Shlomit Goldman

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

BackgroundThe 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.MethodsFirst 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. Students T-test was used for statistical analysis.ResultsIn 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.ConclusionsFirst 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.

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.

Pregnancy and birth after transfer of embryos that developed from single- nucleated zygotes obtained by injection of round spermatids into oocytes

OBJECTIVE To use injection of spermatids into oocytes as a mode of infertility treatment in cases in which spermatozoa are not available. DESIGN Prospective clinical evaluation and case report. SETTING In Vitro Fertilization Unit, Herzliya Medical Centers, Herzliya-on-Sea, Israel. PATIENT(S) Thirteen couples with male factor infertility in which the male partner lacked spermatozoa in the ejaculate or testicular biopsy samples. INTERVENTION(S) Round spermatid injection and elongated spermatid injection into oocytes. MAIN OUTCOME MEASURE(S) Evaluation of the rate of two-pronucleated and single-nucleated zygote development. RESULT(S) The rate of two-pronucleated zygote development after round spermatid injection and elongated spermatid injection was relatively low (27% and 36%, respectively). Single-nucleated zygotes develop more frequently after round spermatid injection and elongated spermatid injection (35% and 17%, respectively) than after intracytoplasmic sperm injection with mature spermatozoa. A normal pregnancy and childbirth resulted from the transfer of 4 cleaving embryos, each of which developed from a single-nucleated zygote in a round spermatid injection treatment cycle with ejaculated spermatids. CONCLUSION(S) Embryos derived from single-nucleated zygotes after spermatid conception can be viable and give rise to an ongoing clinical pregnancy and childbirth.

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 mares 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.

A simplified coculture system with luteinized granulosa cells improves embryo quality and implantation rates: a controlled study.

OBJECTIVE To investigate the use of a simplified short-term coculture system with luteinized granulosa cells (GCs) in patients with failed IVF-ET. DESIGN Controlled clinical study. SETTING IVF unit, Department of Obstetrics and Gynecology, Carmel Medical Center. PATIENT(S) Patients with poor embryo quality in their previous IVF-ET cycles. INTERVENTION(S) Embryos from 40 patients, in which > 50% of the embryos were classified as poor quality in their previous IVF attempts, were grown on autologue GC culture system for a short period (24-48 hours) before being replaced in the uterine cavity. MAIN OUTCOME MEASURE(S) Embryo quality. RESULT(S) Significant decrease in poor quality embryos and increase in the proportion of good quality embryos were observed using a coculture system with autologue human GCs. Pregnancy rates in this groups of patients reached our standard IVF results during the same period. CONCLUSION(S) This study describes a simplified short-term coculture system with human autologue GCs. Poor quality embryos may be rescued to cleave regularly.

p53 Mediates Epidermal Growth Factor (EGF) Induction of MMP-2 Transcription and Trophoblast Invasion

Trophoblast invasion is a highly restricted process, regulated by growth factors, hormones and cytokines. Trophoblast invasion is attainable due to proteolytic degradation of the epithelial basement membrane and the extracellular matrix by proteolytic enzymes like the matrix metalloproteinases (MMPs) particularly MMP-2. Epidermal growth factor (EGF), a major mediator of implantation, has been documented to induce MMP-2 and trophoblast invasion. The aim of this study was to investigate the transcriptional regulation of MMP-2 in EGF- stimulated invasive trophoblast cells, using JAR choriocarcinoma cell line and 6-8w 1st trimester trophoblasts. MMP-2 expression was induced by EGF within 1 hour. Gelshift and supershift assay were used to explore transcription factors involved in MMP-2 regulation. EGF induced binding activity and expression of phophorylated p53, AP-2alpha and -gamma, C/EBPepsilon and -lambda to their responding sequences, found in the MMP-2 promoter. Additionally EGF induced binding activity to SP-1, but reduced the expression of SP-1 and SP-4. Inhibition of p53 by antisense reduced both basic and EGF- induced MMP-2 expression. In summary: MMP-2 transcriptional regulation by EGF in invasive trophoblasts is mediated through several binding sites and transcription factors including p53, AP-2alpha and -gamma, C/EBPepsilon and -lambda and SP-1. p53 mediates both basic and EGF-induced MMP-2 transcription.

Difference in Progesterone-Receptor Isoforms Ratio Between Early and Late First-Trimester Human Trophoblast Is Associated with Differential Cell Invasion and Matrix Metalloproteinase 2 Expression

Abstract The expression profile of the progesterone-receptor isoforms and progesterone regulation of matrix metalloproteinase 2 (MMP2) were investigated in early and late first-trimester trophoblast cells. Human trophoblast cells were obtained from legal abortions (6–12 wk of gestation). Purity of 95–98% was verified using immunohistochemistry with specific antibodies. Evaluation of cell count was performed with XTT Reagent kit, and invasion was tested using Matrigel invasion assay. Zymography was used to detect proteolytic activity, and Western blot immunoassay was used to study protein concentration. Gene expression of PGRB, PGR, and MMP2 was studied using reverse transcription-polymerase chain reaction with the housekeeping gene GAPDH used for normalization. Promoter activity was determined using luciferase reporter assay. Differential progesterone-receptor profile was documented with the dominance of PGRB in early trophoblast and the dominance of PGRA in late trophoblast. This differential profile is compatible with the inverse effect of progesterone on the two cell populations, decreasing invasion and gelatinase expression in the early first-trimester trophoblast and increasing invasion and gelatinase expression in the late first-trimester trophoblast. A decrease in MMP2 promoter activity in early trophoblast cells exposed to progesterone suggests that MMP2 expression is regulated by progesterone at the transcriptional level as well. Early trophoblast cells transfected with expressing vector for PGR encoding PGRA revealed less MMP2 activity and reversal of its response to progesterone similar to the effect observed in late trophoblast cells.

Mitogen-activated protein kinase in human eggs

Mitogen-activated protein (MAP) kinase in human eggs has been investigated by using immunoblotting with both anti-Active MAPK and anti-ERK2 antibodies. The results showed that the main form of MAP kinase was p42ERK2. It was in a dephosphorylated form in oocytes at the germinal vesicle stage, but fully phosphorylated in unfertilised mature eggs. MAP kinase phosphorylation was significantly decreased when pronuclei were formed after intracytoplasmic sperm injection. Neither MAP kinase expression nor activity was detected in morphologically degenerated eggs. Although MAP kinase still existed in early embryos arrested at the 8-cell or morula stages, little, if any, activity could be detected. These data suggest that MAP kinase may play an important role in the cell cycle regulation of human eggs, as in other mammalian species.

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.

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.