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Discovery and Characterization of Human Amniochorionic Membrane Microfractures

2017; Elsevier BV; Volume: 187; Issue: 12 Linguagem: Inglês

10.1016/j.ajpath.2017.08.019

1525-2191

Lauren Richardson, Gracie Vargas, Tyra Brown, Lorenzo Ochoa, Samantha Sheller‐Miller, George R. Saade, Robert N. Taylor, Ramkumar Menon,

Pregnancy and preeclampsia studies

This study obtained visual evidence of novel cellular and extracellular matrix–level structural alterations in term and preterm human fetal amniochorionic membranes. Amniochorions were collected from term cesarean (not in labor) or vaginal (labor) deliveries, preterm premature rupture of membranes, and spontaneous preterm birth. To determine the effect of oxidative stress on membranes at term or preterm labor, term not in labor samples in an organ explant culture in vitro were exposed to cigarette smoke extract. Tissues were imaged using multiphoton autofluorescence and second harmonic generation microscopy. Images were analyzed using ImageJ and IMARIS software. Three-dimensional microscopic analysis of membranes revealed microfractures that were characterized by amnion cell puckering, basement membrane degradation, and tunnels that extended into the collagen matrix with migrating cells. Numbers of microfractures were similar at term regardless of labor status; however, morphometric measures (width and depth) were higher in term labor membranes. Oxidative stress induced higher numbers of microfractures in term not in labor membranes, with morphometry resembling that seen in term labor membranes. Preterm premature rupture of the membranes had the highest number of microfractures compared to membranes from term and other preterm births. Microfractures are structural alterations indicative of areas of tissue remodeling during gestation. Their increase at preterm and in response to oxidative stress may indicate failure to reseal, predisposing membranes to rupture. This study obtained visual evidence of novel cellular and extracellular matrix–level structural alterations in term and preterm human fetal amniochorionic membranes. Amniochorions were collected from term cesarean (not in labor) or vaginal (labor) deliveries, preterm premature rupture of membranes, and spontaneous preterm birth. To determine the effect of oxidative stress on membranes at term or preterm labor, term not in labor samples in an organ explant culture in vitro were exposed to cigarette smoke extract. Tissues were imaged using multiphoton autofluorescence and second harmonic generation microscopy. Images were analyzed using ImageJ and IMARIS software. Three-dimensional microscopic analysis of membranes revealed microfractures that were characterized by amnion cell puckering, basement membrane degradation, and tunnels that extended into the collagen matrix with migrating cells. Numbers of microfractures were similar at term regardless of labor status; however, morphometric measures (width and depth) were higher in term labor membranes. Oxidative stress induced higher numbers of microfractures in term not in labor membranes, with morphometry resembling that seen in term labor membranes. Preterm premature rupture of the membranes had the highest number of microfractures compared to membranes from term and other preterm births. Microfractures are structural alterations indicative of areas of tissue remodeling during gestation. Their increase at preterm and in response to oxidative stress may indicate failure to reseal, predisposing membranes to rupture. Fetal membranes (amniochorionic or placental membranes) comprise a vital intrauterine compartment, where they perform mechanical, immune, and endocrine functions to promote growth of the fetus and protection from environmental adversity.1Menon R. Human fetal membranes at term: dead tissue or signalers of parturition?.Placenta. 2016; 44: 1-5Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Membrane integrity is normally maintained throughout gestation, mediated by a well-balanced homeostasis involving matrix-degrading collagenolytic enzymes and their inhibitors.2Vadillo-Ortega F. Hernandez A. Gonzalez-Avila G. Bermejo L. Iwata K. Strauss III, J.F. Increased matrix metalloproteinase activity and reduced tissue inhibitor of metalloproteinases-1 levels in amniotic fluids from pregnancies complicated by premature rupture of membranes.Am J Obstet Gynecol. 