[SPERM SELECTION TECHNIQUES: VISUAL ASSESSMENT VERSUS THE BINDING POTENTIAL TO HYALURONIC ACID].

BACKGROUND: The human oocyte is surrounded by hyaluronic acid (HA), which acts as a natural selector. Only spermatozoa expressing HA receptors can reach and fertilize the oocyte. This study aims to compare two sperm selection techniques by correlation to fertilization rates and embryo quality. METHODS: Couples undergoing IVF-ICSI treatment due to mild male infertility were enrolled in a prospective study. According to the randomization, the sperm suspensions were put into a polyvinylpyrrolidone (PVP) droplet or an HA-containing medium droplet (Sperm Slow). In the PVP group motile spermatozoa with the best morphology were selected for injection. From the HA-containing medium those sperm demonstrating vigorous tail beating and an absence of progressive motility as well as good morphology, were selected. Primary outcome measures were fertilization rate and embryo quality. RESULTS: Thirty couples were randomized to the PVP group and 24 to the slow sperm group; 353 oocytes were injected. There was no statistical difference in fertilization or cleavage rate. Furthermore, in the PVP group, the mean number of embryos was higher and the average morphology of the best embryo was superior. CONCLUSIONS: Considering that the HA-based sperm selection technique is more expensive and time consuming, the current study does not support using it as a routine method.

Human amniotic epithelial cells differentiate into cells expressing germ cell specific markers when cultured in medium containing serum substitute supplement.

BACKGROUND: Human amniotic epithelial cells (hAECs) maintain the plasticity of pregastrulation embryonic cells, having the potential to differentiate into all three germ layers. The potential of these cells to differentiate into cells expressing germ cell specific markers has never been described before. METHODS: In the present study, hAECs were cultured in medium containing serum substitute supplement (SSS). Gene and protein expression of germ cell and oocyte specific markers was assessed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and flow activated cell sorter analysis (FACS) in hAECs at different time points during the differentiation into cells expressing germ cell specific markers. RESULTS: When cultured with SSS, already at passage 1, hAECs start to express the germ cell specific genes C-KIT, DAZL, VASA and ZP3 and at passage 5 large round cells, resembling oocytes, appeared. The cells express the germ cell specific marker DAZL, the oocyte specific markers GDF9 and ZP3 and the meiosis specific markers DMC1 and SCP3 at the protein level. CONCLUSIONS: From our preliminary results we can conclude that hAECs have the potential to differentiate into cells expressing germ cell specific markers.

Ideal lag time from ovulation to oocyte aspiration using a GnRH agonist trigger.

BACKGROUND: In ART, oocyte maturation (M2) and ovulation is stimulated by a hormonal trigger. For maturation to occur, sufficient "lag time" must elapse between the trigger and aspiration, ranging from 32 to 38 hours. Premature aspiration can result in poor yields; late aspiration risks spontaneous ovulation. AIM: Our study examines optimal lag time using a GnRH antagonist protocol and GnRH agonist trigger for ICSI. METHODS AND MATERIALS: We analyzed data from 220 women undergoing GnRH antagonist protocol using a GnRH agonist trigger for ICSI at our clinic between 02/2012-03/2018. Patients were divided into 4 groups based on lag time: 34.00-34.99 hours (n = 32), 35.00-35.99 hours (n = 113), 36.00-36.99 hours (n = 57) and 37.00 h or more (n = 18). Analyses were performed with the Kruskal-Wallis test, Chi-Square, and Spearman's rho correlation. RESULTS: A positive correlation was found for the number of M2 oocytes aspirated and lag time (ρ = 0.138, p = 0.04) and for the total number of oocytes aspirated and lag time, (ρ = 0.174, p = 0.01). No correlation was found between the proportion of M2 oocytes aspirated and lag time (p = 0.217). The third group (36 h) had significantly more M2 oocytes aspirated than the second group (35 h) (12.4 ± 7.1 vs 9.4 ± 6.2; p = 0.039). The four groups did not differ for the proportion of mature M2 oocytes (H = 2.453, p = 0.484). The four groups differed in the frequency of live births per fresh embryos transferred (χ2 = 9.364, p = 0.025). CONCLUSION: Our study identified a positive correlation between lag time and both the number of M2 oocytes and the total number of oocytes aspirated-factors which lead to an increased rate of successful pregnancies. Further research is necessary.

