Human relaxins (RLNH1, RLNH2), their receptor (RXFP1) and fetoplacental growth.

Relaxin, a systemic and placental hormone, has potential roles in fetoplacental growth. Human placenta expresses two RLN genes, RLNH1 and RLNH2 Maternal obesity is common and is associated with abnormal fetal growth. Our aims were to relate systemic and cord blood RLNH2, placental RLNs and their receptor (RXFP1) with fetoplacental growth in context of maternal body mass index, and associations with insulin-like growth factor 2 (IGF2) and vascular endothelial growth factor A (VEGFA) in the same placentas. Systemic, cord blood and placental samples were collected prior to term labor, divided by prepregnancy body mass index: underweight/normal (N = 25) and overweight/obese (N = 44). Blood RLNH2 was measured by ELISA; placental RLNH2, RLNH1, RXFP1, IGF2 and VEGFA were measured by quantitative immunohistochemistry and mRNAs were measured by quantitative reverse transcription PCR. Birthweight increased with systemic RLNH2 only in underweight/normal women (P = 0.036). Syncytiotrophoblast RLNH2 was increased in overweight/obese patients (P = 0.017) and was associated with placental weight in all subjects (P = 0.038). RLNH1 had no associations with birthweight or placental weight, but was associated with increased trophoblast and endothelial IGF2 and VEGFA, due to female fetal sex. Thus, while systemic RLNH2 may be involved in birthweight regulation in underweight/normal women, placental RLNH2 in all subjects may be involved in placental weight. A strong association of trophoblast IGF2 with birthweight and placental weight in overweight/obese women suggests its importance. However, an association of only RLNH1 with placental IGF2 and VEGFA was dependent upon female fetal sex. These results suggest that both systemic and placental RLNs may be associated with fetoplacental growth.

Protective proteins and telomere length in placentas from patients with pre-eclampsia in the last trimester of gestation.

INTRODUCTION: Visfatin/nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in energy metabolism and sirtuins, SIRT1 and SIRT3, which are NAD-dependent deacetylases, are critical for cellular function. All three either regulate or are regulated by intracellular NAD+ levels and therefore available cellular energy, important for placental cell survival and successful pregnancy. This study investigates whether these protective proteins are involved in the placental pathophysiology of pre-eclampsia (PE) and if they are associated with 8-oxo-deoxyguanosine (8OHdG), a marker of oxidative damage or with placental telomere length. METHODS: Maternal blood and placental samples were collected from 31 patients with PE and 30 controls between 31 and 40 weeks gestation. Quantitative immunohistochemistry was performed on placental specimens for visfatin/Nampt, SIRT1, SIRT3, and nuclear 8OHdG. Plasma visfatin was measured by ELISA and telomere length by Southern blot analysis of telomere restriction fragments. RESULTS: Visfatin/Nampt and SIRT1 in syncytiotrophoblast decreased in PE compared to controls (p < 0.0001, p = 0.004 respectively). SIRT3 decreased in PE most significantly at preterm (p = 0.002). 8OHdG was only significantly lower in preterm controls compared to term controls (p = 0.01) and correlated with SIRT1 in all samples (r = 0.27). Telomere length was not different in PE and controls. DISCUSSION: Decreased visfatin/Nampt, SIRT1 and SIRT3 in syncytiotrophoblast in PE suggests a lack of placental reserve in metabolic energy efficiency, increased inflammation, and lower resistance to environmental stressors. However, there was little effect on nuclear function, or evidence of genomic DNA damage, which would lead to cellular senescence and death.

Systemic and placental α-klotho: Effects of preeclampsia in the last trimester of gestation.

INTRODUCTION: α-klotho is an anti-aging protein, potentially important in preeclampsia (PE). Produced by kidney, brain and placenta, and by mRNA splicing is both a full-length membrane-bound and a truncated soluble protein in the circulation. The membrane-bound protein is an obligate co-receptor for fibroblast growth factor 23 (FGF23) and its action on receptor (FGFR), but ADAM proteinases also cause its shedding. The aims of this study were to investigate levels of maternal plasma, placental, and fetal membrane α-Klotho and their association with placental accelerated villous maturation (AVM) in PE. In addition, placental and membrane levels of ADAM17 and FGFR were measured in the same patients. METHODS: Maternal blood, placenta and fetal membranes from 61 women (31 with PE and 30 controls) between 32 and 40 weeks gestation were collected. Plasma α-klotho was measured by ELISA, and quantitative immunohistochemistry used for α-klotho, ADAM17 and FGFR1 in tissues. Placental AVM was histologically assessed. RESULTS: Maternal plasma levels of α-Klotho were higher in PE compared to controls (p = 0.01) and patients with the highest levels had significantly less AVM (p = 0.03). α-Klotho, ADAM17, and FGFR were all present in syncytiotrophoblast and cytotrophoblast of membranes. Between 32 and 40 weeks gestation, all placental levels decreased in controls respectively (p = 0.04, p = 0.004, p = 0.05), but not in PE. Fetal membrane levels were unchanged. DISCUSSION: Maternal plasma α-Klotho was increased in PE and its levels associated with reduced placental AVM. Changes in placental α-Klotho, ADAM17, and FGFR suggest their involvement in the pathophysiology of PE.

