Increased oxidative stress in human fetal membranes overlying the cervix from term non-labouring and post labour deliveries

Enzymatic breakdown of the collagen-rich extracellular matrix (ECM) that connects the amnion and chorion layers of the fetal membranes is one of the key events leading to rupture of membranes. Oxidant stress caused by increased formation of reactive oxygen species and/or reduced antioxidant capacity may predispose to membrane rupture, a major cause of preterm birth. The aim of this study was to determine the effect of human labour and supracervical (SC) apposition on antioxidant enzymes and 8-isoprostane (a marker of lipid peroxidation). To determine the effect of human labour on oxidative stress status, fetal membranes from the SC site (SCS) were collected from women at term Caesarean section (no labour), and from the site of membrane rupture (SOR) after spontaneous labour onset and delivery (post labour). To determine the effect of SC apposition on oxidative stress status, amnion was collected from the SCS and a distal site (DS) in women at term Caesarean section in the absence of labour. The release of 8-isoprostane was significantly higher in amnion from the SCS compared to DS, and in fetal membranes from the SOR compared to the SCS. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity were lower in amnion from the SC compared to DS. SOD gene expression and enzyme activity were lower in fetal membranes after labour. There was no difference in expression or activity in catalase, GPx and glutathione reductase (GSR) between no labour and post labour fetal membranes. In primary amnion cells, SOD supplementation significantly augmented IL-1β induced MMP-9 expression and activity. In summary, non-labouring SC fetal membranes are characterised by reduced antioxidant enzyme activity when compared to distal membranes, and, as such, may be more susceptible to oxidative damage and thus membrane rupture.     

Fine structure of human chorionic membrane. Ultrastructural and histochemical examinations

Normal human term chorionic membrane was examined by electron microscopy and histochemistry. In comparison with previous studies of the human chorion, our findings showed the following new morphological aspects: (1) The cellular junctions between adjacent trophoblastic cells were made up not only of desmosomes but also of gap junctions and occasional tight junctions. (2) The epithelial basement membrane was stratified and discontinuous. (3) Besides the fibroblasts and macrophages (Hofbauer cells), the connective tissue of the human chorion also contained myofibroblasts. Histochemically, the surface coat of the plasma membrane of the chorionic epithelial cells, the microfibrils, and the proteoglycans associated with the collagen fibrils showed a positive reaction to ruthenium red. This was not so for the basement membrane of the chorionic epithelium. Since the tight junctions are seldom present in the chorionic membrane, they are apparently not significant for the paracellular protein transfer. Similar to the amnion epithelium, it is assumed that the intercellular spaces and the discontinous basement membrane of the chorionic epithelium represent a paracellular route for non-placental protein transfer. As the myofibroblasts in human amnion, we suggest that the myofibroblasts in the chorionic membrane may contribute to the protection of the fetal membranes from overdistension.     

MAPK and AP-1 proteins are increased in term pre-labour fetal membranes overlying the cervix: regulation of enzymes involved in the degradation of fetal membranes