Ets-2 and p53 mediate cAMP-induced MMP-2 expression, activity and trophoblast invasion.

BACKGROUND: We have previously shown that Matrix metalloproteinase (MMP) -2 is a key-enzyme in early trophoblast invasion and that Protein Kinase A (PKA) increases MMP-2 expression and trophoblast invasion. The aim of this study was to examine MMP -2 regulation by PKA in invasive trophoblasts: JAR choriocarcinoma cell-line and 6-8 w first trimester trophoblasts. METHODS: The effect of Forskolin (PKA) on MMP-2 expression was assessed by Northern Blot and RT-PCR. Possible transcription factors binding to consensus MMP-2 promoter sequences in response to Forskolin, were detected by EMSA binding assay and their expression assessed by western blot analysis. Antisense transfection of relevant transcription factors was performed and the inhibitory effect assessed on MMP-2 expression (RT-PCR), secretion (zymography) and trophoblast invasiveness (transwell migration assay). RESULTS: We found that Forskolin increased MMP-2 mRNA in JAR cells within 24 hours, and induced binding to p53, Ets, C/EBP and AP-2. Transcription factors Ets-2, phospho- p53, C/EBP epsilon, C/EBP lambda and AP-2 alpha bound to their respective binding sequences in response to Forskolin and the expressions of these transcription factors were all elevated in Forskolin- treated cells. Inhibition of Ets-2 and p53 reduced MMP-2 expression, secretion and invasiveness of Forskolin treated cells. CONCLUSION: MMP-2 is regulated by PKA through several binding sites and transcription factors including Ets-2, p53, C/EBP, C/EBP lambda and AP-2 alpha. Ets-2 and p53 mediate cAMP- induced trophoblast invasiveness, through regulation of MMP-2.

Plexin-B1, glycodelin and MMP7 expression in the human fallopian tube and in the endometrium.

BACKGROUND: To study the expression of Plexin-B1, Glycodelin, and MMP7 during the menstrual cycle in the endometrium and in the fallopian tube. METHODS: The research included women undergoing hysterectomy, tubal sterilization or salpingo-oophoerectomy. Total RNA from endometrial and fallopian tube tissues was extracted using a total RNA isolation kit. Semi-quantitative RT-PCR was performed to examine mRNA relative expression. RESULTS: Plexin-B1 expression in the endometrium was significantly higher on days 19 - 23 compared to days 12 - 14 (1.166 +/- 0.42 versus 0.523 +/- 0.299), P < 0.005. In the fallopian tube the level of plexin-B1 did not change significantly throughout the menstrual cycle. Glycodelin expression was significantly higher on days 19 - 23 compared with days 12-14, both in the endometrium (0.819 +/- 0.564 versus 0.072 +/- 0.343, P < 0.05) and the fallopian tube (0.796 +/- 0.196 versus 0.329 +/- 0.398, P < 0.05). Although the level of MMP7 secretion was the highest in the secretory phase the difference from the proliferative phase did not reach statistical significance, neither in the endometrium nor in the fallopian tube. This could result from a lack of power. CONCLUSIONS: In the endometrium, both Glycodelin and Plexin-B1 are exhibiting a cyclic pattern suggesting a possible steroid regulation and a role in endometrial receptivity.

Progesterone receptor A and c-Met mediates spheroids-endometrium attachment.

