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Histological Evidence of Oxidative Stress and Premature Senescence in Preterm Premature Rupture of the Human Fetal Membranes Recapitulated in Vitro

      Preterm prelabor rupture of the membranes (pPROM) may lead to preterm births (PTBs). We investigated premature senescence of fetal membranes in women with pPROM and spontaneous PTB with intact membranes (<34 weeks) and the inducibility fetal membrane senescence phenotype by oxidative stress in vitro. IHC was performed for p53, p21, and phospho (p)-p38 mitogen-activated protein kinase (MAPK) as markers of senescence phenotype in pPROM, PTBs, and term births. Term fetal membranes were exposed to cigarette smoke extract to induce oxidative stress. Western blots documented p-p53 and p-p38 MAPK. Transmission electron microscopy assessed cellular morphologic features in clinical and cigarette smoke extract–treated membranes. A total of 80% of pPROM cells and >60% of term cells were positive for all three senescence phenotype markers, and concentrations were higher than in PTBs (P < 0.05). p53 staining was comparable in membranes from PTB and term birth pregnancies, whereas only <30% and <45% of cells were positive for p21 and p38 MAPK, respectively. In vitro cigarette smoke extract exposure increased p-p38 MAPK without any detectable change in p-p53 MAPK. Enlargement of organelles consistent with senescence phenotype was evident in pPROM and term membranes in vivo and after cigarette smoke extract treatment in vitro but was less apparent in PTBs. Histologic and biochemical resemblance of pPROM and term membranes suggests premature senescence of the membranes is a mechanistic feature in pPROM, and this can be phenocopied in an in vitro model.
      Preterm (<37 weeks of gestation) prelabor rupture of the membranes (pPROM) occurs in approximately 4% of pregnancies and is a direct antecedent to 40% to 50% of all spontaneous preterm births (PTBs).
      • Parry S.
      • Strauss III, J.F.
      Premature rupture of the fetal membranes.
      • Gibbs R.S.
      • Blanco J.D.
      Premature rupture of the membranes.
      • Mercer B.M.
      • Crouse D.T.
      • Goldenberg R.L.
      • Miodovnik M.
      • Mapp D.C.
      • Meis P.J.
      • Dombrowski M.P.
      The antibiotic treatment of PPROM study: systemic maternal and fetal markers and perinatal outcomes.
      • Simhan H.N.
      • Canavan T.P.
      Preterm premature rupture of membranes: diagnosis, evaluation and management strategies.
      • Mercer B.M.
      • Lewis R.
      Preterm labor and preterm premature rupture of the membranes: diagnosis and management.
      Although the causes identifying PTB with intact membrane and pPROM have been assumed to be similar, the recognition of differential biomarkers suggest that these two phenotypes may arise from distinct pathophysiologic conditions.
      • Menon R.
      • Fortunato S.J.
      Distinct pathophysiologic pathways induced by in vitro infection and cigarette smoke in normal human fetal membranes.
      • Menon R.
      • Taylor R.N.
      • Fortunato S.J.
      Chorioamnionitis–a complex pathophysiologic syndrome.
      If we are to achieve effective strategies to mitigate the persistent prevalence of PTB in developed and developing nations, it is important to understand the precise pathways involved in pPROM and those responsible for PTB with intact membranes.
      • Menon R.
      • Fortunato S.J.
      Distinct pathophysiologic pathways induced by in vitro infection and cigarette smoke in normal human fetal membranes.
      • Menon R.
      Spontaneous preterm birth, a clinical dilemma: etiologic, pathophysiologic and genetic heterogeneities and racial disparity.
      • Ellestad S.C.
      • Swamy G.K.
      • Sinclair T.
      • James A.H.
      • Heine R.P.
      • Murtha A.P.
      Preterm premature rupture of membrane management–inpatient versus outpatient: a retrospective review.
      In this study, we propose to demonstrate that these clinical phenomena are two independent physiologic events.
      Infection and associated inflammation, particularly cytokine and chemokine activation, are common to both pPROM and PTB; however, a few key findings have led us to propose that different mechanisms are associated with these phenotypes. Using a sensitive and comprehensive gas chromatography/mass spectrometry analysis of amniotic fluid (AF) eicosanoids, we found that higher concentrations of the oxidative stress–induced lipid peroxidation product F2-isoprostane were present in term amniotic fluid samples compared with gestational age–matched PTB amniotic fluid,
      • Menon R.
      • Fortunato S.J.
      • Milne G.L.
      • Brou L.
      • Carnevale C.
      • Sanchez S.C.
      • Hubbard L.
      • Lappas M.
      • Drobek C.O.
      • Taylor R.N.
      Amniotic fluid eicosanoids in preterm and term births: effects of risk factors for spontaneous preterm labor.
      and our unpublished observations indicate that pPROM amniotic fluid samples have F2-isoprostane concentrations similar to those seen in term birth samples. The data suggest that increased oxidative stress is present more in pPROM than in PTB with intact membranes. Reduction of fetal leukocyte and fetal membrane telomere lengths, an indicator of oxidative stress and senescence, was noted in pPROM pregnancies compared with gestational age–matched PTBs.