Fetal membranes overlying the cervix (i.e. supracervical site, SCS) are characterised by increased extracellular matrix (ECM) degradation. In non-gestational tissues, the mitogen activated protein kinase (MAPK) and activator protein (AP)-1 family are involved in the regulation of the ECM degrading enzyme metalloproteinase (MMP)-9. The aims of this study were (i) to compare the expression of AP-1 proteins in fetal membranes from the SCS and a distal site (DS), and (ii) determine if the MAPK/AP-1 pathway is involved in the regulation of MMP-9. Fetal membranes overlying the cervix were identified in situ in women undergoing term elective Caesarean section. Immunohistochemistry (n = 6) was used to localise the expression of the MAPK proteins ERK (total and phosphorylated), JNK (total and phosphorylated) and p38 MAPK (total and phosphorylated), and the AP-1 proteins JunB, cJun (total and phosphorylated), JunD, cFos and FosD. There was no difference in JNK, p38 MAPK, FosB, cJun and JunD protein expression between SC and distal fetal membranes. However, when compared to DS, the intensity and/or extent of staining of ERK, p-ERK, p-JNK, p-p38 MAPK, cFos, JunB and p-cJun were greater in amnion and chorion obtained from the SCS. In order to elucidate a role for these proteins in ECM degradation, pharmacological inhibitors of MAPK protein activation were utilised in primary amnion cells. The ERK inhibitor U0126, JNK inhibitor SP600125 and p38 MAPK inhibitor SB202190 all significantly decreased IL-β-induced MMP-9 gene expression and pro MMP-9 in human primary amnion cells. In summary, at term, non laboured SC fetal membranes are characterised by increased expression of MAPK and AP-1 proteins. MMP-9 expression and production was significantly suppressed by inhibitors of three key enzymes in the signalling cascades leading to AP-1 formation, ERK 1/2, JNK and p38 MAPK. Thus, the MAPK/AP-1 pathway may play a role in the degradation of the ECM at the SCS making it more susceptible to membrane rupture.     

15-Hydroxyprostaglandin dehydrogenase protein expression in human fetal membranes with and without subclinical inflammation

Prostaglandins play a central role in the stimulation and maintenance of both term and preterm labor. 15-Hydroxyprostaglandin dehydrogenase (PGDH), localized primarily to chorion trophoblasts, is the key enzyme responsible for the metabolism of prostaglandins. In preterm chorion, levels of PGDH protein and activity were lower when compared to term and were further reduced with the presence of infection, but effects of subclinical inflammation and membrane rupture on PGDH expression are not known. Our objectives were (1) to determine the relative expression of PGDH in amnion and chorion and (2) to determine the effect of preterm premature rupture of membranes (PPROM) and (3) subclinical inflammation on PGDH protein expression in preterm fetal membranes. Fetal membranes were collected from women with idiopathic preterm labor. Patients were divided into preterm birth (1) <32 weeks with PPROM (n = 6), (2) <32 weeks with intact membranes (n = 11), (3) >or=32 and <37 weeks with PPROM (n = 10), and (4) >or=32 and <37 weeks with intact membranes (n = 10). Different antibodies were used to detect protein expression and localization of PGDH in amnion and chorion from these patients using both Western blotting and immunohistochemistry. Antibody T (AbT) localized PGDH to chorion trophoblasts, whereas antibody C (AbC) detected immunoreactive (ir) PGDH predominantly in the amnion mesenchyme. By Western blot, AbT showed a stronger 29-kDa ir-PGDH band whereas with AbC, a stronger 55-kDa ir-PGDH signal was detected. 55-kDa ir-PGDH was significantly higher in PPROM amnion, specifically in the <32 weeks group (P < .05) and with PPROM >24 hours (P < .05). No change was detected in the 29-kDa ir-PGDH in either amnion or chorion with gestational age or the presence and absence of PPROM. In addition, neither form of ir-PGDH was altered significantly with or without subclinical inflammation. ir-PGDH is detectable in both chorion trophoblasts and amnion, especially in the mesenchyme; however, the predominant form of the enzyme differs in the 2 tissues. PPROM and subclinical inflammation do not appear to affect the levels of 29-kDa ir-PGDH protein in the fetal membranes. The differential expression of 55-kDa ir-PGDH in preterm amnion with and without PPROM supports the need for a better understanding of the different forms of PGDH.     