BACKGROUND: Implantation in humans involves cross talk between an active blastocyst and receptive endometrium. The role of the endometrial receptors in this complex embryo-maternal interaction is still unclear. We tested gene and protein expression of endometrial receptors (Progesterone receptor (PR) and c-Met) and the effect of theses receptors in endometrial receptivity. METHODS: Two endometrial cell lines were used: HEC-1A and RL95-2 considered as being of low and high receptivity, respectively. Western blot and RT-PCR analysis were utilized to study the receptor expression profile.The role of endometrial receptors in endometrial receptivity was studied by attachment and invasion assays of JAR spheroids (made of a trophoblast cell line) on endometrial cells. Different manipulations of inhibition and stimulation of the endometrial receptors were used including: inhibition by specific antibodies against the receptors, or antagonist of the receptors, as well as transfection with antisense for the endometrial receptors, stimulation by specific ligands for the receptors and transfection with the gene for endometrial receptors. RESULTS: Different protein expression patterns of endometrial receptors were observed between the tested endometrial cell lines. The expression levels of PRA ratio to PRB, and the 50 kDa c-MET isoform were significantly lower in HEC-1A as compared with RL95-2. Attachment rates and growth of JAR spheroids into HEC-1A were significantly lower as compared with RL95-2. Stimulation of PR with progesterone altered attachment rates to HEC-1A. Inhibition of PR with RU-486 mildly increased attachment rate to HEC-1A whereas it slightly decreased attachment rate to RL95-2. c-Met inhibition decreased attachment rates only to HEC-1A cells that expressing high levels of Plexin-B1 (PB1). Immunoprecipitation studies revealed that c-Met and PB1 associate in complexes in the endometrial cell lines. CONCLUSION: Differential endometrial receptor profiles are expressed during the receptivity period. The attachment and invasion processes are separately regulated. We suggest a biologically functional role for PRA in endometrial receptivity and in the attachment process. c-Met contribution is minor and related with creation of a complex with PB1.

Mechanisms of matrix metalloproteinase-2 (mmp-2) transcriptional repression by progesterone in jar choriocarcinoma cells.

BACKGROUND: Although the MMP-2 promoter lacks a canonical progesterone response element (PRE), the hormone inhibits MMP-2 expression and is part of treatment protocols in gynecological invasive pathologies, including endometriosis and endometrial hyperplasia. This study aimed to explore the mechanism by which progesterone inhibits MMP-2 expression. METHODS: The effect of progesterone on MMP-2 expression in the JAR human choriocarcinoma cell line was analyzed by gelatin zymography. MMP-2 transcript expression was studied using Northern blot and semi-quantitative RT-PCR. Rat promoter deletion analysis, electrophoretic mobility shift and chromatin immuno-precipitation assays were performed in order to locate the DNA binding site and the transcription factors involved in MMP-2 regulation. RESULTS: Progesterone significantly decreased secretion of pro-MMP-2 and MMP-2 transcript expression level in a dose-dependent manner. Progesterone (1 microM) significantly decreased both human and rat MMP-2 promoter activity (80.1% +/- 0.3 and 81.3% +/- 0.23, respectively). Progesterone acts through the SP1 family transcription factors-binding site, located between -1433 and -1342 bp region from the transcriptional start site of the rat MMP-2 promoter, which are present in the orthologous human MMP-2 promoter. Progesterone receptor (PR), SP2, SP3 and SP4 proteins are constitutively bound to this consensus sequence. CONCLUSION: Progesterone reduces PR and SP4 binding to the MMP-2 promoter, thereby suppressing transcription. Progesterone also promotes SP4 degradation. These novel mechanisms of MMP-2 regulation by progesterone provide the biological rationale for the use of progesterone in clinical settings associated with increased MMP-2 expression.

Matrix metalloproteinase-2 is elevated in midtrimester amniotic fluid prior to the development of preeclampsia.

OBJECTIVE: To evaluate levels of matrix metalloproteinases (MMP) and their inhibitors (TIMP) in second trimester amniotic fluid of women with hypertensive disorders compared to normotensive women. STUDY DESIGN: Amniotic fluid was obtained from 133 women undergoing genetic second trimester amniocentesis. Zymography was performed for MMP characterization and an MMP-2 ELISA kit was used to determine MMP-2 levels. TIMP-2 expression was evaluated using western blot. RESULTS: Mean amniotic fluid MMP-2 and TIMP-2 levels were significantly higher in women who developed a hypertensive disorder compared to normotensive women (P < 0.0004 and P < 0.01, respectively). When subdivided into subgroups, amniotic fluid from women who eventually developed preeclampsia or superimposed preeclampsia showed significantly higher MMP-2 levels than normotensive women (P < 0.05). However, no statistical difference in MMP-2 levels was found between patients with gestational hypertension and normotensive patients. CONCLUSION: Higher amniotic fluid MMP-2 and TIMP-2 levels are found in women who eventually develop preeclampsia.