      • Menon R.
      • Yu J.
      • Basanta-Henry P.
      • Brou L.
      • Berga S.L.
      • Fortunato S.J.
      • Taylor R.N.
      Short fetal leukocyte telomere length and preterm prelabor rupture of the membranes.
      Fetal leukocyte telomeres in pPROM cases at ≤34 weeks of gestation were as short as those of normal term births, supporting an accelerated aging process in pPROM. At term, oxidative stress and fetal membrane senescence are likely normal physiologic responses that lead to labor and delivery. Similar findings in pPROM suggest that exposures, such as infection, cigarette smoking, high body mass index, psychosocial stress, and antioxidant nutrient deficiencies, cause reactive oxygen species (ROS) to accumulate, activating premature fetal membrane senescence. One characteristic of the senescence phenotype (SP) involves the phenomenon of irreversible arrest of cell growth. Unlike apoptosis, these cells persist, alter their function, and change the tissue environment, inducing a unique signature of inflammatory markers similar to that seen in pPROM.
      • Coppe J.P.
      • Desprez P.Y.
      • Krtolica A.
      • Campisi J.
      The senescence-associated secretory phenotype: the dark side of tumor suppression.
      • Menon R.
      • Fortunato S.J.
      Infection and the role of inflammation in preterm premature rupture of the membranes.
      Three key mediators of cell senescence are p53, p21, and p38 mitogen-activated protein kinase (MAPK). The association between senescence and PTB in mice
      • Cha J.
      • Hirota Y.
      • Dey S.K.
      Sensing senescence in preterm birth.
      is suggested by the observation that a functional p53 mutation in decidua results in PTB.
      • Burnum K.E.
      • Hirota Y.
      • Baker E.S.
      • Yoshie M.
      • Ibrahim Y.M.
      • Monroe M.E.
      • Anderson G.A.
      • Smith R.D.
      • Daikoku T.
      • Dey S.K.
      Uterine deletion of Trp53 compromises antioxidant responses in the mouse decidua.
      • Hirota Y.
      • Daikoku T.
      • Tranguch S.
      • Xie H.
      • Bradshaw H.B.
      • Dey S.K.
      Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice.
      Further analysis of these decidual tissues documented a reduction in the antioxidant system that is hypothesized to be the mechanism that results in PTB.
      • Hirota Y.
      • Daikoku T.
      • Tranguch S.
      • Xie H.
      • Bradshaw H.B.
      • Dey S.K.
      Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice.
      • Hirota Y.
      • Cha J.
      • Yoshie M.
      • Daikoku T.
      • Dey S.K.
      Heightened uterine mammalian target of rapamycin complex 1 (mTORC1) signaling provokes preterm birth in mice.
      We selected p53 and its effector p21 for this study given their potent inhibition of cyclin-dependent kinase complexes and interference with cell cycle progression, resulting in senescence.
      • Hirota Y.
      • Cha J.
      • Yoshie M.
      • Daikoku T.
      • Dey S.K.
      Heightened uterine mammalian target of rapamycin complex 1 (mTORC1) signaling provokes preterm birth in mice.
      p38 MAPK
      • Scherle P.A.
      • Jones E.A.
      • Favata M.F.
      • Daulerio A.J.
      • Covington M.B.
      • Nurnberg S.A.
      • Magolda R.L.
      • Trzaskos J.M.
      Inhibition of MAP kinase kinase prevents cytokine and prostaglandin E2 production in lipopolysaccharide-stimulated monocytes.
      activation by phosphorylation of its catalytic site residues, threonine-180 and tyrosine-182, also promotes cell cycle arrest and cellular senescence by targeting the expression of proteins of SP.
      • Brancho D.
      • Tanaka N.
      • Jaeschke A.
      • Ventura J.J.
      • Kelkar N.
      • Tanaka Y.
      • Kyuuma M.
      • Takeshita T.
      • Flavell R.A.
      • Davis R.J.
      Mechanism of p38 MAP kinase activation in vivo.
      On the basis of our observation of telomere length reduction and accelerated senescence in pPROM,
      • Menon R.
      • Yu J.
      • Basanta-Henry P.
      • Brou L.
      • Berga S.L.
      • Fortunato S.J.
      • Taylor R.N.
      Short fetal leukocyte telomere length and preterm prelabor rupture of the membranes.
      the primary purpose of this study was to provide histologic evidence of markers for senescence in human fetal membranes from normal term births and in clinical settings of pPROM and PTB. We examined the levels of expression of p53, p21, and p38 MAPK by immunohistochemistry (IHC). In addition, morphologic changes of senescence were observed through transmission electron microscopy (TEM) in fetal membranes from early pPROM and PTB with intact membranes pregnancies (both <34 weeks) compared with membranes from normal term vaginal deliveries. We also examined the inducibility of ROS-associated senescence markers in normal term, not in labor, fetal membranes in vitro. Water soluble cigarette smoke extract (CSE) was used to induce ROS in fetal membranes. Activation of p-p53, p-p38 MAPK was documented by Western blot analysis, and morphologic changes were documented by TEM to confirm senescence.