Increased apoptosis of human fetal membranes in rupture of the membranes and chorioamnionitis

Apoptosis is thought to participate pathophysiologically in the rupture of human fetal membranes (ROM). The aim of this study was to assess apoptosis of the amnion and the chorion in relation to ROM and chorioamnionitis (CAM). The amnion and chorion at the position of the cervical os and fundus of the uterus were obtained from 44 patients. Apoptotic DNA fragmentation was densitometrically determined, and the relative ratio was used for the quantitative evaluation. Among patients without CAM, the relative ratios of apoptosis in the amnion from patients with ROM were higher than those in patients without ROM (P< 0.05). Among patients without ROM, the apoptotic levels in the amnion from patients with CAM were higher than those in patients without CAM (P< 0.05). These were the cases with the amnion at the position of cervical os and fundus, but not with the chorion. The highest ratio of apoptosis was seen in the amnion from patients with CAM and ROM. Among patients with ROM and no CAM, the apoptotic levels at the cervical os in the amnion (P=0.059) and chorion (P< 0.05) was higher than those at the fundus. The increased apoptosis of human fetal membranes was related to ROM and CAM. Apoptosis plays a role in the pathophysiology of ROM.     

Expression, localisation and activity of ATP binding cassette (ABC) family of drug transporters in human amnion membranes

Placental ATP-binding cassette (ABC) transporters limit fetal exposure to xenobiotics by regulating transplacental passage into the fetal circulation; their expression and function in fetal membranes, however, has not been studied. In the present study the expression, localisation and function of ABC transporters in human amnion was examined to explore their potential role in modulating amniotic fluid drug disposition in pregnancy. Single-assay oligo-microarrays were used to profile amnion gene expression, and drug transporters expressed at significant levels were identified and selected for further studies. The expression of ABCG2/breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRP) 1 (ABCC1), 2 (ABCC2) and 5 (ABCC5) was detected on the arrays, and verified by RT-PCR and immunoblotting. On confocal microscopy of fetal membrane cryosections, MRP1 and MRP5 were immunolocalised to both apical and basolateral surfaces of the amniotic epithelium, while MRP2 was expressed at low levels only in the apical membrane. BCRP in contrast showed cytoplasmic staining throughout the amniotic epithelium. In addition to the amnion, MRP1 and BCRP immunostaining was observed in the chorion and the decidua. Cell accumulation studies using selective MRP and BCRP inhibitors showed the transporters to be functionally active in amnion epithelial monolayer cultures. In contrast, transwell transport studies using intact amnion membranes did not show significant vectorial transport. These findings identify the amnion as a novel site of ABC drug transporter expression. Functional studies indicate that they may act primarily to prevent cellular xenobiotic accumulation, rather than to confer fetal protection through reduced accumulation in amniotic fluid.     

Characteristic differences in immunohistochemical localization of new placental proteins (PP1, PP19, PP21) in the human placenta

The immunohistochemical localization in the human placenta of new placental proteins PP1, PP19, and PP21 was clarified using modified indirect enzyme-labeled antibody method and compared with that of pregnancy-specific beta 1-glycoprotein (SP1). The major results are as follows: positive staining for PP1 was seen at the nucleus and cytoplasm of villous cytotrophoblasts, the X cells at the basal plate, and of chorionic trophoblasts, while the decidua cells and amnion were not stained. PP19 was characteristically seen in the nucleus and cytoplasm of syncytiotrophoblasts. X cells in basal plate, chorionic trophoblasts, and maternal leukocytes. The villous cytotrophoblasts, decidua cells, and amnion were not stained. PP21 localization was found at the microvilli and basal membrane of syncytiotrophoblasts and at the cytotrophoblast plasma membrane of the chorionic villus in early gestation. In late gestation, increased staining was seen at the syncytiotrophoblast microvilli and the villous basement membrane, and moderate staining at plasma membrane of the amniotic epithelium and chorionic trophoblasts. SP1 was found only at the syncytiotrophoblast cytoplasm of chorionic villi. Studies using these four placental proteins simultaneously may therefore provide a new key learning about unknown metabolic functions of trophoblasts.     