1996; 174: 1371-1376Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 3Fortunato S.J. Menon R. Lombardi S.J. MMP/TIMP imbalance in amniotic fluid during PROM: an indirect support for endogenous pathway to membrane rupture.J Perinat Med. 1999; 27: 362-368Crossref PubMed Scopus (108) Google Scholar, 4Athayde N. Romero R. Gomez R. Maymon E. Pacora P. Mazor M. Yoon B.H. Fortunato S. Menon R. Ghezzi F. Edwin S.S. Matrix metalloproteinases-9 in preterm and term human parturition.J Matern Fetal Med. 1999; 8: 213-219Crossref PubMed Scopus (0) Google Scholar Recently, we reported that amniochorionic membrane senescence occurs as a concomitant of fetal growth and tissue aging.5Menon R. Behnia F. Polettini J. Saade G.R. Campisi J. Velarde M. Placental membrane aging and HMGB1 signaling associated with human parturition.Aging (Albany NY). 2016; 8: 216-230Crossref PubMed Scopus (106) Google Scholar Tissue damage arising from amniochorion senescence produces a sterile inflammatory response associated with parturition.6Menon R. Bonney E.A. Condon J. Mesiano S. Taylor R.N. Novel concepts on pregnancy clocks and alarms: redundancy and synergy in human parturition.Hum Reprod Update. 2016; 22: 535-560Crossref PubMed Scopus (153) Google Scholar Thus, amniochorionic membranes play major roles during gestation and parturition. Amniochorionic membranes anatomically consist of a single layer of cuboidal amnion epithelial cells, chorionic trophoblasts, and scattered fibroblasts connected by a layer of type IV collagen-rich extracellular matrix.7Bryant-Greenwood G.D. The extracellular matrix of the human fetal membranes: structure and function.Placenta. 1998; 19: 1-11Abstract Full Text PDF PubMed Scopus (187) Google Scholar Basement membrane degradation by specific matrix degrading enzymes, as well as generalized proteolysis, is a factor predisposing to mechanical rupture of membranes before parturition at term or preterm.7Bryant-Greenwood G.D. The extracellular matrix of the human fetal membranes: structure and function.Placenta. 1998; 19: 1-11Abstract Full Text PDF PubMed Scopus (187) Google Scholar A reported feature of amniochorionic membrane rupture that provides evidence of these structural changes is the so-called zone of extreme altered morphology that overlies the site of rupture.8Malak T.M. Bell S.C. Structural characteristics of term human fetal membranes: a novel zone of extreme morphological alteration within the rupture site.Br J Obstet Gynaecol. 1994; 101: 375-386Crossref PubMed Scopus (172) Google Scholar Morphometric characteristics of the zone of extreme altered morphology is also suggestive of membrane aging because cells and organelles are often enlarged and express sterile inflammatory markers such as matrix metalloproteinase 9. Although these changes may be expected in membranes at term, similar features were seen in cases with early ( 390/7 weeks) with no pregnancy-related complications. Subjects with multiple gestations, placenta previa, fetal anomalies, and/or medical treatment or surgeries (interventions for clinical conditions that are not linked to pregnancy) during pregnancy were excluded. Severe cases of preeclampsia (persistent symptoms (headache, vision changes, right upper quadrant pain), abnormal laboratory findings (thrombocytopenia, repeated abnormal liver function tests, creatinine doubling or >1.2, or HELLP syndrome), or clinical findings (pulmonary edema or eclampsia) were excluded. Subjects who had any surgical procedures during pregnancy or who were treated for hypertension, preterm labor, or for suspected intra-amniotic infection (eg, reports of foul-smelling vaginal discharge, high levels of c-reactive protein, fetal tachycardia) and delivered at term were excluded from the control groups. The in vitro organ explant culture system for human amniochorionic membranes and stimulation of membranes with cigarette smoke extract were as previously reported.9Menon R. Boldogh I. Hawkins H.K. Woodson M. Polettini J. Syed T.A. Fortunato S.J. Saade G.R. Papaconstantinou J. Taylor R.N. Histological evidence of oxidative stress and premature senescence in preterm premature rupture of the human fetal membranes recapitulated in vitro.Am J Pathol. 