The authors have declared that no competing interests exist.
Conceived and designed the experiments: JPV RMM KMAW LR RPK MGG CER CJB. Performed the experiments: KMAW RPK JPV MS MD. Analyzed the data: JPV RMM RPB TKB FMF MS RPK AB MS KMAW CJB. Contributed reagents/materials/analysis tools: RPK TKB RPB FMF. Wrote the paper: JPV RMM TKB LR RPK KMAW CJB.
Early events leading to intrauterine infection remain poorly defined, but may hold the key to preventing preterm delivery. To determine molecular pathways within fetal membranes (chorioamnion) associated with early choriodecidual infection that may progress to preterm premature rupture of membranes (PPROM), we examined the effects of a Group B Streptococcus (GBS) choriodecidual infection on chorioamnion in a nonhuman primate model. Ten chronically catheterized pregnant monkeys (
Group B Streptococcus (GBS) is one cause of preterm birth, stillbirth, and fetal brain injury. GBS is present in the vagina and is thought to ascend into the uterus of some women where it can cause placental inflammation and preterm birth. Understanding the earliest events in the placenta that lead to preterm birth is elusive in humans, because the placenta cannot be studied until after birth. Here, we use a nonhuman primate model to show that an early GBS infection can damage the structural support of the fetal membranes, specifically the cytokeratin network in the epithelium of the amnion (one part of the membranes). Next, we obtained human placentas to show that this cytokeratin network was also damaged in human patients that had preterm premature rupture of the membranes, a major cause of preterm birth. Our work is important in understanding why fetal membranes may rupture prematurely, which may lead to early interventions to prevent membrane damage after placental infection and preterm birth.
Preterm premature rupture of membranes (PPROM) occurs in 1–2% of all pregnancies, but complicates 30% of all preterm deliveries
The underlying pathogenesis of PPROM is thought to be due to a combination of physical stresses and biochemical weakening of the chorioamnion. In association with infection, investigation of PPROM has mainly focused on the catabolic degradation of collagen mediated by matrix metalloproteinases (MMP) with some studies of other pathways including apoptosis and oxidative stress
We hypothesized that in addition to MMP degradation of extracellular collagen (mainly in the chorion), pathways targeting the intracellular cytoskeleton of the amniotic epithelium would be important in creating vulnerability for PPROM. This hypothesis was driven by the observation that the chorion, while thicker, is weaker than the amnion suggesting that biomechanical failure of structural components unique to amnion may be critical for membrane rupture
Our nonhuman primate model had previously demonstrated that a transient GBS infection located within the choriodecidua can induce a cytokine response within the chorioamnion and amniotic fluid (Table S1). This response was associated with labor in two of the GBS animals. Evidence of chorioamnionitis was noted on placental histology of only two of the five GBS animals whereas the remaining animals had normal histology (
We previously reported uterine contraction, microbial culture, and cytokine (amniotic fluid and fetal plasma) results in our model of early choriodecidual infection in association with
To identify early genomic events following a GBS choriodecidual infection, we performed microarray analysis of chorioamnion from the site of inoculation. Of the differentially expressed genes, a subset is shown in