      Materials and Methods

      This study was approved by the Western Institutional Review Board (Seattle, WA) and the institutional review board at the University of Texas Medical Branch (Galveston, TX; protocol 11-251 UTMB). Placental tissues from pPROM, PTBs, and normal-term births were obtained from individuals after obtaining written consent.

       Fetal Membrane Collection for in Situ Analysis

      Fetal membranes were collected from participants with normal birth after vaginal delivery at term after spontaneous onset of labor and artificial rupture of the membranes (between 39 and 41 weeks with no prior history of PTB or pPROM), PTB with intact membranes, or pPROM (both <34 weeks) (n = 8 in each group). Preterm labor was defined as the presence of ≥2 regular uterine contractions every 10 minutes accompanied by documented cervical change. pPROM was confirmed by pooling, funneling, or AmniSure test. Membranes were dissected from the placenta, washed three times in normal saline, and cleansed of decidua and blood clots using cotton gauze. Sections were cut from the midzone of the reflected membranes, avoiding the regions overlaying the cervix. Although this area may contribute to pPROM, our objective was to document evidence of senescence as a generalized phenomenon distributed throughout the membrane and not uniquely localized at the rupture site. Specimens were stored in 3.7% buffered formaldehyde.

       Tissue Processing for IHC

      Tissue sections were cut at 3 to 5μm, mounted on positively charged slides, and dried in a slide oven at 60°C for 30 minutes to ensure adherence to the slides. Sections were deparaffinized in four changes of xylene for 5 minutes each and then rehydrated through a series of graded alcohols with a final rinse in distilled water. Endogenous peroxides were quenched by soaking sections in two changes of 0.3% H2O2 in methanol.

       Antigen Retrieval and Immunostaining for p53, p21, and p38

      Before IHC, some sections were treated with antigen retrieval solution to facilitate antibody binding. Briefly, slides were incubated in a Black and Decker Vegetable Steamer for 20 minutes in Target Retrieval Solution (catalogue no. S1699; Dako Corporation, Carpinteria, CA) preheated to 99°C. They were removed from the steamer and placed on an open counter to cool for 20 minutes in the solution. The slides were then rinsed in three changes of distilled water and placed into a container of Tris-buffered saline with Tween 20 (catalogue no. 2380; Signet Pathology Systems, Inc, Dedham, MA) for 5 minutes to decrease the surface tension of the slides and facilitate coating of the IHC reagents as described below.

       Primary Antibody Reaction

      The following antibodies and dilutions were used for immunostaining: mouse monoclonal p53 antibody (1:200; catalog no. ab1101; Abcam, Cambridge, MA), which recognizes a 6-aminoacid 20-25 epitope at the N-terminus; rabbit polyclonal antibody to p21 (1:800; catalog no. ab18209; Abcam); and rabbit polyclonal antibody to p38 MAPK (1:200; catalog no. ab7952; Abcam), which recognizes both the native and phosphorylated p38α isoform. Treatments with primary antibodies were performed for 1 hour. Human colon tissue was used as a positive control and PBS as a negative control for IHC experiments.

       Streptavidin-Biotin–Based Detection

      Slides were loaded horizontally onto the Dako Autostainer, where each of the following steps was automated. All incubations occurred at room temperature, and sections were coated with 200 μL of each reagent unless otherwise specified. Tris-buffered saline was used to rinse the sections between each of the IHC steps. Both streptavidin and biotin (catalog no. SP2001; Avidin Biotin blocking kit; Vector Laboratories, Burlingame, CA) were diluted in antibody diluent (Dako) at a ratio of 1 mL of avidin and biotin to 5 mL of diluent. Diluted avidin was applied to sections and incubated for 7 minutes. The primary antibody was diluted to specified concentrations in the biotin solution and applied for 1 hour. Sections were then incubated in universal secondary antibody (catalog no. K0675; LSAB2 kit; Dako) for 15 minutes followed by label from the same kit for 15 minutes. Finally, the chromogen liquid diaminobenzidine (Dako, Carpinteria, CA) was applied for 5 minutes. The slides were manually rinsed in distilled water, counterstained with Harris hematoxylin (Fisher Scientific, Hampton, NH) for 1 minute, rinsed in distilled water followed by 0.25% ammonia water to blue, and then rinsed in distilled water. The slides were dehydrated through a graded series of alcohols, cleared in four changes of xylene, and coverslipped with synthetic glass and Permount mounting medium.

       Assessment of Immunostaining

      Eight sections each for p53, p21, and p38 MAPK were examined independently by two laboratory staff members, one of whom was blinded to the case/control status. For each antigen, staining was assessed by evaluating 10 randomly selected high-power fields (40× objective). The number of positively stained cells per field was determined, and the mean number of antigen positive cells per 100 cells was calculated.