2014; 184: 1740-1751Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar In this study, cigarette smoke extract was used to mimic the oxidative stress experienced by amniochorionic membranes at term before labor that transition the membrane into a labor phenotype.5Menon R. Behnia F. Polettini J. Saade G.R. Campisi J. Velarde M. Placental membrane aging and HMGB1 signaling associated with human parturition.Aging (Albany NY). 2016; 8: 216-230Crossref PubMed Scopus (106) Google Scholar In short, 6-mm biopsies of amniochorionic membranes were collected from term not in labor cesarean deliveries and placed in an organ explant system for 24 hours. Cigarette smoke extract 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), which was then filter sterilized through a 0.22-mm filter (Millipore, Bedford, MA) to remove contaminant microbes and insoluble particles. Amniochorionic membranes were then stimulated with cigarette smoke extract (1:25 dilution) for 48 hours (n = 5), whereas the term not in labor control medium was replaced with tissue culture medium (n = 5). After a 48-hour treatment, the explants were removed and placed into 500 μL of 10% formalin in 1.5-mL Eppendorf tubes for fixation before imaging. Optical clearing of whole tissue explants to render the amniochorionic membranes optically transparent was performed by incubation of fixed amniochorionic membrane explants in 2,2′-thiodiethanol (refractive index 1.52), (Sigma-Aldrich St. Louis, MO). 2,2′-Thiodiethanol solutions (50% and 100% w/w) were prepared in Milli-Q water (Merck Millipore, Billerica, MA) at room temperature. Fixed tissue was completely immersed in 50% 2,2′-thiodiethanol for 2 hours, and then in 100% 2,2′-thiodiethanol overnight at room temperature. Samples were then switched to fresh 100% 2,2′-thiodiethanol for mounting and imaging as described above. For mounting, the tissue was placed with the amnion side facing a #1.5-μm cover glass in a mounting chamber (30-mm cage plate; ThorLabs, Newton, NJ), with a second cover glass placed on the chorion side; 300 μL of phosphate-buffered saline was added to the tissue, and the coverslips were tightened to secure the phosphate-buffered saline and tissue in the imaging chamber. The amnion epithelium was then oriented toward the objective on the upright microscope and centered with the help of an aiming beam from the excitation source. To determine the cellular and structural alterations in detail, a combination of multiphoton autofluorescence microscopy and second harmonic generation microscopy was used. This approach was used to investigate the multilayered three-dimensional micro-organization of human amniochorionic membranes without the use of exogenous contrast agents, as previously applied by our group.17Richardson L. Vargas G. Brown T. Ochoa L. Trivedi J. Kacerovský M. Lappas M. Menon R. Redefining 3Dimensional placental membrane microarchitecture using multiphoton microscopy and optical clearing.Placenta. 2017; 53: 66-75Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Multiphoton autofluorescence microscopy and second harmonic generation microscopy were conducted using a Prairie Ultima IV upright microscope (Prairie Technologies/Bruker, Middleton, WI) using excitation from a Ti:sapphire femtosecond laser (Mai Tai; Spectra-Physics, Santa Clara, CA). A 25× objective with 1.05 numerical aperture (XLPlanN; Olympus, Tokyo, Japan) was used for image collection. Samples were illuminated at 840 nm for generation of both multiphoton autofluorescence microscopy and second harmonic generation microscopy, collected in an epi-illumination geometry. Collected autofluorescence and second harmonic generation microscopy was split into two detection paths using a dichroic mirror, and autofluorescence emission was detected at >500 nm, whereas second harmonic generation microscopy collected at 420 nm. Multiphoton autofluorescence microscopy and second harmonic generation microscopy were collected simultaneously in a 2-channel configuration using Gallium arsenide phosphide photomultiplier tubes (Hamamatsu Photonics, Hamamatsu, Japan) for detection. Regions of interest were obtained (1 second per frame; 512 × 512 pixels or 1024 × 1024 pixels). Depth scans were obtained using a z-interval of 1 μm, with imaging depth ranging from 110 to 400 μm depending