Description | Symbol | log2 fold change |
Similar to CK 24 | LOC701667 | −5.90 |
CK 3 | KRT3 | −5.31 |
Similar to CK 6A | LOC718942 | −5.27 |
Similar to CK 6A | LOC718942 | −5.25 |
Zinc finger, BED-type containing 2 | ZBED2 | −4.48 |
Solute carrier family 27, member 6 | SLC27A6 | −4.24 |
Similar to Laminin 5 beta 3 | LOC717602 | −4.10 |
Laminin, gamma 2 | LAMC2 | −3.95 |
Similar to CK 16 | LOC707236 | −3.85 |
Similar to CK 6A | LOC718942 | −3.82 |
Similar to breast cancer membrane protein 11 | LOC714517 | −3.80 |
Similar to Desmocollin 2 isoform A (DSC2A) | LOC712942 | −3.79 |
Similar to CK 14 | LOC703932 | −3.75 |
Platelet/endothelial cell adhesion molecule | PECAM1 | 1.85 |
Colony stimulating factor 3 receptor | CSF3R | 1.87 |
Chemokine ligand 12 | CXCL12 | 1.88 |
Myosin, heavy chain 11, smooth muscle | MYH11 | 1.91 |
Colony stimulating factor 2 receptor, beta | CSF2RB | 1.94 |
Similar to microfibrillar-associated protein 4 | LOC709521 | 1.95 |
Similar to DCP2 decapping enzyme | LOC694225 | 1.99 |
Similar to DCP2 decapping enzyme | LOC694225 | 2.04 |
Fatty acid binding protein 4, adipocyte | FABP4 | 2.15 |
Similar to DCP2 decapping enzyme | LOC694225 | 2.18 |
Matrix metallopeptidase 1 | MMP1 | 2.28 |
Myosin, heavy chain 11, smooth muscle | MYH11 | 2.32 |
Similar to HspB1 | HSPB1 | 2.42 |
Placenta-specific 8 | PLAC8 | 2.57 |
Genes significantly downregulated by choriodecidual GBS exposure included multiple cytokeratins (CK) such as
Both a three-factor analysis
To further explore the relationships between differentially expressed single genes, we identified gene sets and pathways with concordant changes in expression using GSA
Genes shown were filtered for interactions reported in humans only. Direct and indirect interactions are shown by solid lines and dashed lines respectively. Green indicates gene downregulation and red depicts upregulation. Color intensity represents the average of log2 fold change with brighter colors representing a more significant difference between GBS and controls. Symbols for each molecule are presented according to molecular functions and type of interactions. Functional assignations attributed by IPA software. An asterisk indicates that there were multiple probesets for the same gene on the Affymetrix array. The black arrows indicate the GBS cases with chorioamnionitis.
Gene set description | Gene set length | Gene Ontology ID | p value |
morphogenesis of an epithelium | 23 | GO:0002009 | 0.006 |
ectoderm development | 18 | GO:0007398 | 0.006 |
epidermis development | 89 | GO:0008544 | 0.006 |
positive regulation of peptidase activity | 12 | GO:0010952 | 0.006 |
peptide cross-linking | 22 | GO:0018149 | 0.006 |
galactose catabolic process | 7 | GO:0019388 | 0.006 |
negative regulation of actin filament polymerization | 7 | GO:0030837 | 0.006 |
negative regulation of epithelial cell differentiation | 6 | GO:0030857 | 0.006 |
keratinization | 26 | GO:0031424 | 0.006 |
hemidesmosome assembly | 12 | GO:0031581 | 0.006 |
cell junction assembly | 74 | GO:0034329 | 0.006 |
intermediate filament-based process | 7 | GO:0045103 | 0.006 |
intermediate filament organization | 15 | GO:0045109 | 0.006 |
SNARE binding | 19 | GO:0000149 | 0.006 |
structural constituent of cytoskeleton | 93 | GO:0005200 | 0.006 |
thrombin receptor activity | 5 | GO:0015057 | 0.006 |
peptidase activator activity | 10 | GO:0016504 | 0.006 |
oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen | 77 | GO:0016702 | 0.006 |
oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors | 33 | GO:0016706 | 0.006 |
L-ascorbic acid binding | 21 | GO:0031418 | 0.006 |
protein kinase A catalytic subunit binding | 10 | GO:0034236 | 0.006 |
heterotrimeric G-protein binding | 6 | GO:0032795 | 0.01 |
chemorepellent activity | 5 | GO:0045499 | 0.011 |
neurotransmitter transporter activity | 5 | GO:0005326 | 0.012 |
MHC class II protein binding | 5 | GO:0042289 | 0.012 |
structural molecule activity | 223 | GO:0005198 | 0.013 |
AMP-activated protein kinase activity | 7 | GO:0004679 | 0.015 |
oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen | 47 | GO:0016705 | 0.015 |
sarcoplasm | 6 | GO:0016528 | 0.006 |
rough endoplasmic reticulum membrane | 7 | GO:0030867 | 0.006 |
pseudopodium | 14 | GO:0031143 | 0.006 |
intermediate filament cytoskeleton | 45 | GO:0045111 | 0.006 |
flagellar axoneme | 9 | GO:0035086 | 0.007 |
platelet alpha granule lumen | 46 | GO:0031093 | 0.009 |
cell periphery | 28 | GO:0071944 | 0.01 |
AMP-activated protein kinase complex | 7 | GO:0031588 | 0.012 |
pyruvate dehydrogenase complex | 7 | GO:0045254 | 0.014 |
photoreceptor outer segment | 38 | GO:0001750 | 0.016 |
basement membrane | 91 | GO:0005604 | 0.016 |
microtubule associated complex | 34 | GO:0005875 | 0.016 |
costamere | 20 | GO:0043034 | 0.019 |
intermediate filament | 97 | GO:0005882 | 0.02 |
acrosomal membrane | 11 | GO:0002080 | 0.021 |
Gene set description | Gene set length | Gene Ontology ID | p value |
myeloid leukocyte differentiation | 5 | GO:0002573 | <0.002 |
apoptotic DNA fragmentation | 25 | GO:0006309 | <0.002 |
protein monoubiquitination | 17 | GO:0006513 | <0.002 |
pinocytosis | 5 | GO:0006907 | <0.002 |
regulation of cellular pH | 5 | GO:0030641 | <0.002 |
T cell receptor V(D)J recombination | 5 | GO:0033153 | <0.002 |
response to laminar fluid shear stress | 10 | GO:0034616 | <0.002 |
T cell homeostasis | 21 | GO:0043029 | <0.002 |
regulation of bone resorption | 7 | GO:0045124 | <0.002 |
positive regulation of erythrocyte differentiation | 16 | GO:0045648 | <0.002 |
vagina development | 10 | GO:0060068 | <0.002 |
neural precursor cell proliferation | 6 | GO:0061351 | <0.002 |
erythrocyte maturation | 11 | GO:0043249 | 0.002 |
positive regulation of survival gene product expression | 12 | GO:0045885 | 0.002 |
positive regulation of macrophage differentiation | 8 | GO:0045651 | 0.004 |
gland development | 5 | GO:0048732 | 0.004 |
Rho GTPase activator activity | 24 | GO:0005100 | 0.006 |
ARF GTPase activator activity | 25 | GO:0008060 | 0.006 |
cytokine binding | 20 | GO:0019955 | 0.006 |
RS domain binding | 7 | GO:0050733 | 0.006 |
telomeric DNA binding | 11 | GO:0042162 | 0.007 |
Rac guanyl-nucleotide exchange factor activity | 8 | GO:0030676 | 0.009 |
eukaryotic cell surface binding | 27 | GO:0043499 | 0.009 |
SH3/SH2 adaptor activity | 51 | GO:0005070 | 0.01 |
oxidoreductase activity, acting on the CH-CH group of donors, NAD or NADP as acceptor | 6 | GO:0016628 | 0.01 |
GTPase activator activity | 183 | GO:0005096 | 0.011 |
organic anion transmembrane transporter activity | 15 | GO:0008514 | 0.011 |
sodium-independent organic anion transmembrane transporter activity | 5 | GO:0015347 | 0.011 |
L-amino acid transmembrane transporter activity | 7 | GO:0015179 | 0.013 |
chromo shadow domain binding | 6 | GO:0070087 | 0.013 |
scavenger receptor activity | 45 | GO:0005044 | 0.014 |
nuclear chromosome | 29 | GO:0000228 | 0.006 |
integral to nuclear inner membrane | 5 | GO:0005639 | 0.006 |