       In Vitro Fetal Membrane Organ Culture and Stimulation with Water-Soluble CSE

      The in vitro culture system and stimulation of membranes with CSE have been described in our prior reports.
      • Fortunato S.J.
      • Menon R.
      • Swan K.F.
      • Lyden T.W.
      Organ culture of amniochorionic membrane in vitro.
      • Menon R.
      • Fortunato S.J.
      • Yu J.
      • Milne G.L.
      • Sanchez S.
      • Drobek C.O.
      • Lappas M.
      • Taylor R.N.
      Cigarette smoke induces oxidative stress and apoptosis in normal term fetal membranes.
      Briefly, we used normal-term placentas from women not in labor at term undergoing elective cesarean sections to obtain reflected fetal membranes that were cut into 6-mm disks (n = 5). The means ± SD gestational age of these women were 38.5 ± 1.2 weeks. Fetal membrane disks were placed in an organ explant system for 48 hours. CSE was prepared by bubbling smoke drawn from a single lit commercial cigarette (unfiltered Camel; R.J. Reynolds Tobacco Co., Winston Salem, NC) through 50 mL of tissue culture medium (Ham's F12/Dulbecco’s modified Eagle’s medium mixture with antimicrobial agents). The extract was filter sterilized through a 0.22-μm filter (Millipore, Bedford, MA) to remove contaminant microbes and insoluble particles. Fetal membranes were stimulated with CSE (1:10 dilution) for 24 hours, and media and tissue samples were frozen for analysis. In some experiments, membranes were co-stimulated with 10 μmol/L N-acetyl cysteine (an antioxidant) to determine the contribution of ROS-mediated activation.

       Western Blot Analysis

      Total p53, p-p53, total p38 MAPK, and p-p38 MAPK protein levels were assessed by Western blot analysis. Briefly, fetal membranes were homogenized in radioimmunoprecipitation assay buffer with protease inhibitors using the bullet blender (Next Advance, Averill Park, NY). Protein quantification was performed using the Pierce bicinchoninic acid kit (Thermo Scientific, Rockford, IL). Samples containing 45 μg of protein were separated using SDS-gel electrophoresis (Bio-Rad, Hercules, CA) according to the manufacturer’s suggestions, and proteins were transferred to polyvinylidene difluoride membranes using the iBlot dry blotting system (Life Technologies, Grand Island, NY). The membranes were blocked for 2 hours in 5% fat-free milk in Tris-buffered saline and Tween-20 and incubated with primary antibody to p38 (catalog no. 9212; Cell Signaling, Danvers, MA) or p-p38 (catalog no. 9211; Cell Signaling) overnight at dilutions of 1:400 or 1:1000, respectively. Blots were washed and incubated with secondary antibody for 1 hour. After the final washings, the blots were incubated with Pierce ECL2 chemiluminescence detection reagent (1:100 dilution; catalogue no. 8019; Thermo Scientific) followed by autoradiography. Detected bands were analyzed densitometrically using the ImageJ software version 1.46r (NIH, Bethesda, MD), and results were normalized to β-actin (A5441; Sigma, St. Louis, MO).

       3-NT Modified Protein Staining as a Marker of Oxidative Stress

      To document oxidative stress in clinical specimens and in tissues treated with CSE in vitro, we used IHC to localize 3-nitrotyrosine (3-NT) based on the principle that ROS produce highly reactive peroxynitrite radicals that react with exposed tyrosine residues on proteins, resulting in a stable polypeptide-bound 3-NT.
      • Webster R.P.
      • Brockman D.
      • Myatt L.
      Nitration of p38 MAPK in the placenta: association of nitration with reduced catalytic activity of p38 MAPK in pre-eclampsia.
      • Stanek J.
      • Eis A.L.
      • Myatt L.
      Nitrotyrosine immunostaining correlates with increased extracellular matrix: evidence of postplacental hypoxia.
      • Darwish R.S.
      • Amiridze N.
      • Aarabi B.
      Nitrotyrosine as an oxidative stress marker: evidence for involvement in neurologic outcome in human traumatic brain injury.
      • Tarpey M.M.
      • Wink D.A.
      • Grisham M.B.
      Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations.
      • Beal M.F.
      Oxidatively modified proteins in aging and disease.
      • Ischiropoulos H.
      Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species.
      IHC was performed using the anti–3-NT antibody (39B6; catalog no. ab61392; Abcam) at a dilution of 1:200. The color was developed as described above in the IHC section.

       Ultrastructural Morphology of Membranes from Clinical Conditions and after CSE Exposure

      Fetal membranes from term birth, PTB, and pPROM pregnancies (n = 3 in each group) and fetal membrane disks from normal-term pregnancies with or without CSE exposure (n = 3 each) were fixed, stained, and embedded in PolyBed 812. Initial fixation was for 24 hours at 4°C in a fixative with 2.5% paraformaldehyde, 0.2% glutaraldehyde, and 0.03% picric acid in 0.05 mol/L cacodylate buffer. After fixation, samples were rinsed three times with cacodylate buffer and postfixed with 1% osmium tetroxide in 0.1 mol/L cacodylate buffer. Osmicated tissue was rinsed twice with deionized water and stained en bloc with 2% aqueous uranyl acetate for 1 hour at 60°C. The samples were then dehydrated by a series of ethanol-water solutions (50%, 75%, 95%, and 100% ethanol for three exchanges). Dehydrated tissue was infiltrated with two exchanges of propylene oxide, then with propylene oxide–diluted PolyBed resin at 1:1 ratio and 1:2 ratio, and then twice with pure PolyBed 812. Finally, the samples were embedded in PolyBed 812 and cured overnight at 60°C. Because precise tissue orientation could not be maintained during curing of the resin, the first resin blocks were cut to give a wide flat face the desired sectioning plane, replaced into new embedding molds, and cured again. Samples were cut as 90-nm sections, placed on Formvar-coated slotted grids, and poststained for 3 minutes with a solution of Reynold's lead citrate. Images were taken with a JEM 1400 electron microscope (JEOL, Tokyo, Japan).