Association of type II apoptosis and 92-kDa type IV collagenase expression in human amniochorion in prematurely ruptured membranes with tumor necrosis factor receptor-1 expression

OBJECTIVE: We assessed the presence of tumor necrosis factor receptor-1 (TNF-R1), apoptosis, and simultaneous expression of 92-kDa collagenase type IV (MMP-9) in samples of human chorioamnion from women with premature rupture of membranes (PROM). METHODS: Amniotic membranes from women who underwent normal labor, cesarean delivery, or had PROM at term were studied by immunohistochemistry for localization of TNF-R1 and R2. Transmission electron microscopy and DNA fragmentation analyses by agarose gel electrophoresis were performed to identify apoptosis characteristics. Zymography and in situ zymography were used to assess gelatinolytic activity. RESULTS: We found that TNF-R1 was abundant in membranes from subjects who had normal labor and very abundant in those who had PROM. By contrast TNF-R2 was abundant only in membranes from subjects who had cesarean delivery. Gelatinolytic activity was associated with extracellular matrix rather than cells and was higher in extracts from fetal membranes from PROM and normal labor than in extracts obtained from cesarean deliveries. Transmission electron microscopy of fetal membranes from PROM revealed ultrastructural characteristics in amnion epithelium consistent with type II apoptosis. DNA laddering in agarose gel electrophoresis corroborated results from DNA fragmentation. CONCLUSION: During PROM the fetal membranes undergo type II apoptosis and extracellular matrix degradation in association with TNF-R1 expression.     

Role of apoptosis,bcl-2 and bax protein expression in premature rupture of fetal membranes

OBJECTIVE: To examine the degree of apoptosis in human fetal membranes associated with premature rupture of membranes (PROM) as compared with normal pregnancies and to evaluate the expression of proapoptotic bax and antiapoptotic bcl-2 gene products. STUDY DESIGN: Fetal membranes from 50 pregnancies were included in the study. Thirty of 50 pregnancies had PROM. Twenty pregnancies with intact membranes served as controls. Chorioamniotic membrane biopsies were taken from the rupture site of the membrane and periphery of the rupture side. In the control group, membrane biopsies were taken from the artificial rupture site, cervical pole of the membranes and membranes close to the edge of the placenta. In recognizing apoptotic figures, routinely processed samples were stained with hematoxylin and eosin for light microscopic evaluation. Quantification of the apoptotic cells was performed with high-power fields and expressed as the number per 100 cells. The membranes of both groups were then stained with bcl-2 and bax antibodies by using the standard steptavidin-biotin-immunoperoxidase method. Staining with both antibodies were compared between two groups. RESULTS: Apoptotic cells were detected in the amniotic epithelium, in chorionic cells and fibroblastic layer of the fetal membranes. Apoptotic cells were found mostly in the chorionic cells. There was a statistically significant difference between the apoptotic index in PROM and the control group in both rupture and peripheral sites (P < .05), although within each group peripheral and rupture sites showed no difference in terms of apoptotic cell counts. Both bax and bcl-2 expression was observed in 40% of control cases and in 57% and 50% of cases with PROM, respectively, mostly in the chorionic trophoblastic cells. The PROM and control groups showed no statistically significant difference in terms of bcl-2 and bax protein expression. CONCLUSION: Apoptosis may play a role in the pathogenesis of PROM, but the changes in apoptosis do not seem to be mediated by bcl-2 and bax genes in the amniotic membrane. Other regulatory mechanisms must be investigated.     

Cellular localization of vascular endothelial growth factor in ovine placenta and fetal membranes

To further understand the role of vascular endothelial growth factor (VEGF) in mediating angiogenesis and vascular permeability during development in the sheep placenta and fetal membranes, we examined the localization of VEGF mRNA and protein in placental, chorionic and amniotic tissues by in situ hybridization and immunohistochemistry in ovine fetuses at 62, 102 and 141 days gestation (term=150 days). In the placenta, VEGF mRNA expression and VEGF protein immunostaining were strong in cytotrophoblasts surrounding the villi. In addition, VEGF protein was localized in smooth muscle cells around fetal and maternal blood vessels and in the maternal epithelium. There was no apparent difference in placental VEGF mRNA or protein levels associated with advancing gestation. In the fetal membranes, VEGF mRNA was detected in the amniotic epithelium and the chorionic cytotrophoblastic cell layer. The intensity of the hybridization signals in both amnion and chorion appeared low at 62 days, moderate at 102 days and high at 141 days gestation. VEGF protein was detected in amniotic epithelium and chorionic cytotrophoblasts at all gestational ages studied. The increase in VEGF gene expression in fetal membranes as term approaches suggests that during fetal development VEGF may promote the vascularity and permeability of the microvessels which perfuse the fetal membranes, as well as permeability of the amniotic membrane itself. Thus VEGF may participate in the regulation of amniotic fluid volume.     

