Gillian D. Bryant-Greenwood

The extracellular matrix of the human fetal membranes: Structure and function

The human fetal membranes are genetically identical to the fetus and form a highly specialized interface between mother and fetus, of considerable significance to the successful maintenance and termination of pregnancy in the higher vertebrates. Additionally, the upright posture of women presents these tissues with a greater mechanical challenge than in other species. The major extracellular matrix components providing tensile strength and elastic recoil are reviewed, as well as the key enzyme, activator/inhibitor system responsible for their remodelling and breakdown. However, this fails to convey the important concept that the matrix components are bound to each other and to the cells involved in their formation and organization. These matrix components are collectively responsible for the biomechanical properties of the tissue, but they must also be considered as dynamic elements of a broader signalling system, which include hormonal autocrine/paracrine systems. A unifying hypothesis is presented, which attempts for the first time to bring these two facets of the matrix together, which permits a potential coordination of local events at the maternal-fetal interface leading to parturition. In order to understand fully both the normal biology and the pathobiology of these tissues, such integration of the cellular and extracellular signalling pathways must be achieved.

Control of peripartal collagenolysis in the human chorion-decidua

OBJECTIVE This study was designed to observe the in vivo status of chorion-decidua gene expression for some major metalloproteinases, an inhibitor (tissue inhibitor of metalloproteinase-1), and an activator (tissue plasminogen activator) and the hormone relaxin at accessible time points in the peripartal period. STUDY DESIGN Chorion-decidua from patients at cesarean section with and without labor and after spontaneous labor and delivery was used for preparation of poly (A)+ ribonucleic acid and quantitative Northern analyses with a series of oligo and complementary deoxyribonucleic acid probes. RESULTS In the period designated as the period before parturition, relaxin and matrix metalloproteinase-1 (interstitial collagenase) gene expression were relatively high, reflecting the controlled loss of amniotic collagen necessary for fetal membrane expansion without rupture as the uterine volume increases. When active labor has begun, but before delivery, matrix metalloproteinase-3 (stromelysin) and matrix metalloproteinase-9 (type V collagenase) gene levels significantly increased. After normal spontaneous labor and delivery, tissue plasminogen activator and tissue inhibitor of metalloproteinase-1 messenger ribonucleic acids significantly increased, together with a marginally increased expression of the genes for matrix metalloproteinase-1, matrix metalloproteinase-2 (type IV collagenase), and relaxin. CONCLUSION The expression of the genes for some major collagenolytic enzymes, an inhibitor, activator, and relaxin in the chorion-decidua, is different at the different stages of parturition.

The LOXL2 Gene Encodes a New Lysyl Oxidase-like Protein and Is Expressed at High Levels in Reproductive Tissues

We have reported in this paper the complete cDNA sequence, gene structure, and tissue-specific expression of LOXL2, a new amine oxidase and a member of an emerging family of human lysyl oxidases. The predicted amino acid sequence, from several overlapping cDNA clones isolated from placenta and spleen cDNA libraries, shared extensive sequence homology with the conserved copper-binding and catalytic domains of both lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) protein. These conserved domains are encoded by five consecutive exons within the LOX,LOXL, and LOXL2 genes that also maintained exon-intron structure conservation. In contrast, six exons encoding the amino-terminal domains diverged both in sequence and structure. Exon 1 of the LOXL2 gene does not encode a signal sequence that is present in LOX and LOXL, suggesting a different processing and intracellular localization for this new protein. Expression of the LOXL2 gene was detected in almost all tissues with the highest steady state mRNA levels in the reproductive tissues, placenta, uterus and prostate. In situ hybridization identified placental syncytial and cytotrophoblasts responsible for the synthesis of LOXL2mRNA and demonstrated a spatial and temporal expression pattern unique to the LOXL2 gene.

Sequential appearance of relaxin, prolactin and IGFBP-1 during growth and differentiation of the human endometrium.

Relaxin (RLX) is a product of the human corpus luteum, pregnancy decidua and placenta, prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) are products of the cyclic endometrium and of the pregnancy decidua. All three proteins are thought to function interdependently in endometrium/decidua as local factors within the uterus without reaching the systemic circulation. In this study, the avidin-biotin immunoperoxidase method for immunolocalization with monoclonal or polyclonal antibodies has been applied to serial sections of endometria obtained from patients at different stages of the menstrual cycle and in early and late gestation. This allowed the cellular localization of the three proteins to be followed simultaneously through the reproductive stages from cyclic endometrium to term gestational decidua. The production, as opposed to sequestration of RLX from an ovarian source was demonstrated by the application in parallel of an antibody to the processed hormone and its connecting peptide. RLX was shown localized to the glandular and luminal epithelia in the proliferative and secretory phases. The decidualized stromal cells also immunostained for RLX in the late secretory phase and in early and late pregnancy. PRL was localized first to the glandular epithelium and then stroma, appearing after RLX, IGFBP-1 appeared later in the secretory phase and predominantly in the decidualized stromal cells confirming previous studies. In contrast, all three proteins were immunostained in early pregnancy and increased to term gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Visfatin/Pre-B cell colony-enhancing factor in amniotic fluid in normal pregnancy, spontaneous labor at term, preterm labor and prelabor rupture of membranes: An association with subclinical intrauterine infection in preterm parturition