       Statistical Analysis

      Western blot experiments were replicated a minimum of 6 times. Densitometric units of p-p38 MAPK and total p38 MAPK were compared using the Kruskal-Wallis test followed by Dunn's multiple comparisons test. Data are expressed as arithmetic means ± SEM. For the quantitative analysis of IHC data, we used analysis of variance followed by Tukey's multiple comparisons test to correct for pairwise treatment effects or t-test, as appropriate. All data were analyzed using GraphPad Prism software version 6 (GraftPad Software, San Diego, CA). P < 0.05 was considered significant.

      Results

      Demographic details of the study participants are provided in Table 1. We examined 24 tissue samples from three different groups (term, PTB, and pPROM). Maternal age, marital status, ethnicity, prevalence of clinical and histologic chorioamnionitis, number of cigarettes smoked during pregnancy, and gestational age were similar between the pPROM and PTB with intact membranes groups. We excluded cigarette smokers from our term group to avoid any confounding effects.
      Table 1Demographic and Gestational Characteristics of Studied Patients
      CharacteristicTerm (n = 8)PTB (n = 7)pPROM (n = 8)P
      Maternal age (years)
      Analysis of variance (Tukey's multiple comparisons test).
      32 ± 629 ± 527 ± 80.35
      Marital status
      Fisher’ s exact test.
       Single3 (37.5)03 (50.0)0.14
       Married5 (62.5)7 (100.0)5 (62.5)
      Ethnicity
       White5 (62.5)5 (83.3)5 (62.5)0.53
       Black3 (37.5)1 (16.7)3 (37.5)
      Smoked during pregnancy01 (14.3)00.30
      Gestational age at birth (weeks/days)39 ± 133 ± 332 ± 4<0.0001
      Chorioamnionitis002 (25.0)0.12
      Gravidity
       Primiparous1330.38
       Multiparous745
      Data are presented as means ± SD or number (percentage).
      Analysis of variance (Tukey's multiple comparisons test).
      Fisher’ s exact test.
      Amnion, chorion, and scattered fibroblast cells embedded in the extracellular matrix of the fetal membranes had positive staining for p53, p21, and p38 MAPK (Figure 1, A–F). p53 staining was seen in 72% and 80% of amnion and chorion cells at term and 58% and 65% in PTB, respectively (Figure 1, A and D). The cells in amnion (79%) and cells in chorion (89%) were positive for p53 in pPROM samples (Figure 1A). The percentage of p53-positive cells was significantly higher in term and pPROM compared with PTB (both P < 0.05) in amnion cells, but no significant differences were seen between term and pPROM (P = 0.15) (Figure 1D). In chorion, pPROM membranes had significantly more p53 staining cells than either PTB or term (P < 0.05). For p21, the fractions of positively staining cells in amnion and chorion, respectively, were as follows: term birth, 58% and 62%; PTB, 15% and 24%; and pPROM, 69% and 75% (Figure 1, B and E). Staining was significantly more widespread in term (Figure 1B) and pPROM (Figure 1B) groups compared with the PTB group (Figure 1, B and E) (P < 0.05); however, no difference was seen between the term and pPROM groups (Figure 1E). For p38 MAPK, the fractions of cells (amnion and chorion, respectively) that had positive staining were as follows: term birth, 59% and 72%; PTB, 24% and 48%; and pPROM, 80% and 73% (Figure 1, C and F). Both amnion and chorion layers in pPROM membranes had a significantly higher number of p38 MAPK staining cells than term or PTB (both P < 0.05), whereas no difference was seen between term and pPROM. In summary, fetal membranes from pPROM pregnancies revealed significantly more numbers of immunopositive cells compared with either term birth or PTB, with PTB cells scoring the lowest for all three SP markers. Chorion cells had the highest staining among all three groups for all markers except p38 MAPK (where amnion cells in pPROM had the strongest reactivity). Two pPROM cases had histologic chorioamnionitis, and three each of our cases in the pPROM and PTB groups had microbial invasion of the amniotic cavity documented by microbial culture. We did not observe any specific differences in immunostaining patterns when data were stratified for infection, although sample size was not sufficient to draw any major conclusion. Analysis of markers based on race was also not significant, but a larger sample size is needed to address this question.