nuclear lamina | 9 | GO:0005652 | 0.006 |
clathrin coated vesicle membrane | 13 | GO:0030665 | 0.006 |
clathrin coat | 9 | GO:0030118 | 0.011 |
T cell receptor complex | 12 | GO:0042101 | 0.014 |
intercellular bridge | 6 | GO:0045171 | 0.015 |
phagocytic vesicle | 14 | GO:0045335 | 0.015 |
extracellular vesicular exosome | 7 | GO:0070062 | 0.019 |
heterogeneous nuclear ribonucleoprotein complex | 20 | GO:0030530 | 0.022 |
IkappaB kinase complex | 10 | GO:0008385 | 0.023 |
spindle midzone | 12 | GO:0051233 | 0.024 |
phosphatidylinositol 3-kinase complex | 10 | GO:0005942 | 0.027 |
chromocenter | 8 | GO:0010369 | 0.028 |
actin filament | 44 | GO:0005884 | 0.03 |
To place the genomics findings of cytostructural downregulation, apoptosis, and inflammation within a visual construct, IPA's Core Analysis feature was used to map functional networks of relevant genes. The top five molecular and cellular function pathways identified by IPA were cell death and survival, cellular movement, cellular growth and proliferation, cellular development, and cell morphology (
Number of Molecules | p value | |
Cancer | 326 | 4.45×10−18–8.90×10−4 |
Endocrine System Disorders | 84 | 1.57×10−11–3.45×10−4 |
Reproductive System Disease | 146 | 1.57×10−11–6.19×10−4 |
Gastrointestinal Disease | 135 | 6.35×10−9–5.07×10−4 |
Dermatological Diseases and Conditions | 126 | 6.96×10−8–8.90×10−4 |
Cell Death and Survival | 271 | 6.57×10−16–8.80×10−4 |
Cellular Movement | 196 | 4.41×10−15–8.05×10−4 |
Cellular Growth and Proliferation | 271 | 8.84×10−12–9.30×10−4 |
Cellular Development | 265 | 1.25×10−11–9.30×10−4 |
Cell Morphology | 152 | 2.37×10−8–6.19×10−4 |
Cardiovascular System Development and Function | 144 | 1.06×10−11–7.32×10−4 |
Hematologic System Development and Function | 188 | 1.25×10−11–9.30×10−4 |
Lymphoid Tissue Structure and Development | 98 | 1.22×10−9–7.32×10−4 |
Tumor Morphology | 84 | 1.29×10−9–7.47×10−4 |
Organismal Development | 166 | 1.48×10−9–8.65×10−4 |
Top Canonical Pathways | Ratio | p value |
Glioblastoma Multiforme Signaling | 24/164 | 1.33×10−7 |
Role of Macrophages, Fibroblasts, and Endothelial Cells in Rheumatoid Arthritis | 34/332 | 9.04×10−7 |
Macropinocytosis Signaling | 13/76 | 2.67×10−5 |
Colorectal Cancer Metastasis Signaling | 27/258 | 2.70×10−5 |
Sphingosine-1-phosphate Signaling | 15/120 | 1.64×10−4 |
Top Transcription Factors | Predicted Activation State | p-value of overlap |
TGFB1 | Not specified | 3.34×10−15 |
TNF | Not specified | 3.34×10−15 |
Lipopolysaccharide | Not specified | 3.34×10−15 |
TP53 | Not specified | 3.34×10−14 |
HIF1A | Inhibited | 3.34×10−13 |
*The Functional Analysis of a Network identified biological functions and/or diseases that were most significant to the molecules in the network using a right-tailed Fisher's exact test.
**Canonical Pathway Analysis identified pathways from the IPA library that were most significant to the data set.
Significance of the association was measured in two ways: (1) as the ratio of the number of molecules from the focus gene set that map to the pathway to the total number of molecules that map to the canonical pathway and (2) using Fisher's exact test.
***Transcription factor analysis is based on prior knowledge of expected effects between transcription factors and their target genes stored in the IPA library.
The overlap p-value measures whether there is a statistically significant overlap between the dataset genes and the genes regulated by a transcription factor using Fisher's Exact Test.