on sample preparation as specified below. Images were obtained using a digital zoom factor of 1.19, resulting in a field of view of 408 × 408 μm. Image stacks were analyzed for epithelial characteristics, epithelial shedding and gaps, and collagen alterations using ImageJ software bundled with 64-bit Java version 1.8.0_112 (NIH, Bethesda, MD; http://imagej.nih.gov/ij), whereas IMARIS software version 7.6.5 (Bitplane, Concord, MA) was used for three-dimensional reconstructions of multiphoton autofluorescence microscopy and second harmonic generation microscopy stacks. The total area of a 6-mm explant can be imaged by taking 25 to 30 individual images (mosaic tiling as reported earlier17Richardson L. Vargas G. Brown T. Ochoa L. Trivedi J. Kacerovský M. Lappas M. Menon R. Redefining 3Dimensional placental membrane microarchitecture using multiphoton microscopy and optical clearing.Placenta. 2017; 53: 66-75Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) and stitching them together with ImageJ. The center nine images, captured with a 25× objective, were used as the normalizing surface area to serve as the common denominator for microfracture quantification. Microfractures in membranes were counted in a 1.50 × 106-μm3 area with ImageJ. This allowed us to normalize the surface area scanned for determining microfracture quantities. Microfractures were sliced and three-dimensionally reconstructed with IMARIS software to determine depth and width for each category studied. Kruskal-Wallis followed by Dunn's comparison between groups and U-test statistics were used to compare the clinical groups; significant differences were defined as P < 0.05 in two-tailed tests. The multilayered architecture of the human amniochorionic membranes were analyzed by reconstructed multiphoton autofluorescence microscopy and second harmonic generation microscopy methods as shown in Figure 1, A and B . Multiphoton autofluorescence microscopy was generated from endogenous cytoplasmic fluorophores and pseudocolored red in all images; fibrillar collagen autofluorescence in the extracellular matrix was captured by second harmonic generation microscopy and pseudocolored green (Figure 1B). Features associated with each layer are shown, with the signal arising from the amnion being due to cellular autofluorescence because no fibrillar collagen to produce second harmonic generation microscopy is found in that layer (thus, no green signal). Multiphoton autofluorescence microscopy allowed for visualization of cells in the fibroblast and reticular layers embedded within the extracellular matrix. The amnion, stromal cells, and chorion cells were studied in the context of their extracellular matrix interfaces. As previously described, this method allows the identification of surface topography, collagen distribution, and cellular density within unique cell layers.17Richardson L. Vargas G. Brown T. Ochoa L. Trivedi J. Kacerovský M. Lappas M. Menon R. Redefining 3Dimensional placental membrane microarchitecture using multiphoton microscopy and optical clearing.Placenta. 2017; 53: 66-75Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Characterization of the amnion epithelial layer topography specifically revealed areas of surface disturbances (Figure 1C), epithelial shedding (Figure 1D), and epithelial gaps (voids lacking an epithelial cell, that extend through the single amnion layer, exposing the extracellular matrix) (Figure 1E). Analysis of amnion topography identified areas within the epithelial monolayer that showed intercellular gaps (Figure 2, A and B ). This is consistent with epithelial cell shedding. Morphological puckering (Figure 2C) or indentation of the monolayer surface was also noted. Analysis of orthogonally sliced sections showed areas of degraded basement membrane (Figure 2C) containing migrating epithelial cells (Figure 2D). These are shed or migratory cells that appear to invade degraded collagen (visualized as tunnels) in the extracellular matrix, where proteolysis of collagen appeared to extend deeply into the extracellular matrix (Figure 2E). This constellation of morphological features, with tunnels extending through damaged basement membrane containing migrating cells, is termed microfracture, because it appears to represent detachments between the epithelium and