      Figure thumbnail gr1
      Figure 1Amniochorion membranes from normal-term vaginal delivery in the term, PTB, and pPROM conditions. For each of the three conditions, amnion and chorion layers are shown separately for three senescence/cell cycle proteins: p53 (A), p21 (B), and p38 MAPK (C). D: Quantitative assessment of p53 staining in amnion and chorion from term (white bars), PTB (gray bars), and pPROM (black bars). E: Quantitative assessment of p21 staining in amnion and chorion from amnion and chorion from term, PTB, and pPROM. F: Quantitative assessment of p38 staining in amnion and chorion from amnion and chorion from term, PTB, and pPROM. P < 0.05. Original magnification: ×20 (A–C).
      We examined total and p-p53 in membranes treated with CSE; total p53 was higher after CSE treatment compared with controls (P < 0.05) and was decreased after N-acetyl cysteine (NAC) treatment compared with controls (Figure 2A). We did not see p-p53 in any of our membrane preparations, suggesting that CSE does not activate p53 (data not shown). However, in vitro CSE stimulated p-p38 MAPK in normal-term membranes compared with controls. The mean gestational age of the placentas used for this study was 38 weeks. Total p38 produced no difference during a 24-hour period (Figure 2B); however, p-p38 MAPK was increased in CSE-treated fetal membranes after 24-hour stimulation (P < 0.05) (Figure 2C). The effect appears to be mediated by ROS because CSE-induced p-p38 MAPK expression was abrogated by NAC (P = 0.037) (Figure 2C). In a related study, we examined the kinetics of ROS induction in fetal amnion cells from term pregnancy in culture. ROS production kinetics were documented by the release of 2′,7′-dichlorodihydro-fluorescein diacetate, when amnion cells were exposed to CSE extract. ROS generation was seen as early as 2 minutes after exposure to CSE and was significantly higher at all time points tested compared with unstimulated controls. This effect was also inhibited by NAC treatment (data not shown).
      Figure thumbnail gr2
      Figure 2Western blot analysis of total p53 (A), total p38 MAPK (B), and p-p38 MAPK (C). Bar graphs show densitometric quantitation of Western blot analysis normalized to β-actin expression. Total p53 expression was higher in CSE-treated fetal membranes than unstimulated controls. Co-stimulation of CSE with NAC significantly reduced CSE-induced total p53. Total p38 MAPK expression was not different in fetal membranes exposed to CSE compared with untreated controls. Co-stimulation with NAC also had no effect on total p38 MAPK. C: Densitometric quantitation of p-p38 MAPK reveals an increase in CSE-treated membranes that are significantly higher than untreated membranes. Simultaneous treatment of NAC significantly reduced p-p38 (both P < 0.05). P < 0.05.
      Oxidative stress in fetal membranes from clinical specimens and after in vitro exposure to CSE was confirmed by qualitative IHC for 3-NT (Figure 3, A–G). As shown in Figure 3A, membranes from term and pPROM pregnancies (Figure 3E) had intense 3-NT staining compared with PTB (Figure 3C), confirming that oxidative stress is more dominant in those conditions. Similarly, and as expected, CSE-treated fetal membranes (Figure 3, D and F) had strong 3-NT staining compared with controls, particularly those involving the chorion cells (Figure 3, B and F).
      Figure thumbnail gr3
      Figure 3A, C, and E: 3-NT staining of fetal membranes from term (A), PTB (C), and pPROM pregnancies (E). The intensity of staining is similar in membranes from pPROM and term but higher than that seen in PTB membranes. Term and pPROM membranes have a similar number of cells with oxidative stress, and PTB membranes has slightly less intense staining. B and D: Oxidative stress in CSE-treated membranes in vitro. Compared with untreated controls in culture (B), CSE-treated fetal membranes (D) have strong staining of 3-NT, confirming ROS in these tissues. F: Representation of staining in controls and CSE-treated fetal membranes. G: Amnion, chorion, and cells in the extracellular matrix region reveal 3-NT-positive cells that produce oxidative stress quantitation of 3-NT staining in clinical specimens. Original magnification: ×20.