To validate our microarray data, we performed quantitative RT-PCR on 17 genes of interest. We directly compared levels of gene expression obtained with amplified RNA samples using GAPDH expression as a control for input cDNA. Concordance between mRNA generated microarray data and the quantitative RT-PCR data was noted in all samples (
Genes that were significantly up- or downregulated by qRT-PCR between GBS and controls are indicated by an asterisk (two-sided t-test, p<0.05). All genes shown were also significant in the unadjusted microarray analysis.
Next, we performed immunofluorescence and confocal microscopy to localize CK distribution within the chorioamnion and identify whether morphologic changes typical of CK network dysfunction and depletion were present within the amniotic epithelium. We chose to investigate CK6, because it was significantly downregulated within the microarray data and is expressed in amniotic epithelium. Similar to prior studies, the distribution of CK6 appeared limited to amniotic epithelium, myofibroblasts and macrophages within the extracellular matrix of the chorioamnion as well as the chorion
CK retraction from the cellular periphery was significantly associated with GBS exposure (p = 0.006) as was the combined test of both retraction and speckling (p = 0.006).
As CK network dysfunction is a novel finding in amniotic epithelium after choriodecidual infection, we further investigated the amniotic epithelium ultrastructure with transmission electron microscopy in both macaque (GBS and saline; N = 2 each) and human chorioamnion (normal term, N = 6; PPROM, N = 11). Clinical information from the human pregnancies is listed in Table S5 and electron microscopy images shown in
Intermediate filament aggregates (black arrowheads) are evident in the macaque GBS and human PPROM case, but not the normal controls. The basal amnion processes are broader and appear shorter with prominent central IF aggregates (black arrowheads) in the GBS-infused sample (D) and after PPROM (H). In some human PPROM cases, the basal amnion processes are nearly obliterated. Images shown were obtained from macaque (GBS-1, Saline-5) and human tissues (Control-1, PPROM-7) that correlate with the data shown in Tables S1 and S5. The measurement bar in panel D may be applied to panels A, B and C. The measurement bar in panel H may be applied to panels E, F, G and H.
We hypothesized that intermediate filaments would be retracted from the periphery leading to shortening of the amnion foot processes along the basement membrane as dysfunctional intermediate filaments retract from peripheral organizing centers. At the basal surface of the amnion, foot processes in the macaque control were thin and elongated with relatively dispersed cytoplasmic intermediate filament networks. In the GBS cases, amniocyte foot processes were much broader with prominent central intermediate filament aggregates. In human amnion obtained from normal pregnancies at term, the amniocyte foot processes were somewhat broader than in the macaque, but also demonstrated a highly folded undulating profile along the basement membrane. Human amniocyte foot processes were on average 594 nm shorter in the PPROM cases than controls, which was highly significant after controlling for processing site (p = 0.002,
Measure | PPROM | Term Births | ||
Mean | SE | Mean | SE | |
Foot Process Length at both sites combined (nm) (NPPROM = 6, NControl = 6) | 558.9 | 112.6 | 1152.4 | 244.4 |
Foot Process Length from “Site 1” (nm) (NPPROM = 1, NControl = 3) | 435.4 | NA | 621.2 | 105.8 |
Foot Process Length from “Site 2” (nm) (NPPROM = 5, NControl = 3) | 583.7 | 134.6 | 1683.6 | 73.1 |
Human amniocyte foot processes were on average 594 nm shorter in the PPROM cases than controls, which was highly significant after controlling for processing site (p = 0.002). A histogram depicting the distribution of amniocyte foot process lengths at both sites is shown in
Our results demonstrated a large-scale downregulation of amniocyte CK synthesis, retraction of the intermediate filament network from the basement membrane and formation of dense intermediate filament aggregates after GBS exposure or PPROM. CK are components of intermediate filaments vital for maintaining cellular structure, elasticity, and resisting shear stress. Weakening of the intermediate filament network, which occurs in several human disorders with mutated CK genes (e.g. epidermolysis bullosa simplex), increases the risk of membrane dysfunction
First, bacteria from the vagina traffics into the choriodecidual space (panel A). Inflammatory mediators (e.g. IL-8, IL-6, TNF-α) are produced by the decidua and/or membranes, which diffuses through the chorioamnion and into the amniotic fluid. CK synthesis becomes downregulated and intracellular pools of cytokeratins gradually become depleted. When the reserves of cytokeratins are depleted, peripheral retraction of the cytokeratin network occurs along with formation of aggresomes. Perturbations in amnion CK networks may act synergistically to increase vulnerability to membrane weakening including other pathways mediated by the pathogen, MMP, or oxidative stress. Panel B illustrates the histologic changes that begin to occur in the chorioamnion. Neutrophils infiltrate the decidua and chorion. The chorion thins and nuclei in the amniotic epithelium begin to undero apoptosis appearing pyknotic in our illustration. The side-bar lists selected differentially expressed genes identified by IPA analysis IL, Interleukin; CK, cytokeratin, MMP matrix metalloprotease. Gene symbol color indicates significantly differentially expressed up- (red) or down- (green) regulation. Note: neutrophils and macrophages are included in this conceptual model as these cell types contribute to chorioamnionitis histopathology and were observed in our results. We do not mean to infer that these are the responsible cell types for the changes observed.
