase of endometrium. Chinese Journal of Obstetrics and Gynecology
(in Chinese), 2000, 35(9):544-6.
11. Stavreus-Evers A, Aghajanova L, Brismar H, et al. Co-existence of heparin-binding epidermal growth factor- like growth factor and pinopodes in human endometrium at the time of implantation. Mol Hum Reprod,
2002, 8(8): 765-9.
12. Yoo HJ, Barlow DH & Mardon HJ. Temporal and spatial regulation of expression of heparin-binding epidermal growth factor-like growth factor in the human endometrium: a possible role in blastocyst
implantation. Dev Genet, 1997, 21(1): 102-8.

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CONSERVATION OF TELOMERE FUNCTION AND THEThe concept of a healing factor for chromosome ends or “telomeres” was evoked80 years ago owing to the recognition by Barbara McClintock and Hermann Mullerthat the natural end of a linear intact chromosome differs from that of a brokenchromosome. Using fruit flies and corn as model organisms, they observed thatnatural chromosome ends, unlike broken ones,

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Abortion Reporting ICD-9 Procedure Codes used on the PROCEDURE REPORT SURGICAL Procedure Performed: 69.01- Sharp Curettage (D&C) 69.51- Suction Curettage69.93- Insertion/Laminaria74.91- Hysterotomy/Hysterectomy75.0- Insertion/Intra-amniotic (saline)96.49- Insertion/ProstaglandinDialation and Evacuation (D&E)* NON-SURGICAL/Medical Procedure Performed* Methotrexate and misoprostol

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