Genetic associations of relaxin: preterm birth and premature rupture of fetal membranes.

OBJECTIVE: Relaxin H2 (RLN2) is a systemic hormone (sRLN) that is produced by the corpus luteum, whereas decidual RLN (dRLN) acts only locally. Elevated sRLN is associated with spontaneous preterm birth (sPTB) and elevated dRLN with preterm premature rupture of membranes (PPROM). Associations were sought between single nucleotide polymorphisms (SNPs) in the RLN2 promoter with levels of dRLN and sRLN in Filipino patients with sPTB, PPROM, or normal term delivery. STUDY DESIGN: Stringent selection of women with sPTB (n = 20) or PPROM (n = 20) and term control subjects (n = 20) was made from >8000 samples from Filipino patients who delivered at 34-36 weeks' gestation. Twelve SNPs were genotyped on maternal blood, with 9 excluded based on the high linkage disequilibrium or being the same as in the control population. Quantitative immunocytochemistry on parietal decidual tissue was performed (n = 60); sRLN was measured by enzyme-linked immunosorbent assay in a subset of patients (n = 21). RESULTS: SNP rs4742076 was associated significantly with PPROM (P < .001) and increased expression of dRLN (P < .001). The genotype TT had increased dRLN in PPROM (P < .05). SNP rs3758239 was associated significantly with both PPROM and sPTB (P < .01), and genotype AA had increased dRLN expression (P < .05). The sRLN showed a trend of higher levels in PPROM and sPTB, but was not significant. CONCLUSION: SNP rs4742076 in the RLN2 promoter was associated with increased dRLN expression and PPROM; SNP rs3758239 was associated with both PPROM and sPTB in these Filipino patients. Specific homozygous genotypes were identified for both SNPs and were shown to be associated with increased dRLN tissue expression.

Relaxin, its receptor (RXFP1), and insulin-like peptide 4 expression through gestation and in placenta accreta.

This study was designed to show whether placental relaxin (RLN), its receptor (RXFP1), or insulin-like peptide 4 (INSL4) might have altered expression in patients with placenta accreta. The baseline expression of their genes through gestation (n = 34) was quantitated in the placental basal plate (BP) and villous trophoblast (TR), and compared to their expression in placenta accreta (n = 6). The proteins were also immunolocalized and quantitated in the accreta tissues. The messenger RNAs (mRNAs) of matrix metalloproteinase 9, -2, and tissue inhibitors of matrix metalloproteinase (TIMP)-1 were also measured. Results demonstrated that the BP and TR expressed low levels of RLN/RXFP1 and INSL4 through gestation. In accreta, increased RLN gene and protein in BP were associated with antepartum bleeding whereas INSL4 expression decreased throughout the TR. There were no changes in mRNAs for MMPs, but TIMP-1 was increased only in the invasive TR.

Relaxin and preterm birth.

Preterm birth (PTB) is a global problem with a high incidence in the developing world. Relaxin (RLN) has classically been associated with parturition, but its role(s) in the human have been difficult to determine. For the first time, we bring together the systemic (ovarian) and autocrine/paracrine (intrauterine) sources of RLN, in an attempt to understand how RLN contributes to PTB in women.

Mechanisms of relaxin receptor (LGR7/RXFP1) expression and function.