Whether cytokeratin downregulation is a direct cytotoxic effect of GBS in the chorioamnion or an indirect effect of cytokine production by cells in the placenta is unknown. GBS and other gram-positive cocci are known to bind cytokeratin 8
Any benefit of CK downregulation in preventing bacterial invasion of the amniotic fluid may be short-lived and at the expense of the cytoskeletal support of the cell within several days of the initial insult
Multiple genes and pathways associated with apoptosis were upregulated in our model (e.g.
Our Ingenuity Pathway Analysis also suggested a modest increase in
Mechanisms triggering PPROM
Study limitations include a modest sample size, which is a necessary constraint of nonhuman primate research due to the costly nature of the experiments and for issues of conservation. These experiments are extremely expensive, which makes a study design involving prospective sampling of the chorioamnion in multiple animal groups with adequate power not possible even in the context of a large federally funded scientific grant. Two additional limitations biased our electron microscopy findings towards the null hypothesis, which makes our highly significant findings more remarkable. First, we excluded the most severe cases of PPROM due to poor histology. Secondly, as the basement membrane became flatter in our PPROM cases (with loss of CK and amniocyte foot processes), we tended to measure foot process length in areas that still had some folding of the membrane creating selection bias towards areas that appeared more normal. Overall, our findings suggest that even in the absence of AF invasion, bacteria in the choriodecidua induce an inflammatory response that is associated with downregulated expression of CK and many cytostructural elements important for chorioamnion integrity and support, which correlates with evidence of CK network dysfunction.
Changing our study design to inoculate a different organism with greater pathogenicity may have resulted in bacterial trafficking through the chorioamnion into the amniotic fluid and rapid preterm labor
Our study suggests that the loss of intermediate filament cytostructural support within the amniotic epithelium is another major contributor to chorioamnion weakening that should be considered alongside other mechanisms such as apoptosis, MMP activation, and oxidative stress. The length of time between initial infection and final depletion of the cytokeratin reserves of intermediate filaments may represent a “PPROM clock” that corresponds with the gestational length from the choriodecidual infection until membrane rupture. Future directions in understanding PPROM should focus on the relevance of CK networks to the integrity of amniotic epithelium over gestation and in the setting of infection. A better understanding of the time course of keratin assembly, disassembly and perturbations in CK networks within the amniotic epithelium may provide insight into how long choriodecidual infections are generally present prior to membrane rupture. More work is also needed to understand the regulation of cytostructural mRNA within the chorioamnion and the contribution of changes in miRNA. Therapies that stabilize CK networks and promote keratin assembly may lead to new therapeutic targets for the prevention of PPROM and preterm birth.
Many of the methods related to our animals and study groups, uterine activity, GBS and bacterial cultures, quantitation of inflammatory mediators (cytokines, prostaglandins, matrix metalloproteinases), and performance of the microarray, qRT-PCR, microarray and IPA analysis have been previously published
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council and the Weatherall report, “The use of non-human primates in research”. The protocol was approved by the Institutional Animal Care Use Committee of the University of Washington (Permit Number: 4165-01). All surgery was performed under general anesthesia and all efforts were made to minimize suffering.