Abstract Objective: Visfatin, a novel adipokine originally discovered as a pre-B-cell colony enhancing factor, is expressed by amniotic epithelium, cytotrophoblast, and decidua and is over-expressed when fetal membranes are exposed to mechanical stress and/or pro-inflammatory stimuli. Visfatin expression by fetal membranes is dramatically up-regulated after normal spontaneous labor. The aims of this study were to determine if visfatin is detectable in amniotic fluid (AF) and whether its concentration changes with gestational age, spontaneous labor, preterm prelabor rupture of membranes (preterm PROM) and in the presence of microbial invasion of the amniotic cavity (MIAC). Methods: In this cross-sectional study, visfatin concentration in AF was determined in patients in the following groups: 1) mid-trimester (n=75); 2) term not in labor (n=27); 3) term in spontaneous labor (n=51); 4) patients with preterm labor with intact membranes (PTL) without MIAC who delivered at term (n=35); 5) patients with PTL without MIAC who delivered preterm (n=52); 6) patients with PTL with MIAC (n=25); 7) women with preterm PROM without MIAC (n=26); and 8) women with preterm PROM with MIAC (n=26). Non-parametric statistics were used for analysis. Results: 1) The median AF concentration of visfatin was significantly higher in patients at term than in mid-trimester; 2) Among women with PTL who delivered preterm, the median visfatin concentration was significantly higher in patients with MIAC than those without MIAC; 3) Similarly, patients with PTL and MIAC had a higher median AF visfatin concentration than those with PTL who delivered at term; 4) Among women with preterm PROM, the median AF visfatin concentration was significantly higher in patients with MIAC than those without MIAC. Conclusions: 1) Visfatin is a physiologic constituent of AF; 2) The concentration of AF visfatin increases with advancing gestational age; 3) AF visfatin concentration is elevated in patients with MIAC, regardless of the membrane status, suggesting that visfatin participates in the host response against infection.

Fetal membrane distention

OBJECTIVE This study was undertaken to determine which genes were up-regulated by acute distention in an amniotic epithelial cell line and in human fetal membranes. STUDY DESIGN WISH cells, a human amniotic epithelial cell line, were grown on silicone elastomer sheets coated with extracellular matrix and reproducibly distended by 40% in a novel device for 4 hours. Differential gene expression was analyzed by means of suppression subtractive hybridization. Expression of the identified genes was then quantitated by Northern blot analysis in fetal membrane explants after distention in the same device for 4 hours. The effect of distention on apoptosis of the cells and tissue samples was concomitantly studied by means of the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method. RESULTS The genes for interleukin 8 and pre-B-cell colony-enhancing factor were found to be up-regulated in both the WISH cells and the distended fetal membranes. The apoptotic index values in both the cells and the tissue samples were unaffected by distention. CONCLUSIONS Acute distention induces the up-regulation of interleukin 8 and pre-B-cell colony-enhancing factor in both WISH cells and human fetal membranes and does not cause apoptosis.

The effects of pre-B-cell colony-enhancing factor on the human fetal membranes by microarray analysis

OBJECTIVE Our purpose was to show the effects of pre-B-cell colony-enhancing factor on the genes that are expressed by the human fetal membranes. STUDY DESIGN Explants of fetal membranes (amnion, chorion, and decidua) from three term patients were treated with 100 ng/mL recombinant human pre-B-cell colony-enhancing factor for 4 hours. RNAs were hybridized to gene chips that contained >18,000 known genes. One experiment was done in triplicate to assess replication. Data were analyzed to quantitate the signal intensities of each complementary DNA on the array. Confirmation of the results was carried out on tissues from nine other patients by the measurement of the proteins or quantitative real-time reverse transcriptase-polymerase chain reaction. RESULTS Replication gave <92.6% identical results, which showed high method reproducibility. Pre-B-cell colony-enhancing factor treatment caused a significant increase in 103 genes and decrease in 139 genes. Only 8 genes were up-regulated consistently and significantly in all three patients (three key inflammatory cytokines [tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta], four important chemokines [macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, macrophage inflammatory protein-3alpha, and growth-related oncogene-gamma], and prostaglandin-endoperoxide synthase 2). These data were confirmed by the measurement in the media with the use of specific enzyme-linked immunosorbent assays for tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and macrophage inflammatory protein-3alpha and by quantitative real-time reverse transcriptase-polymerase chain reaction for growth-related oncogene-gamma and prostaglandin-endoperoxide synthase 2. CONCLUSION Pre-B-cell colony-enhancing factor appears to be at the proximal end of the pathway to labor initiation and may link sterile distention-induced labor with that of infection-induced labor.