matrix. Four characteristic features for a microfracture in human amniochorionic membranes are reported. This description is based on the analysis of membranes from term not in labor (Figure 3, A–E ) and term labor (Figure 3, F–J) membranes, as well as in vitro cultures in response to cigarette smoke extract (Figure 3, K–O) that mimicked the oxidative stress conditions experienced in utero before and during term labor. The main features of microfracture are: i) altered amnion epithelial layer (puckering) or site of epithelial shedding (Figure 3, B, G, and L); ii) deterioration and damage of the basement membrane (Figure 3, C, H, and M); iii) tunnels representative of collagen degradation in the extracellular matrix that extend from the basement membrane through the spongy layer (Figure 3, D, I, and N); and iv) the presence of migrating cells in the tunnel (Figure 3, E, J, and O). Identification of microfractures, regardless of condition, suggests that microfractures are a normal constituent of membranes at term. Additionally, membranes from each clinical group also contained microfractures based on the characteristics described above; however, the density, depth, and width of the microfractures differed among the groups. Multiphoton autofluorescence microscopy and second harmonic generation microscopy membrane images (1.50 × 106 μm3 area) analyzed by ImageJ allowed the quantification of differences between term not in labor, term labor, and cigarette smoke extract–treated tissues. Microfracture numbers were similar between the term not in labor and term labor groups (Figure 3P).Figure 3Characteristics and quantification of microfractures in term fetal membrane samples and in membranes exposed to oxidative stress by cigarette smoke extract: A–O: Microfractures in term not in labor (A–E), term labor (F–J), and cigarette smoke extract–treated tissue in vitro in an organ explant system for 24 hours (K–O). The yellow cross hairs pinpoint the migrating cell of interest throughout each group of images. A, F, and K: Orthogonal views of a microfracture. B, G, and L: Amnion morphology above the microfracture. C, H, and M: Degraded basement membrane around the migrating cell. D, I, and N: Three-dimensional collagen reconstruction by IMARIS software of a sliced microfracture showing a tunnel of degraded collagen. E, J, and O: Degraded collagen tunnel with migratory cell. P–R: Quantitation and morphometric measures of microfractures in term not in labor, term labor, and cigarette smoke extract exposed fetal membranes in vitro. P: Number of microfractures: Multiphoton images add up to a 1.5008 × 106 μm3 area that was analyzed for microfracture quantity. Numbers of microfractures were higher after cigarette smoke exposure (CSE) of term not in labor (TNIL) membranes in culture, followed by term labor (TL) membranes. Q: Depth of microfractures: Term labor explants contained deeper microfractures then term not in labor membranes. Cigarette smoke extract treatment produced significantly deeper microfractures than term not in labor controls in vitro or term labor membranes (P = 0.002). R: Width of microfractures: Cigarette smoke extract treatment induced wider microfractures compared to term not in labor control cultures (P = 0.0325) in vitro. Term labor microfractures were only slightly wider that the term not in labor controls, and they were not significantly different. ∗∗∗P < 0.001. Scale bars: 30 μm (B, C, E, G, H, J, L, M, O). All images obtained with a 25× objective.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether oxidative stress can increase microfractures, term not in labor samples were exposed in culture to cigarette smoke extract (n = 5). Cigarette smoke extract treatment increased the number of microfractures by 1.62-fold compared to term not in labor (control) (Figure 3P). IMARIS software was used to create the three-dimensional reconstruction of the collagen matrix region, allowing microfractures to be sliced showing width and depth. Cigarette smoke extract induced deeper (P < 0.001) (Figure 3Q) and wider (P = 0.03) fractures (Figure 3R) than what was seen in term not in labor controls, suggesting that oxidative stress can cause an increased number of microfractures with increased morphometry. Although the numbers of microfractures are similar in width and de