[Progesterone receptor's profile and the effect of the hormone and its derivatives on invasiveness and MMP2 secretion in endometrial carcinoma cell lines].

INTRODUCTION: Progesterone inhibits endometrial proliferation and is used for the treatment of early stage endometrial carcinoma in women interested in fertility preservation or for advanced or recurrent disease. Better responses and prognosis were documented in women who are progesterone receptor positive. The receptor has 2 main isoforms and the ratio between these two is responsible for progesterone activity OBJECTIVES: To evaluate the effect of progesterone and its derivative on invasion and matrix metalloproteinase 2 (MMP2) secretion in two endometrial carcinoma cell lines having different progesterone receptor isoform profiles. METHODS: HEC-1A and RL95-2 cell Line cultures were used. Tests for invasion using Matrigel and zymography for MMP secretion were conducted after the exposure of the cells to different derivatives of progesterone. RESULTS: RL95-2 cell are PR-A dominant and were found to be more invasive than HEC-1A which are PR-B dominant. After exposure to progesterone and medroxyprogesterone but not to hydroxyprogesterone invasion and MMP2 secretion were decreased significantly only in the HEC-1A cell line. CONCLUSION: PR-B dominance may predict better responses to progesterone therapy in women with endometrial carcinoma.

Progesterone receptor profile in the decidua and fetal membrane.

Whereas in most mammals the onset of labor is preceded by a rapid fall in the maternal progesterone levels, in humans and in higher primates, maternal, fetal and amniotic fluid concentrations of progesterone are sustained before the onset of labor. Therefore, the mechanism for parturition, which has been proposed for humans, is 'functional' progesterone withdrawal. This review is focused on the expression profile, activity and interaction of the progesterone receptor (PR) isoforms in the decidua and the fetal membrane during the initiation of labor. Binding of progesterone to PR induces a significant conformational change on the receptor proteins. These changes result in dimerization, increased receptor phosphorylation and binding of receptor dimers to specific hormone responsive DNA elements in the promoter of target genes. Interaction with specific co-activator proteins and general transcription factors are responsible for the formation of a productive transcription initiation complex. The PR also mediates the activation of cytoplasmic signaling pathways, participating in the induction of signal transduction pathway in the cytoplasm. Balanced expression of the two major progesterone receptors isoforms is crucial for progesterone function as uterine muscle inhibitor. Change in PR isoforms profile seems to be responsible for decidual activation. Decidua without contractions shows consistent profile with PR-B being the dominant isoform. PR-A, PR-C and two additional truncated isoforms are also detected but in significantly smaller concentration. After initiation of contractions, a sharp decline in PR-B shifts the PR-A/PR-B ratio toward PR-A dominance. This shift in the decidua towards increase expression of progesterone receptor isoform A and decrease in PR isoform B is having a pivotal role in decidual activation and initiation of labor.

The matrix metalloproteinases (MMPS) in the decidua and fetal membranes.