       Ultrastructure of Amnion and Chorion in Placental Membranes from Clinical Specimens

      To correlate ultrastructural morphologic findings with IHC findings, we performed TEM on three specimens per group from the term birth, PTB, and pPROM groups. A detailed description of morphologic findings, with emphasis on mitochondria and endoplasmic reticulum, which are strong responders of oxidative stress, is provided below, summarized in Table 2, and shown in Figure 4, A–D.
      Table 2Summary of TEM Characteristics of Organelles in Term, PTB, and pPROM Pregnancies
      CharacteristicTermPTBpPROM
      AmnionChorionAmnionChorionAmnionChorion
      MicrovilliIrregular and branchedShort projected into gaps and attached to desmosomesIrregular and branchedShort microvilliIrregular and branchedShort and some branched projected into gaps and attached to desmosomes
      NucleusOccasional irregular contoursPredominantly round and some contoured nucleiContoured nucleusContoured nucleusOccasional Irregular contoursMildly irregular and some contoured nuclei
      Endoplasmic reticulumEnlargedDilated cisternaeCompact cisternae with very little internal spaceCompact cisternae with very little internal spaceEnlargedEnlarged and dilated cisternae
      MitochondriaEnlarged with pale matricesVery large, rounded with pale matricesSmall, round with pale matricesSmall, round and pale matricesEnlarged with pale matricesVery large, rounded with pale matrices
      Figure thumbnail gr4
      Figure 4TEM documentation of morphologic changes associated with senescence in vivo at term, PTB, and pPROM (A–D) and in vitro after CSE treatment of fetal membranes from normal term pregnant women not in labor (E and F). Amnion and chorion cells from term, PTB, and pPROM pregnancies. A: TEM view of amnion cells. The surfaces of microvilli were branched and irregular in shape regardless of the condition. An overall loss of microvilli was seen, which was more prominent at term and in pPROM. Microvilli are shown insets. B: TEM view of chorion nucleus from term, PTB, and pPROM. In both term and pPROM, the chorionic cell's nuclei are predominantly ovoid. PTB reveals consistent irregularity of the nuclear contour. Chromatin are condensed toward the membrane in all three conditions. C: TEM view of chorion endoplasmic reticulum from term, PTB, and pPROM. Endoplasmic reticulum (ER) reveals maximum swelling or dilation at term and in pPROM, whereas PTB membranes have more normal-appearing ER (arrow). Oxidative stress–associated increased responsiveness of the ER is evident in membranes from term birth and pPROM. D: TEM view of chorion mitochondria from term, PTB, and pPROM. Enlarged mitochondria (arrow) are seen in all three conditions, but term births and pPROM births have more swollen mitochondria compared with PTBs, which had a mixture of both. CSE-exposed fetal membranes (E) and control (F, untreated) fetal membranes. CSE-exposed amnion cells have smaller deflated-looking cells (thin arrow) with elongated and branched microvilli. Untreated amnion cells are fairly uniform with short and regular microvilli. Chorion cells exposed to CSE have focal projections and dense condensation of chromatin (arrow), whereas normal chorion cell (unstimulated) have well-defined round nucleolus and minimal chromatin condensation. In CSE-treated membranes, rough ER in chorion cells are separated completely and are swollen and enlarged (arrow). In control chorion, ER is well ordered. Mitochondria are enlarged in CSE-exposed chorion cells, whereas they are smaller and elongated in control chorion cells. Original magnification: ×600 (A, term; and C, term) and ×400 (A, pPROM; B; C, pPROM and PTB; and D–F).

       Amnion Epithelium

       Normal-Term Birth

      The specimens of membranes from placentas delivered at term had the least alteration overall compared with the other two groups (Figure 4A). The amniotic epithelial cells had short microvilli that often appeared branched. The mitochondria appeared small and rounded with pale matrices. Endoplasmic reticulum was dilated, and cisternae of rough endoplasmic reticulum appeared open with lucent contents. The nuclei generally appeared lucent and distinct single nucleoli were present.

       PTB

      The amniotic epithelium from the PTB with intact membranes had ultrastructural features similar to the term group overall (Figure 4A). Endoplasmic reticulum and mitochondrial profiles were small, round, and appeared pale. The nuclei had mild irregularity of the nuclear contour with more condensation of nuclear chromatin at the inner nuclear envelope than in the term birth group.

       pPROM

      Amniotic epithelial cells appeared finely vacuolated at low magnification, with pale irregular vacuoles standing out in contrast to a moderately electron dense cytoplasm (Figure 4A). This was unique to the pPROM group compared with the term and PTB groups. Unlike PTB, in some cells the nuclear envelope was dilated. Nuclei consistently had irregular nuclear contours with frequent infolding of the envelope into the nucleus, and the chromatin had moderately dense condensation at the inner surface of the nuclear envelope. Similar to that seen in the term birth group, dilated segments of rough endoplasmic reticulum were found in the cytoplasm. The mitochondria appeared enlarged and had pale matrices.

       Chorion

       Normal-Term Birth

      The nuclei of the cells of the chorion were generally ovoid with slight irregularity and contained a light gray meshwork of chromatin (Figure 3B) with a simple cytoplasm. Cisternae of rough endoplasmic reticulum were numerous and appeared open, with pale contents, and in some cells the rough endoplasmic reticulum was clearly dilated (Figure 3C). Mitochondria were round or swollen with pale matrices (Figure 3D). Both endoplasmic reticulum and mitochondria at term resembled those seen in pPROM membranes (Figure 3, C and D).

       PTB with Intact Membranes

      Chorionic cells included a mixture of cells that appeared contracted or shrunken (Figure 3, B–D). In the contracted cells, the cytoplasm appeared moderately electron dense, and long, thin, undulating electron dense mitochondria were seen throughout the cytoplasm. The nuclei had irregular nuclear contours with frequent infolding of the nuclear envelope (Figure 3B). Cisternae of the endoplasmic reticulum appeared compact (Figure 3C), with little internal space, and were markedly different from those in the term birth group. Mitochondria had an electron-dense matrix (Figure 3D).

       pPROM

      The chorion cells in the pPROM group were generally swollen and flattened, with only rare condensed cells (Figure 3D). The nuclei had mildly irregular shapes and a degree of nuclear condensation intermediate between that of the term and PTB groups. Cisternae of the rough endoplasmic reticulum generally appeared open or dilated (Figure 3C). Mitochondrial profiles were moderately large, round, and pale and resembled those of term membranes (Figure 3D).
      In summary, ultrastructural morphologic features of both amnion and chorion cells from term and pPROM had signs of senescence characterized by overall swelling of cells and endoplasmic reticulum and mitochondria. PTB membranes had more normal-appearing organelles compared with the other two groups; however, irregular nuclear contours were more dominant in both amnion and chorion cells in this group than in the pPROM and term groups.

