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.

[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.

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.

Distribution of estrogen and progesterone receptors isoforms in endometrial cancer.

BACKGROUND: 70-80% of sporadic endometrial carcinomas are defined as endometrioid carcinoma (EC). Early-stage, well differentiated endometrial carcinomas usually retain expression of estrogen and progesterone receptors (ER and PR, respectively), as advanced stage, poorly differentiated tumors often lack one or both of these receptors. Well-described EC prognosis includes tumor characteristics, such as depth of myometrial invasion. Therefore, in the current study, we evaluated the expression profile of ER and PR isoforms, including ER-α, PR-A and PR-B, in correlation to EC tumor histological depth. METHODS: Using immunohistochemistry and image analysis software, the expression of ER-α, PR-A, PR-B and Ki67 was assessed in endometrial stroma and epithelial glands of superficial, deep and extra-tumoral sections of 15 paraffin embedded EC specimens, and compared to 5 biopsies of non-malignant endometrium. RESULTS: Expression of PR-A and ER-α was found to be lower in EC compared to nonmalignant tissue, as the stromal expression was dramatically reduced compared to epithelial cells. Expression ratios of both receptors were significantly high in superficial and deep portions of EC; in non-tumoral portion of EC were close to the ratios of nonmalignant endometrium. PR-B expression was low in epithelial glands of EC superficial and deep portions, and high in the extra-tumoral region. Elevated PR-B expression was found in stroma of EC, as well. CONCLUSIONS: The ratio of ER-α and PR-A expression in the epithelial glands and the stroma of EC biopsies may serve as an additional parameter in the histological evaluation of EC tumor. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1155060506119016.

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.

The effects of decidual injury on the invasion potential of trophoblastic cells.

OBJECTIVE: To investigate the effect of a decidual incision on trophoblastic invasion potential in vitro. METHODS: Human trophoblast cells were obtained from first-trimester legal terminations of pregnancy. Decidual tissue was retrieved from healthy, low-risk women who underwent an elective cesarean delivery at term. Each dissected decidual sample was divided into four similar-sized samples. The first slice was not treated, the second was incised with a surgical blade to mimic an in vivo injury, the third was incised and immediately repaired with medical adhesive material. This model was used to investigate trophoblastic invasion through a fully repaired decidua. The fourth slice was covered with medical adhesive material only, to exclude any effect of the adhesive material on the decidua. The percent of invasion was calculated as: absorbance of invaded cell×100=invasion index (%). Invasion was expressed as invasion index. The mean and standard deviation of the invasion index were then calculated. RESULTS: Eight decidual samples were retrieved from eight women. Incised decidua showed a significantly higher mean invasion index (83.3% [±8.1%], P=.012) than the other three models (intact decidua, 69.9% [±5.1%]; incised decidua repaired with adhesive, 66.6% [±8.2%]; intact decidua with adhesive, 58.3% [±11.3%]. There was no significant difference in the invasion index between the other models (P=.4). CONCLUSION: Induced decidual injury significantly increased the invasion potential of trophoblastic cells compared with intact decidua. Complete re-approximation of the incised edges reversed this effect in vitro.

Human amniotic epithelial cells cultured in substitute serum medium maintain their stem cell characteristics for up to four passages.

BACKGROUND AND OBJECTIVES: The common applied culture medium in which human amniotic epithelial cells (hAECs) maintain their stem cell characteristics contains fetal calf serum (FCS) and thus is not compatible with possible future clinical applications due to the danger of animal derived pathogens. To overcome this problem, we replaced FCS with serum substitute supplement, a serum substitute used in the in vitro fertilization for embryo development, in the common applied culture medium and cultured hAECs in this substitute serum medium (SSM). METHODS AND RESULTS: Purity validation and characterization of freshly isolated and cultured hAECs was assessed through the expression of stem cell specific markers by RT-PCR (gene expression), by immunofluorescence staining and FACS (protein expression). Furthermore, karyotype was performed at passage four in order to exclude possible chromosome anomalies in hAECs cultured in SSM. The differentiation potential of hAECs into the cardiomyogenic lineage was tested through cardiac Troponin T expression by immunohistochemistry. hAECs cultured in SSM maintained expression of all the major pluripotent genes Sox-2, Oct-4 and Nanog as well as the expression of the embryonic stem cell specific surface antigens SSEA-4, SSEA-3 and TRA-1-60 over four passages. Using cardiac differentiation medium containing 10% serum substitute supplement, hAECs differentiated into cardiac troponin T expressing cells. CONCLUSIONS: We can conclude that, hAECs maintain their stem cell characteristics when cultured in SSM for up to 4 passages. This makes possible future clinical applications of these cells more feasible.