The LGR7/RXFP1 and LGR8/RXFP2 receptors are unique receptors among the G-protein-coupled receptors (GPCRs) in having a low-density lipoprotein class A (LDL-A) module. Their complex gene organization, among the intron-richest of the GPCRs, suggests that alternative splicing is a common occurrence. We have therefore investigated the role of the LDL-A module and shown the identity, expression, and functions of three LGR7 splice variants in the human decidua. Point mutations of conserved residues or complete deletion of the LDL-A module resulted in loss of the cAMP response to relaxin. Its glycosylation also impacted LGR7 cell surface delivery and therefore receptor activation. The wild-type (WT) LGR7 was expressed as both precursor and mature forms, but deletion of the LDL-A module resulted in expression of only the mature form. Three new alternatively spliced variants of LGR7 were identified, all containing a truncated extracellular region. Their functional characterization showed them exerting dominant negative effects on the WT LGR7 by preventing its homodimerization, maturation, and subsequent trafficking to the cell surface, resulting in loss of function. In summary, different mechanisms have been identified for controlling the cell surface expression and function of the LGR7 protein which are likely to be significant for the role of relaxin in human parturition.

Identification of relaxin-responsive cells in the human choriodecidua at term.

The decidua of the human maternal-fetal interface is a local source of intrauterine relaxin, and the choriodecidua expresses its receptor (LGR7). Since these tissues consist of a variety of cells, we sought to identify the primary cell(s) responsible for LGR7 expression and relaxin responsiveness.

Characterization of relaxin receptor (RXFP1) desensitization and internalization in primary human decidual cells and RXFP1-transfected HEK293 cells.

We report here the desensitization and internalization of the relaxin receptor (RXFP1) after agonist activation in both primary human decidual cells and HEK293 cells stably transfected with RXFP1. The importance of beta-arrestin 2 in these processes has also been demonstrated. Thus, in HEK-RXFP1 cells the desensitization of RXFP1 was significantly increased when beta-arrestin 2 was overexpressed. After relaxin activation, beta-arrestin 2 was translocated to the cell membrane and RXFP1 underwent rapid internalization. We have previously shown that RXFP1 forms dimers/oligomers during its biosynthesis and trafficking to the plasma membrane, we now show that internalization of RXFP1 occurs through this dimerization/oligomerization. In nonagonist stimulated cells, it is known that the majority of the RXFP1 is located intracellularly and was confirmed in the cells used here. Constitutive internalization of RXFP1 could account for this and indeed, slow but robust constitutive internalization, which was increased after agonist stimulation was demonstrated. A carboxyl-terminal deleted RXFP1 variant had a similar level of constitutive agonist-independent internalization as the wild-type RXFP1 but lost sensitivity to agonist stimulation. This demonstrated the importance of the carboxyl terminus in agonist-stimulated receptor internalization. These data suggest that the autocrine/paracrine actions of relaxin in the decidua are under additional controls at the level of expression of its receptor on the surface of its target cells.

Cloning, expression, and functional characterization of relaxin receptor (leucine-rich repeat-containing g protein-coupled receptor 7) splice variants from human fetal membranes.

The relaxin receptor [leucine-rich repeat-containing G protein-coupled receptor 7 (LGR7)] belongs to the leucine-rich repeat containing G protein-coupled receptors subgroup C. Three new LGR7 splice variants have been cloned from the human fetal membranes and shown to be truncated versions of the full-length receptor, encoded by different lengths of the extracellular domain. The expression of their mRNAs has been confirmed by both qualitative and quantitative PCR and shown to be higher in the chorion and decidua before, compared with after, spontaneous labor. When HEK293 cells were transfected with each LGR7 splice variant, their proteins were retained within the endoplasmic reticulum. However, the protein for the shortest variant was also secreted into the medium. We have characterized the intracellular functions and effects of these LGR7 variants on the function of the wild-type (WT)-LGR7. In coexpression studies, each splice variant interacted directly with the WT-LGR7 and exerted a dominant-negative effect on cAMP accumulation by the WT-LGR7 after relaxin treatment. This interaction resulted in the sequestration of the WT-LGR7 inside the cells by down-regulation of its maturation and cell surface delivery. The constitutive homodimerization of WT-LGR7 has been shown here to take place in the endoplasmic reticulum, and the presence of any one of the splice variants decreased this by the formation of heterodimers with the WT-LGR7, supporting the view that homodimerization is a prerequisite for receptor trafficking to the cell surface. These data suggest that the dominant-negative effects of the LGR7 splice variants expressed in the chorion and decidua could be functionally significant in the peripartal period by inhibiting the function of WT-LGR7 and dampening the responsiveness of these tissues to endogenous relaxin.

The low-density lipoprotein class A module of the relaxin receptor (leucine-rich repeat containing G-protein coupled receptor 7): its role in signaling and trafficking to the cell membrane.