Fetal membrane distention: II. Differentially expressed genes regulated by acute distention in vitro.

OBJECTIVE This study was undertaken to identify genes with expression up-regulated by acute distention in the human fetal membranes. STUDY DESIGN Fetal membrane explants were distended reproducibly in a novel device in vitro for 4 hours, and suppression subtractive hybridization was used to identify the candidate genes for up-regulation of expression in response to this stimulus. The up-regulation in response to distention was confirmed by quantitative Northern blot analysis both after a 4-hour in vitro distention and after labor in vivo. RESULTS Suppression subtractive hybridization identified 3 genes with expression up-regulated by acute distention: an interferon-stimulated gene encoding a 54-kd protein, the gene for huntingtin-interacting protein 2 (a ubiquitin-conjugating enzyme), and a novel transcript. Expression of each of the distention-responsive genes found to be up-regulated in vitro was also up-regulated in fetal membranes in association with labor. CONCLUSIONS Suppression subtractive hybridization was successfully applied to a complex tissue, the human fetal membranes, and 3 novel distention-responsive genes were identified. Both acute in vitro distention and labor in vivo up-regulate expression of at least 3 genes in the human fetal membranes.

Detection of elastin in the human fetal membranes: Proposed molecular basis for elasticity

The human fetal membranes provide a sterile biomechanical container which adjust by growth to mid-pregnancy to the increase in fetal size, and by elasticity to the forceful movements of the fetus. The molecular basis for this elasticity is not known, yet reduced elasticity may lead to their premature rupture and preterm birth, a major problem in perinatal medicine. Classically, elastin confers the property of elastic recoil to elastic fibres which are assembled from a family of tropoelastin precursors. These are covalently cross-linked to form insoluble elastin by formation of desmosine and isodesmosine, catalysed by the enzyme lysyl oxidase. The amnion, chorion and decidua were shown by Northern analysis and RT-PCR to contain detectable levels of tropoelastin mRNA and the mRNA encoding lysyl oxidase. The proteins encoded by these mRNAs were also identified by Western blotting and immunolocalization. Further, insoluble elastin was extracted from the human fetal membranes and shown by comparison to elastin preparations from other elastic tissues to have a reasonable desmosine content. Finally, scanning electron microscopy confirmed the presence of multiple layers of an apparently very thin elastic system in this tissue. This biochemical and histopathologic study has demonstrated therefore that the human fetal membranes synthesize and deposit a novel elastic fibre. The presence of such an elastic system in these tissues provides, for the first time, a probable molecular basis for the elastic properties of this tissue.

A relaxin-mediated pathway to preterm premature rupture of the fetal membranes that is independent of infection.

OBJECTIVE This study was designed to show whether the overexpression of relaxin in the decidua of patients with preterm premature rupture of the membranes is independent of or a consequence of chorioamnionitis. STUDY DESIGN Two experiments were conducted. In the first experiment fetal membranes and decidua were collected from patients with preterm premature rupture of the membranes (n = 17) or preterm labor (n = 17) and were divided according to their degree of histologic infection. Messenger ribonucleic acid was isolated from the tissues and quantitative, sequential Northern analyses were carried out for the expression of human relaxin, interleukin-1beta, interleukin-6, and interleukin-8. The second experiment was aimed at increasing the numbers of messenger ribonucleic acid preparations in the two extreme categories, uninfected and severely infected tissues, with preterm premature rupture of the membranes and preterm labor. Some samples of messenger ribonucleic acid from the first experiment were rerun with the Northern analyses in the second experiment. These repeat samples showed no statistical differences in the results run at different times. Therefore the data from the respective groups of patients in both experiments were pooled for statistical analysis. RESULTS In both the first experiment and in the pooled data of the two experiments the expression of the relaxin genes was significantly greater (P < .005) in the tissues from patients with preterm premature rupture of the membranes compared with those with preterm labor, in the absence of infection. No effect of the level of infection on the expression of relaxin was noted. In contrast, interleukin-6 gene expression was significantly increased (P < .05) in severely infected tissues, which was independent of whether the delivery was from preterm premature rupture of the membranes or preterm labor. The expression of the interleukin-1beta and interleukin-8 genes were only marginally increased even in severe infection. Marked patient variability in expression of the interleukin genes, especially in severe infection, was noted. CONCLUSION A relaxin-mediated pathway that leads to preterm premature rupture of the membranes may exist independent of infection.