Expression and cellular localisation of chloride intracellular channel 3 in human placenta and fetal membranes

Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular channel (CLIC) family. Evidence suggests that several CLICs are expressed in human placenta, although their roles in this tissue are not certain. Northern blot analysis has shown that CLIC3 is highly expressed in placenta relative to other human tissues; however, its cellular distribution is not known. This study used microarray expression profiling to clarify which CLICs are expressed in human placenta and RT-PCR, Western blot and immunohistochemistry to determine the expression pattern of CLIC3 in human placenta and fetal membranes. Placentas and fetal membranes were obtained from term pregnancies after delivery and placental tissue was obtained from first trimester following either chorionic villous sampling or elective pregnancy termination. Trophoblast cells were isolated from first trimester and term placentas and placental endothelial cells were isolated from term placentas. Microarray expression profiling identified high expression of mRNA for CLICs 1, 3 and 4 in the isolated first trimester and term trophoblast cells. High mRNA expression in the isolated endothelial cells was also found for CLICs 1 and 4, but not CLIC3. Low expression was found for CLIC5 in all three types of isolated cells. RT-PCR confirmed that CLIC3 mRNA was expressed in trophoblast cells at both gestational ages, but was not present in endothelial cells. CLIC3 mRNA was also identified in whole placental extracts at both gestational ages and in term amnion and choriodecidua. Immunohistochemistry using a chicken anti-human CLIC3 antibody localised strong CLIC3-specific staining to the syncytiotrophoblast and villous cytotrophoblast cells in both first trimester and term placentas, and weaker staining in extravillous trophoblast cells in first trimester. In fetal membranes at term strong CLIC3-specific staining was localised to chorionic trophoblast cells, with weaker staining in amniotic epithelial and decidual cells. It was previously shown that chloride uptake was increased into cells that had been transfected with CLIC3. CLIC3 may facilitate chloride ion movement and the regulation of cellular processes associated with the movement of chloride in the placental and fetal membrane cells in which it is expressed.     

Expression of Erythropoietin mRNA, Protein and Receptor in Ovine Fetal Membranes

Erythropoietin and its receptor have been identified in human, murine and ovine placentas. Based on the common embryonic origin of the placenta and fetal membranes, we postulated that erythropoietin is similarly expressed in the fetal membranes. Using in situ hybridization and immunohistochemistry, we tested the hypothesis that ovine fetal membranes are sites of erythropoietin production and action. At 86, 103 and 138 days gestation, erythropoietin mRNA and protein were present in the amnion localized to the cell layer consisting largely of amniotic epithelium and in the chorion localized to the chorionic columnar cells consisting of cytotrophoblasts. Binucleate cells, differentiated cytotrophoblasts known to produce hormones, were identified in the chorion in the region of erythropoietin expression but were not observed in amniotic tissue. The erythropoietin receptor protein was present in the amnion and chorion at 103 and 138 days gestation but was not observed in either tissue at 86 days. In summary, erythropoietin appears to be produced as well as utilized within the ovine amnion and chorion. Within the amnion, the amniotic epithelial cells express the erythropoietin gene whereas, within the chorion, either the cytotrophoblasts or the binuclear cells may be the source. Due to the presence of the receptor, we speculate that the erythropoietin produced in the membranes may mediate fetal membrane function and/or growth through an autocrine and/or paracrine mechanism. Further, the fetal membranes may be the source of erythropoietin in the amniotic fluid.     