The role of the matrix metalloproteinases (MMPs) in the decidua, fetal membranes and amniotic fluid (AF) has been receiving more and more attention. The MMPs are not only important intermediaries in pathological processes leading to preterm labor but it seems that they also play a crucial role in the activation of labor at term. During normal gestation MMP-1, -2, -3, -7 and -9 are found in the amniotic fluid and fetal membranes. MMP-2 and MMP-3 are expressed constitutively while MMP-9 is barely detectable until labor. At labor, while MMP-9 is the major MMP responsible for gelatinolytic activity in the membranes, MMP-2 is dominant in the decidua. MMP-7 (AF) increases with gestation but does not appear to play a major role in labor. The expression of MMPs is attenuated through the expression of relaxins, integrins and extracellular matrix metalloproteinase inducer (EMMPRIN). Spontaneous preterm delivery (PTD) may be a product of preterm labor (PTL), preterm premature rupture of membranes (P-PROM) or placental abruption. Each of these processes may have differing pathways but the presence of an intrinsic inflammatory response with or without infection seems to involve all etiologies. The inflammatory response is mediated with cytokines such as interleukins -1, -6 and -8 and tumor necrosis factor alpha. MMP-3, MMP-7 and MMP-8 appear to be important in these processes. MMP-9, which is the major MMP involved in normal labor, plays an important role in pathological labor as well. Finally, apoptosis seems to play a role in pathological labor, particularly deliveries involving P-PROM. African-American are at greater risk of PTD than white or Hispanic Americans. Environmental differences may not suffice to explain this phenomenon. Genetic polymorphisms of the MMP genes may help explain the greater risk among this population. Finally, manipulating MMPs may have a role in the prevention of PTD. Agents suggested include indomethacin, N-acetylcysteine, progesterone and specific inhibitors of phosphodiesterase 4.

Semaphorin-4D (Sema-4D), the Plexin-B1 ligand, is involved in mouse ovary follicular development.

BACKGROUND: Human Plexin-B1 is expressed in two truncated forms. The long form encodes a trans-membranal protein, while the short form, which is bound to the cell surface and partially secreted, possibly serves as a decoy receptor. Plexin receptors are trans-membrane proteins. The sema domain, found in the extracellular region, is common to all plexins, semaphorins, and the scatter factor receptors and is crucial for the biological activity and plexin receptor specificity. Semaphorin-4D/Plexin-B1 binding provides attractive and repulsive cues for the navigation of axonal growth cones, and new studies suggest that this system also plays a role in the regulation of the biological functions of endothelial cells, specifically in the control of angiogenesis. In a previous study, we have demonstrated the expression and possible role of Plexin-B1 in the mouse ovary. The present study was designed to test the hypothesis that Plexin-B1 effects are mediated by Semaphorin-4D. METHODS: In vivo expression and localization of mouse ovarian Sema-4D were tested by immunohisto-chemistry. The role of Sema-4D in follicular development was examined by in vitro growth of preantral follicles in the presence or absence of Semaphorin-4D, with or without neutralizing antibodies against Plexin-B1. Follicular growth and steroid hormone secretion rates were tested. RESULTS: Semaphorin-4D is expressed in the mouse ovary in vivo mostly in the granulosa cells and and its expression is modulated by PMSG and hCG. In the presence of Semaphorin-4D, in-vitro constant growth was observed as indicated by follicular diameter during the culture period and elevated steroid hormone secretion rates compared with control. These effects were abolished after addition of neutralizing antibodies against Plexin-B1. CONCLUSION: In the ovarian follicle, the effect of Plexin-B1 is mediated by sema-4D.

Progesterone receptor isoforms profile, modulate matrix metalloproteinase 2 expression in the decidua.

OBJECTIVE: This study explores the effect of progesterone and the role of the progesterone receptor (PR) profile on matrix metalloproteinase (MMP)-2 expression in human decidua. STUDY DESIGN: Zymography was conducted for MMP secretion. Semiquantitative reverse transcriptase-polymerase chain reaction was performed to examine MMP2 transcripts. Progesterone's effect on the MMP2 promoter was determined testing luciferase activity. The role of PR isoform on MMP2 expression was studied using human PR complementary DNA encoding PR isoforms PRA, PRB, or PRC. RESULTS: In decidua with overexpressed PRB, progesterone decreased MMP2 expression. Progesterone increased pro-MMP2 expression in decidua with overexpressed PRA or PRC. MMP2 promoter activity was unchanged following transfection with human PRA in the absence or presence of progesterone. Decreased promoter activity was observed following transfection with human PRB or human PRC. Progesterone increased promoter activity with overexpressed human PRC. CONCLUSION: Progesterone hampers MMP2 expression in the decidua via PRB. PRA has a repressive effect on PRB, whereas PRC seems to have a repressive effect on both PRA and PRB.