       Changes in Amnion Exposed to Cigarette Smoke

      In the CSE amniotic epithelial cells (Figure 3E), the cytosol generally appeared more electron dense than in untreated controls (Figure 3F). The microvilli were elongated, branched, and flattened and had an overall height less than that of controls. Nuclear chromatin appeared more densely granular (Figure 3E).

       Amnion in Untreated Controls

      The amniotic epithelial cells from untreated membranes were cuboidal with rounded apices and had short, irregular microvilli (Figure 3F). Nuclei were rounded and often contained moderate-sized nucleoli. The cells were joined laterally by multiple desmosomes. Lateral intercellular spaces extend irregularly into adjacent cells.

       Chorion Exposed to Cigarette Smoke

      In most cells, the nuclear envelop appeared dilated, the cytoplasm appeared pale, and the rough endoplasmic reticulum and nuclear envelope appeared dilated (Figure 3E). Mitochondria appeared swollen and had pale matrices (Figure 3E). Rarely, the mitochondrial matrix contained amorphous dense bodies. In other cells, the mitochondrial matrix appeared more electron dense than normal, and the intermembranous space was enlarged, with a condensed mitochondrial configuration.

       Chorion in Untreated Controls

      Electron micrographs revealed a loose collagenous extracellular matrix, and the nuclei of the chorionic epithelial cells were oval and contained small nucleoli (Figure 3F). The cytoplasm included rough endoplasmic reticulum, ellipsoid mitochondria, and normal-appearing Golgi apparatus and sometimes contained homogeneous, moderately electron dense material similar to material in the extracellular space (Figure 3F). The structure of organelles appeared normal in untreated controls.
      In summary, CSE-treated membranes demonstrated similarities between pPROM and term birth clinical samples, indicating morphologic evidence of senescence.

      Discussion

      In this study, we compared histologic evidence of cellular senescence and related biochemical markers in fetal membranes from term, PTB, and pPROM pregnancies. These data confirm our prior findings of high oxidative stress and accelerated senescence in pPROM and support our postulation that pPROM (especially early pPROM <34 weeks) is a syndrome of premature aging of the membranes. ROS-inducing risk factors can trigger senescence-associated pathways in normal fetal membranes, likely causing inflammation, proteolysis,
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      A comparison between the morphologic features of membranes from women not in labor used for in vitro studies and membranes from women in labor at term used for clinical data comparisons revealed distinct morphologic features under TEM. Although 3-NT staining was not distinguishable between the two, senescence features are markedly present in membranes from women in labor at term. We predict that the development of SP and the subsequent generation of the senescence-associated secretory phenotype (SASP) are major physiologic changes associated with the initiation of labor besides the disruption of the endocrine clock. In addition, the change from quiescence to active labor is likely to include the development of senescence of fetal membranes and placenta acting as termination signals from the fetus.
      We further verified that SP could be induced in normal fetal membranes at term, before the onset of labor, using CSE. We report activation of p38 MAPK by CSE in normal fetal membranes. This finding provides further evidence of the physiologic significance of the activation of the SP to adverse challenges. Activation of p-38 MAPK to p-p38 MAPK by CSE could be prevented by antioxidant NAC, confirming the influence of oxidative stress. We have previously reported that the exposure of fetal membrane explants to CSE increases total p53 and apoptotic cell death of other markers.
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      One of the limitations of this study is the use of crude CSE for stimulation in in vitro cultures. This is not calibrated to the number and duration of cigarettes smoked by a individual. This extract included water soluble toxicants from cigarette smoke, whereas the fetal membranes are likely exposed to metabolites of these toxicants. Another limitation is the use of membranes from women not in labor with full-term pregnancies as controls that are naturally and physiologically expected to be aged. Although natural senescence is expected at term, the effect of CSE was significant in our experiment as indicated by morphologic and biochemical changes justifying the use of these tissues for such studies.
      In summary, we found that human fetal membranes from pPROM pregnancies reflect SP in situ and report the inducibility of such a phenotype in normal-term membranes in response to ROS. Senescence-associated markers suggest a mechanism for ROS-associated damage to the membranes that may result in premature aging and SASP generation, which is depicted by inflammatory markers normally seen in pPROM. Further characterization of SP and SASP in pPROM is necessary to understand the biomarkers associated with senescence development during adverse pregnancy conditions.

      Acknowledgments

      We acknowledge the contributions of The Perinatal Research Laboratory staff, Esther Tamayo for laboratory management, Rheanna Urrabaz-Garza for histostaining quantitation, Talar Kechichian for Western blots, Michael Sherman for electron microscopy, Kenneth Escobar, and Kerry Graves for histopathology and IHC analysis.

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