The relaxin receptor (LGR7, relaxin family peptide receptor 1) is a member of the leucine-rich repeat containing G protein-coupled receptors subgroup C. This and the LGR8 (relaxin family peptide receptor 2) receptor are unique in having a low-density lipoprotein class A (LDL-A) module at their N termini. This study was designed to show the role of the LDL-A in LGR7 expression and function. Point mutants for the conserved cysteines (Cys(47) and Cys(53)) and for calcium binding asparagine (Asp(58)), a mutant with deleted LDL-A domain and chimeric LGR7 receptor with LGR8 LDL-A all showed no cAMP response to human relaxins H1 or H2. We have shown that their cell surface delivery was uncompromised. The mutation of the putative N-linked glycosylation site (Asn(36)) decreased cAMP production and reduced cell surface expression to 37% of the wild-type LGR7. All point mutant, chimeric, and wild-type receptor proteins were expressed as the two forms. The immature or precursor form of the receptor was 80 kDa, whereas the mature receptor, delivered to the cell surface was 95 kDa. The glycosylation mutant was also expressed as two forms with appropriately smaller molecular masses. Deletion of the LDL-A module resulted in expression of the mature receptor only. These data suggest that the LDL-A module of LGR7 influences receptor maturation, cell surface expression, and relaxin-activated signal transduction.

Relaxin and the human fetal membranes.

The human fetal membranes are complex tissues that perform many important functions during gestation. The extracellular matrix provides their strength to withstand the forces directed from the fetus and myometrium. Relaxin is a collagenolytic hormone that causes increased production of the matrix metalloproteinases. Its expression from the decidua is increased in patients with preterm premature rupture of the membranes, and its leucine-rich G receptor 7 is upregulated at preterm. The authors previously showed that relaxin is not involved in the infection-mediated cytokine response, but in the absence of infection, it causes increased secretion of both interleukin -6 and interleukin-8 from the membranes. In this article, the authors propose that relaxin is one of a number of sterile stimuli capable of causing increased proinflammatory cytokines, similar to but less robust than the effects of infection. These probably represent distinct inflammatory pathways involving different intracellular signaling events, which can result in either preterm premature rupture of the membranes or preterm labor. The current challenge is to fully understand these pathways and to clarify their similarities and differences.

Pre-B-cell colony-enhancing factor is a secreted cytokine-like protein from the human amniotic epithelium.

OBJECTIVE: The purpose of this study was to determine whether pre-B-cell colony-enhancing factor is a secreted cytokine in the human amnion and to study its chemotaxic and antiapoptotic properties. STUDY DESIGN: Pre-B-cell colony-enhancing factor secretion was studied from amniotic epithelial-like WISH cells and primary amniotic epithelial cells that were seeded on squares of immobilon-P membrane and stimulated with lipopolysaccharide or tumor necrosis factor-alpha, respectively. The pre-B-cell colony-enhancing factor protein was detected both intracellularly and after secretion, as bound to the membrane, by immunostaining and densitometry. Medium and cell lysates that were obtained from WISH cells that were treated with lipopolysaccharide alone or together with a pre-B-cell colony-enhancing factor antisense oligonucleotide to block pre-B-cell colony-enhancing factor translation were also analyzed for secreted pre-B-cell colony-enhancing factor by Western blotting and densitometry. A chemotaxic effect of pre-B-cell colony-enhancing factor on human neutrophils was compared with the chemoattractants interleukin-8 and N-Formyl-Met-Leu-Phe methyl ester in a rapid fluorescence-based neutrophil migration assay. Apoptosis was induced in primary amniotic epithelial cells and fibroblasts by actinomycin D (1 microg/mL); the antiapoptotic effects of pre-B-cell colony-enhancing factor on early apoptosis were measured by the annexin V assay, and the late effects were determined by measurement of nuclear matrix protein in the media. RESULTS: Treatment of amnion cells that adhered to immobilon-P membrane to induce the secretion of pre-B-cell colony-enhancing factor showed significantly (P<.05) more pre-B-cell colony-enhancing factor protein surrounding the cells compared with the controls. Although the addition of lipopolysaccharide to cultured WISH cells caused the secretion of pre-B-cell colony-enhancing factor into the medium, co-treatment with an antisense oligonucleotide to pre-B-cell colony-enhancing factor obliterated it. Analysis of the cell lysates showed no significant change, which suggests that most of the pre-B-cell colony-enhancing factor protein had been secreted. No significant chemotaxic effects of pre-B-cell colony-enhancing factor were observed; however, pre-B-cell colony-enhancing factor treatment (100 ng/mL), together with actinomycin D, cancelled the early induction of apoptosis, although there was a dose-dependent and significant late antiapoptotic effect on primary amnion epithelial cells (P<.001) and fibroblasts (P<.01). CONCLUSION: Pre-B-cell colony-enhancing factor is a secreted protein from amniotic epithelial cells. Although it had no chemotaxic effects, it was antiapoptotic for both amniotic epithelial cells and fibroblasts and may protect these cells against apoptosis that is induced by chronic distension, labor, or infection.