The effect of progesterone on gelatinase expression in the decidua and fetal membranes before and after contractions.

OBJECTIVE: This study was aimed to explore the effect of progesterone on gelatinase expression in the decidua and fetal membranes before and after contractions. STUDY DESIGN: Zymography was conducted for matrix metalloproteinase (MMP) secretion. Semiquantitative reverse transcriptase-polymerase chain reaction was performed to examine MMP2 transcripts, and the effect of progesterone on MMP2 promoter activity was determined with the use of luciferase activity. RESULTS: Progesterone decreased pro-MMP2 secretion, expression, and promoter activity in decidua before contractions began. The effect of progesterone was reversed completely by mifepristone (RU486). Progesterone failed to inhibit MMP2 expression in the amnion and chorion before contractions began. After contractions, progesterone failed to inhibit MMP2 expression in both the decidua and fetal membranes. CONCLUSION: MMP2 expression is inhibited by progesterone only in the decidua and only before contractions begin.

Characterization of irreversible physio-mechanical processes in stretched fetal membranes.

We perform bulge tests on live fetal membrane (FM) tissues that simulate the mechanical conditions prior to contractions. Experimental results reveal an irreversible mechanical behavior that appears during loading and is significantly different than the mechanical behavior that appears during unloading or in subsequent loading cycles. The irreversible behavior results in a residual strain that does not recover upon unloading and remains the same for at least 1h after the FM is unloaded. Surprisingly, the irreversible behavior demonstrates a linear stress-strain relation. We introduce a new model for the mechanical response of collagen tissues, which accounts for the irreversible deformation and provides predictions in agreement with our experimental results. The basic assumption of the model is that the constitutive stress-strain relationship of individual elements that compose the collagen fibers has a plateau segment during which an irreversible transformation/deformation occurs. Fittings of calculated and measured stress-strain curves reveal a well-defined single-value property of collagenous tissues, which is related to the threshold strain εth for irreversible transformation. Further discussion of several physio-mechanical processes that can induce irreversible behavior indicate that the most probable process, which is in agreement with our results for εth, is a phase transformation of collagen molecules from an α-helix to a ß-sheet structure. A phase transformation is a manifestation of a significant change in the molecular structure of the collagen tissues that can alter connections with surrounding molecules and may lead to critical biological changes, e.g., an initiation of labor. STATEMENT OF SIGNIFICANCE: This study is driven by the hypothesis that pre-contraction mechanical stretch of the fetal membrane (FM) can lead to a change in the microstructure of the FM, which in turn induces a critical biological (hormonal) change that leads to the initiation of labor. We present mechanical characterizations of live FM tissues that reveal a significant irreversible process and a new model for the mechanical response of collagen tissues, which accounts for this process. Fittings of calculated and measured results reveal a well-defined single-value property of collagenous tissues, which is related to the threshold strain for irreversible transformation. Further discussion indicates that the irreversible deformation is induced by a phase transformation of collagen molecules that can lead to critical biological changes.

MMPS and TIMPS in ovarian physiology and pathophysiology.

The matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) have been postulated to play a critical role in the extracellular matrix (ECM) remodeling associated with follicular development. The gelatinases were localized to the theca of developing follicles and in the stroma of the rodent ovary. Gelatinolytic activity corresponded with the localization of MMP-2 and MMP-9 around the developing follicles and at the apex of preovulatory follicles. The TIMPs-1, -2, and -3 were localized to the stroma and theca of developing follicles and correlation between MMPs and the quality of the developing follicles was found. During the process of ovulation, MMP-1 protein was found in the theca interna and externa, interstitial glands, and germinal epithelium. Synthetic inhibitor of mammalian tissue collagenases was documented to be inhibitory to ovulation in perfused rat ovaries. MMP-19 and TIMP-1 messenger RNA were localized to the granulosa and thecal-interstitial cells of large preovulatory and ovulating follicles. Both were induced and upregulated 5-10 fold by human chorionic gonadotropin (hCG). MMP-2 mRNA found in theca-interstitial cells and membrane-type (MT) 1-MMP mRNA found in granulosa and theca-interstitial cells were both induced after stimulation with pregnant mare's serum gonadotropin (PMSG). Gelatinolytic activity was observed throughout the formation of the corpus luteum. At 12 h after hCG, luteinizing granulosa cells expressed TIMP-1 and TIMP-3 mRNA. In the newly forming corpus luteum at 24 h after hCG administration, the luteal cells expressed TIMP-1, -2, and -3 mRNA with unique pattern of cellular expression for each of the TIMPs. Regression of the corpus luteum is associated with a significant increase in the activity of the metalloproteinases. In luteinized granulosa cells from women with polycystic ovarian syndrome (PCOS) the MMP-TIMP balance is shifted towards greater MMP activity. Cultured luteinized granulosa cells obtained from PCOS patients secrete higher levels of MMP-9 and MMP-2 compared to granulosa cells from normal ovulatory patients whereas the secreted basal level of TIMP-1 was similar in both types of granulosa cells. These results indicate a higher net gelatinolytic activity within the luteinizing granulosa cells of patients with PCOS. It has been shown that in sheep, diversion of normal follicles to atresia by hypophysectomy is followed by a significant increase of intrafollicular levels of MMP-2 and MMP-9 and the disappearance of connexin-43. It is therefore reasonable to speculate that MMP-9 and MMP-2 may be associated with inappropriate atresia in PCOS.

Expression and importance of matrix metalloproteinase 2 and 9 (MMP-2 and -9) in human trophoblast invasion.

BACKGROUND: The aim of this study was to examine the invasiveness of first trimester trophoblasts according to the secretion profile of MMP-2 and -9 at different gestational stages, and to test the similarity between primary trophoblast cell-culture and the JAR choriocarcinoma cell-line. METHODS: First trimester trophoblasts were divided into two groups: 6-8 weeks (early) and 9-12 w (late) of gestation. The two trophoblast groups and JAR cells were cultured in medium, with various concentrations of forskolin and Epidermal Growth Factor (EGF). Proteolytic activity was detected by zymography and invasiveness was assessed by Matrigel invasion assay. Student's T-test was used for statistical analysis. RESULTS: In 6-8 w trophoblast, proMMP-2 was only slightly dominant over proMMP-9 (53.2% vs. 46.8% respectively), whereas in 9-12 w, proMMP-9 was clearly dominant over proMMP-2 (61.7% vs.38.3% respectively). In JAR cells proMMP-2 was strongly dominant (90.2% vs.9.8% respectively). In JAR cells forskolin significantly increased proMMP-2 and -9 secretion (128.5% +/- 12 and 183.2% +/- 27.9 of control, respectively). EGF had a dual effect on JAR cells: at 8 ng/ml both proMMP-2 and -9 were increased (133.5% +/-15 and 223.9% +/- 32.4 of control, respectively) while at 80 ng/ml both proMMP-2 and -9 were decreased (65.1% +/- 18.3 and 66.6% +/- 37 of control, respectively). Forskolin significantly increased both proMMP-2 and -9 secretion in 6-8 w and 9-12 w trophoblasts (125.9% +/- 6.3,128.4% +/- 6.4; 169.7% +/- 20.3, 120.3% +/- 4.5 of control, respectively). EGF also significantly increased both proMMP-2 and -9 secretion in 6-8 w and 9-12 w trophoblasts (141.22% +/- 14.8, 138.8% +/- 10.3; 168.3% +/- 18.2, 117.3 +/- 3.8 of control, respectively). Both forskolin and EGF increased trophoblast cells invasiveness in all groups. The invasive ability of trophoblast cells, induced by forskolin, was reduced by MMP-2 antibody in: JAR cells, 6-8 w and 9-12 w trophoblasts. Likewise trophoblast invasion induced by EGF was reduced by MMP-2 antibody in all groups. However the invasive ability induced by forskolin or EGF was inhibited by MMP-9 antibody only in trophoblasts from 9-12 w. CONCLUSIONS: First trimester trophoblasts express differential gelatinase secretion profile according to the gestational week. In JAR and early trophoblasts (6-8 w) MMP-2 is the main gelatinase and the key enzyme in trophoblast invasion. Thereafter in late first trimester trophoblasts (9-12 w), both MMP-2 and -9 participate in trophoblast invasion.