Scavenger receptor for hemoglobin in preterm prelabor rupture of membranes pregnancies complicated by histological chorioamnionitis

Objective: We sought to evaluate the distribution of scavenger receptor for hemoglobin positive (CD163⁺) cells in the placenta and fetal membranes from pregnancies complicated by preterm prelabor rupture of membranes with respect to the presence and absence of histological chorioamnionitis. Methods: Sixty-two women with singleton pregnancies with a gestational age between 24+0 and 36+6 weeks were included in a prospective cohort study. CD163 was evaluated by immunohistochemistry in the placenta and fetal membranes. The number of CD163⁺ cells and neutrophils was counted in the following locations: fetal membranes’ amnion, chorion, and decidua, as well as the placenta’s amnion, chorionic plate, subchorionic fibrin, stem villi, terminal villi, and decidua. Results: CD163⁺ cells were found in all compartments of the placenta and the fetal membranes regardless of the inflammatory status. A positive correlation between the number of CD163⁺ cells and neutrophils in the subchorionic fibrin and the chorionic plate was found. The number of CD163⁺ cells was higher in the placental subchorionic fibrin and chorionic plate when histological chorioamnionitis was present. Conclusion: The presence of histological chorioamnionitis affected the number of CD163⁺ cells in the placental chorionic plate and in the subchorionic fibrin but not in the fetal membranes.     

The concentration of collagen and the collagenolytic activity in the amnion and the chorion

Fetal membranes obtained from second-trimester abortions, elective Caesarean sections and after normal deliveries were studied. The hydrolytic activity against a DNP-peptide expressing the collagen turn-over was found to be very high in the second trimester and relatively high at term of pregnancy. The activity in the chorion was twice that in the amnion. The concentration of another collagen-degrading enzyme, leukocyte elastase, which is present during inflammatory reactions was high at the site of rupture. The collagen concentration given as micrograms hydroxyproline per mg dry weight was unchanged throughout pregnancy and labour in both fetal membranes. The concentration in amnion was twice that in the chorion. After delivery, the collagen content given as microgram/cm2 in the chorion was decreased at the rupture line. This was due to decreased thickness of the membrane, whereas the hydroxyproline concentration (microgram/mg dry weight) was unchanged. The amnion was thinner than the chorion, but the decreasing collagen content (micrograms/cm2) at the rupture line was less pronounced. The thinning of especially the chorionic membrane might be secondary to the mechanical stress of pregnancy and labour and/or, as our results suggest, due to enhanced catabolism.     

The physiology of fetal membrane rupture: insight gained from the determination of physical properties

Premature rupture of the fetal membranes is a major cause of preterm birth and its associated infant morbidity and mortality. Recently, it has become clear that rupture of the fetal membranes, term or preterm, is not merely the result of the stretch and shear forces of uterine contractions, but is, in significant part, the consequence of a programmed weakening process. Work in the rat model has demonstrated that collagen remodeling, with activation of matrix metalloproteinases (MMPs), and apoptosis increase markedly in the amnion at end-gestation, suggesting that these processes are involved in fetal membrane weakening. We have developed fetal membrane strength testing equipment and a systematic tissue sampling methodology that has allowed us to demonstrate that term, non-labored, fetal membranes have a zone of weakness overlying the cervix, which contains biochemical markers of both collagen remodeling and apoptosis. These findings provide strong support for the concept of programmed fetal membrane weakening prior to labor. Our model has also been used to establish the physical properties of individual fetal membrane components (amnion, chorion), determine the sequence of events during the fetal membrane rupture process, and demonstrate that treatment of fetal membranes with TNF or IL-1beta, in vitro, induces weakness and the identical biochemical markers of collagen remodeling and apoptosis seen in the physiological weak zone. The ability to simultaneously correlate macroscopic physical properties with histological and biochemical fetal membrane characteristics, presents a unique perspective on the physiology of fetal membrane rupture.     

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.