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.

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.

[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.

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.

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.

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.

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.

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 efficacy of specific IVIG anti-idiotypic antibodies in antiphospholipid syndrome (APS): trophoblast invasiveness and APS animal model.

OBJECTIVES: Administration of intravenous Ig (IVIG) is a recognized, safe and efficient mode of immunomodulatory therapy for many autoimmune diseases. Anti-idiotypic antibody binding to pathogenic autoantibodies and hence inhibition of binding to the corresponding antigen is one postulated mechanism of the beneficial effect of IVIG. The aim of this study was to fractionate the anti-beta-2-glycoprotein-I (beta(2)GPI) anti-idiotypic antibodies from a commercial IVIG preparation and use it as a treatment in an experimental antiphospholipid syndrome (APS) mouse model. METHODS: Anti-beta(2)GPI polyclonal antibodies were purified on a beta(2)GPI column. The purified antibodies were bound to CN-Br-activated sepharose and employed for purification of IVIG-anti-anti-beta(2)GPI (anti-idiotypic antibodies), defined as specific intravenous Ig (sIVIG). The idiotype specificities were confirmed by competition assays. The effect of sIVIG in vitro was tested in a trophoblast and choriocarcinoma matrigel/invasion assay (i.e. proliferation and metalloproteinase (MMP)2/MMP9 expression) and in vivo in a fetal loss model of APS. RESULTS: Anti-beta(2)GPI antibodies inhibited human trophoblast cell invasion in vitro. The function was attributed to the Fab portion of the anti-beta(2)GPI Igs, since beta(2)GPI-related synthetic peptides specific for the Fab part of the anti-beta(2)GPI antibodies neutralized its activity. APS sIVIG fraction reduce human trophoblast invasion in vitro by 560 times more than the whole IVIG compound and improved the MMP2 and MMP9 production by trophoblast cells. sIVIG improved significantly (200 times more) the pregnancy outcome in BALB/c mice passively infused with anti-beta(2)GPI antibodies, in comparison to treatment with IVIG (P < 0.02). CONCLUSIONS: Based on the current results, we propose that APS sIVIG may be considered as potential specific therapeutic safe compound for developing a treatment for APS patient's early fetal loss.

Human uterine epithelial RL95-2 and HEC-1A cell-line adhesiveness: the role of plexin B1.

OBJECTIVE: To study the expression of plexin-B1 in high- and low-receptive epithelial-endometrial cell lines, and its possible role in endometrial adhesiveness. DESIGN: Controlled, laboratory study. SETTING: Laboratory for Research in Reproductive Sciences, Department of Obstetrics and Gynecology, Ha'Emek Medical Center, Afula, Israel. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): This study was designed to explore and compare the expression and role of plexin-B1 in endometrial cell lines RL95-2 and HEC-1A, used as models of receptive and nonreceptive cells, respectively. The expression of plexin-B1 was analyzed by Western blotting and reverse-transcriptase polymerase chain reaction. The possible role of plexin-B1 in endometrial-trophoblast adhesiveness was studied with attachment and invasion assays. For further validation, we transfected HEC-1A cells with an expressing vector encoded for plexin-B1. RESULT(S): Significant differences in spheroid attachment was observed between RL95-2 and HEC-1A cells. Western blotting and reverse-transcriptase polymerase chain reaction revealed that in RL95-2 cells, the expression of plexin-B1 was significantly higher. An attachment assay that used RL95-2 cells in the presence of inhibiting antibodies against plexin-B1 significantly decreased the attachment rates of spheroids. A comparison between HEC-1A and transfected HEC-1A (HEC-1A-2) cells showed significant differences in spheroid attachment. No significant difference was found between HEC-1A-2 and RL95-2. An attachment assay using inhibitory antibodies against plexin-B1 significantly decreased the spheroid-attachment rate. CONCLUSION(S): Based on our results, we think that plexin-B1 contributes to trophoblast-endometrium interactions, most likely by enhancing adhesion properties.

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 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.