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.

OBJECTIVE: To investigate the regulation of MMP-9, TIMP-1, and progesterone via three signal transduction pathways in luteinized granulosa cells from normal ovulatory and PCOD women. DESIGN: In vitro study. SETTING: Laboratory for Research in Reproductive Sciences, Department of Obstetrics and Gynecology, Ha'Emek Hospital, Afula, Israel. PATIENT(S): Ten normal ovulatory and 10 women with polycystic ovary disease (PCOD) treated in an assisted reproduction program. INTERVENTION(S): Cultured cells were exposed to phorbol 12-myristate 13-acetate (TPA), acting via protein kinase C (PKC), to epidermal growth factor (EGF), acting via protein tyrosine kinase (PTK), and to forskolin, acting via protein kinase A (PKA). MAIN OUTCOME MEASURE(S): Secretion of MMP-9, TIMP-1, and progesterone. RESULT(S): Phorbol 12-myristate 13-acetate elicited an increase in MMP-9 and TIMP-1 secretion in both groups and apparently did not affect progesterone secretion. Epidermal growth factor did not change significantly neither MMP-9 nor TIMP-1 secretion but dose dependently decreased MMP-9-TIMP-1 ratio and increased progesterone secretion in the PCOD group. Forskolin inhibited MMP-9 activity and increased TIMP-1 and progesterone secretion in both groups. Progesterone production was inversely related to the ratio of MMP-9-TIMP-1 regardless of cell origin. CONCLUSION(S): In this preliminary study, similar and divergent patterns have emerged in the regulation of MMP-9 and TIMP-1 in human luteinized granulosa cells. Repressing MMP-9-TIMP-1 ratio may have an important modulatory effect on progesterone secretion.

Presence of matrix metalloproteinases and tissue inhibitor of matrix metalloproteinase in human sperm.

The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade protein components of the extra-cellular matrix. The necessity of breakdown of physical barriers in the fertilization process suggests that MMPs, along with their tissue inhibitors (TIMPs), might be involved in this task. We have examined the presence of MMP and TIMP in normal and abnormal human sperm samples by gel zymography and Western blot analysis. Thirty-five normal sperm samples and 35 abnormal sperm samples were examined in this study. Gel zymography showed 92-, 72-, 62-, and 28-kd molecular-weight bands exhibiting gelatin-degrading activity in both normal and abnormal sperm samples. The 92-, 72-, and 62-kd bands with gelatinolytic activity are consistent with pro-MMP-9, pro-MMP-2, and active MMP-2, respectively (pro-MMP being the zymogen of MMP). Western blot analysis showed the presence of TIMP-1 in both normal and abnormal sperm samples. A higher 28-kd activity and a lower 92-kd MMP activity in normal sperm samples relative to abnormal samples were detected. No marked difference in TIMP-1, 72-kd, and 62-kd release was observed between normal and abnormal sperm samples. In conclusion, this is the first report of MMP activity in normal and abnormal human sperm samples and of TIMP presence in sperm samples. The data indicate a different MMP profile between normal and abnormal sperm samples, with a higher 28-kd activity and a lower 92-kd MMP activity in normal relative to abnormal samples.

The role of the matrix metalloproteinases in human endometrial and ovarian cycles.

The matrix metalloproteinases (MMPs) are part of an expanded family of proteins called the astacin family of zinc metalloproteinases. The MMPs, probably balanced by their tissue inhibitors of metalloproteinases (TIMPs), are essential effectors of developmental processes participating in cell migration, cell proliferation, apoptosis and tissue morphogenesis. The MMPs regulate the function of biologically active molecules as well as fulfilling an important role in endothelial cell invasion, angiogenesis and in tumor progression. The dynamic normal physiology of the human reproductive system involves almost all of the above-mentioned aspects of MMPs activity. This review presents and discusses new insights into the role of MMPs, and their TIMPs, in human endometrial cycle and ovarian function.