Human decidual relaxin and preterm birth.

Relaxin in human pregnancy is both a systemic hormone from the corpus luteum and an autocrine/paracrine hormone at the maternal-fetal interface formed by the decidua/placenta and fetal membranes. We have focused our studies on the autocrine/paracrine roles of relaxin, especially in the preterm premature rupture of the fetal membranes, which causes 30-40% of preterm births. By using different techniques and different tissue collections, our laboratory has shown that expression of the relaxin genes and proteins in the decidua and placenta is increased in patients with preterm premature rupture of the fetal membranes. Relaxin binding and the expression of LGR7 are primarily in the chorion and decidua and are downregulated after spontaneous labor and delivery both at term and preterm. However, expression of LGR7 in the fetal membranes is significantly greater in all clinical situations at preterm than term, suggesting an important role for relaxin in these tissues at that time. The roles of the relaxin system in three potential causes of preterm birth are discussed: in the growth and proliferation of the membranes important for fetal membrane accommodation to fetal and placental growth, in acute infection, and in the inflammatory response leading to the initiation of labor.

Regulation of the human relaxin genes H1 and H2 by steroid hormones.

Relaxin, a peptide hormone important to the outcome of human pregnancy is expressed in a tissue specific manner as two genes known as relaxins H1 and H2, in addition to a third human relaxin H3, expressed primarily in the brain. The H1 and H2 genes are highly homologous, differentially expressed in reproductive tissues and appear to activate the same receptor, but their regulation is poorly understood. Based upon the known physiology of these hormones and the response elements in their 5'- and 3'-flanking regions, the possibility that progesterone and/or the glucocorticoids might influence their differential expression was therefore investigated. The changes in the mRNA levels of the relaxin genes in response to either medroxyprogesterone acetate (MPA) or dexamethasone (Dex) were analyzed by RT-PCR using a choriocarcinoma cell line (JAR) as a model system, because the expression of these genes in any primary human cell type is too low for such a study. The addition of 0.5 microM MPA to JAR cells, significantly upregulated the mRNA of only the relaxin H2, while the addition of 0.5 microM Dex significantly upregulated the mRNAs for both the relaxins, after 6h of treatment. Promoter assays indicated an early activation of transcription (1 h), which by 6 h had decreased. Progesterone and/or glucocorticoids could exert their effects via the GRE motif found on the 5'-flanking region of the relaxin genes. The H1-GRE differs from the H2-GRE by a single nucleotide, which may affect H1-GRE binding to the progesterone receptor (PR) but not the glucocorticoid receptor (GR). The antiprogestin RU486 inhibited the binding of the GR to both H1-GRE and H2-GRE, while it enhanced the binding of the PR to these GREs. As determined by gel shift assays, this GRE motif could bind to both the PR and GR and was therefore considered to be functional. Thus, both progesterone and glucocorticoids are capable of differentially regulating the expression of the two human relaxin genes in a model system.

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.

Relaxin causes proliferation of human amniotic epithelium by stimulation of insulin-like growth factor-II.

OBJECTIVE: The study was conducted to determine whether relaxin has a proliferative effect on amniotic epithelial cells and to show that this effect is caused by its stimulation of the insulin-like growth factor-II (IGF-II) gene. STUDY DESIGN: Immunolocalization and Northern analysis were used to confirm the expression of IGF-II by the fetal cells in the membranes. Human amniotic epithelial (WISH) cells were treated with doses of IGF-II or human relaxin and their proliferative effects measured. The mechanism of the effect of relaxin on cellular proliferation was studied with the use of an IGF-II-blocking antibody and Northern analysis for IGF-II gene expression after treatment with relaxin. An in vivo correlate was sought by quantitation of relaxin gene expression in 10 fetal membranes from women with normally grown and large for gestational age infants. RESULTS: The amniotic epithelial and cytotrophoblast cells of the fetal membranes expressed IGF-II, as did the amniotic epithelial-like (WISH) cell line. Treatment of WISH cells with IGF-II or relaxin caused a significant (P <.03) and dose-related increase in WISH cell proliferation over 5 days. The concurrent treatment with a blocking antibody to IGF-II significantly decreased the proliferative response to IGF-II (P <.002) and relaxin (P <.002). Treatment with relaxin caused a significant increase (P <.003) in the transcription of IGF-II in 24 hours. In fetal membranes, the levels of relaxin gene expression correlated with fetal membrane surface area (r = 0.76) and was significantly greater (P <.008) in the membranes from macrosomic infants (4020-4729 g) compared with those normally grown (2855-3830 g). CONCLUSION: IGF-II and relaxin both caused the proliferation of WISH cells. Concurrent treatment with an IGF-II-blocking antibody abrogated the proliferative effects of both hormones. Relaxin increased the transcription of IGF-II, and its expression levels in the fetal membranes correlated with the membrane surface area as well as neonatal birth weight. These data suggest that relaxin is a growth factor for the fetal membranes.

Pre-B-cell colony-enhancing factor, a novel cytokine of human fetal membranes.

OBJECTIVE: Our purpose was to determine whether pre-B-cell colony-enhancing factor (PBEF) is expressed in the human fetal membranes during normal gestation and parturition in the absence of infection and to show its effects on the expression of interleukin (IL)-6 and IL-8. STUDY DESIGN: PBEF was immunolocalized in the fetal membranes from early pregnancy, at preterm, and at term. Its expression was quantitated by Northern analysis in separated uninfected amnion, chorion, decidua, and placenta of patients at term before labor and in full-thickness membranes before and after spontaneous labor at preterm and at term. Amnion-like epithelial (WISH) cells and fetal membrane explants were treated with recombinant PBEF (rhPBEF), and the expression of IL-6 and IL-8 was quantitated. RESULTS: PBEF was immunolocalized throughout gestation in the amniotic epithelium and mesenchymal cells as well as the chorionic cytotrophoblast and parietal decidua. Northern analysis showed significantly more (P <.01) PBEF expressed in the amnion than in either chorion or placenta. Its expression increased after labor at both preterm and term and correlated with that of IL-8 (r = 0.87). rhPBEF treatment of WISH cells significantly increased IL-6 (P <.05) and IL-8 (P <.01) gene expression after 4 hours and of IL-8 protein after 24 hours (P <.01); similar 4-hour treatment of fetal membrane explants significantly increased IL-6 (P <.01) and IL-8 (P <.05) gene expression. CONCLUSION: PBEF is a novel cytokine constitutively expressed by the fetal membranes during pregnancy. It increased the expression of IL-6 and IL-8 and may be important in both normal spontaneous labor and infection-induced preterm labor.

Decidual relaxins: gene and protein up-regulation in preterm premature rupture of the membranes by complementary DNA arrays and quantitative immunocytochemistry.

OBJECTIVE: This study was undertaken to show both decidual relaxin gene and protein up-regulation in preterm premature rupture of the fetal membranes. STUDY DESIGN: Membranes after preterm premature rupture (n = 4) have been matched in pairs with preterm intact membranes (n = 4). These tissues were from patients without infection, labor, preeclampsia or intrauterine growth restriction, and none of the patients had a latency period of more than 8 hours. The messenger RNAs from these tissues were used on complementary DNA expression arrays; 488 genes were analyzed. Relaxin gene expression was quantitated from the arrays and in additional tissues by Northern analysis. The two relaxin proteins, H1 and H2, in the decidual cells were immunolocalized and quantitated by microdensitometry with the use of specific antisera that were raised to decapeptides over the region of least homologic features. The expression of five other genes that were selected from the arrays were quantitated by Northern analysis. RESULTS: Relaxin gene expression was up-regulated 3.4-fold on the complementary DNA arrays but was not confirmed on Northern analysis. On the other hand, protein analysis for relaxin H1 and H2 in the decidual cells showed them to be significantly up-regulated (P <.0001, for both proteins) in patients with preterm premature rupture of the membranes compared with control subjects. The 20 most highly expressed genes at preterm in tissues without rupture were determined. In addition, analysis of the genes that were up-regulated with preterm rupture of the membranes showed 30 differentially expressed genes. CONCLUSION: Relaxin gene expression in the decidua is up-regulated, and its protein expression is significantly increased with preterm rupture of the fetal membranes.