Human placental samples were obtained specifically for this study under the approval of the University of Washington IRB 34004 and through the Global Alliance for the Prevention of Prematurity and Stillbirth (GAPPS, Seattle, WA, USA) Tissue Repository under the approval of Seattle Children's Hospital IRB 12879 and 13975. For both the University of Washington and GAPPS samples, subjects provided written informed consent.
Ten chronically catheterized pregnant monkeys (
In our model, pregnant pigtail macaques were time-mated and fetal age determined using early ultrasound. Temperature in the animal quarters was maintained at 72–82°F. Animals were fed a commercial monkey chow, supplemented daily with fruits and vegetables and drinking water was available at all times. The tethered chronic catheter preparation was used for all
After surgery, the animal was placed in the jacket and tether with the catheters/electrodes tracked through the tether system. Cefazolin and terbutaline sulfate were administered to reduce postoperative infection risk and uterine activity. Both cefazolin and terbutaline were stopped at least 72 hours before experimental start (∼13 half-lives for terbutaline, 40 half-lives for cefazolin, >97% of both drugs eliminated), which represented approximately a 7–10 day period of postoperative terbutaline administration. Cefazolin 1 gram was administered intravenously each day in saline controls to minimize chances of a catheter-related infection. Experiments began approximately two weeks after catheterization surgery to allow recovery (∼30–31 weeks human gestation). At our center, term gestation in the non-instrumented pigtail macaque population averages 172 days.
Intraamniotic pressure was continuously recorded, digitized, and analyzed by previously described methods
After cesarean section, placenta samples underwent tissue fixation in 10% neutral buffered formalin. Complete gross and histopathologic examination was performed on placentas. H&E-stained, full-thickness paraffin sections (placental disc, umbilical cord, fetal membrane roll) were examined from each case to exclude inflammation, necrosis, fetal vascular thrombosis, or other histopathological findings. Chorioamnionitis was diagnosed by the presence of a neutrophilic infiltrate at the chorion-decidua junction (mild) or amniochorion junction (moderate or severe). Funisitis denoted neutrophils in the umbilical vessels and/or surrounding connective tissue.
To study genetic pathways in
The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar
Next, the data was analyzed using GSA in order to investigate categories of genes
We used the Ingenuity Pathway Analysis (IPA) software (Ingenuity® Systems,
Quantitation of mRNA levels was performed by the CHDD Genomics Core Laboratory using fluorogenic 5′ nuclease-based assays and has been previously described
After cesarean section, fetal membranes from placental samples for immunohistochemistry underwent freezing in OCT embedding compound, fresh-frozen and stored at −70°C. Fresh-frozen sections were serially cut with microtome to 5 micron standardized width and mounted on slides. After a 15-minute acetone fixation, slides were air dried and then permeabilized (1% Triton X-100) for 30 minutes prior to blocking (5% donkey serum) for 1 hour. Slides were then immunostained with primary rabbit anti-CK6 antiserum (1∶750, ab24646, Abcam, Cambridge MA), in an overnight incubation, and subsequently with a secondary donkey anti-rabbit 488 antibody (1∶400). Counterstaining with DAPI was performed prior to coverslip application.
Images were acquired with a FV-1000 laser scanning confocal microscope coupled to an IX-81 inverted microscope (Olympus, Center Valley, PA) and controlled by Fluoview software, version 3.1. DAPI labeling was imaged using a 405 nm laser line and 425 to 475 nm bandpass diffraction filter. Alexa488 was detected with the 488 nm line using a diffraction 500 to 550 nm filter. Images for quantitation of intensities and area were obtained with a 20×/NA0.75 UPLAPO dry objective lens, zoom of 2 and a 512×512 box size for a final pixel size of 0.621 µm. High-resolution images for qualitative analysis were collected with a 60×/NA1.35 UPLSAPO oil immersion objective, zoom of 3 and a 1024×1024 box size for a final pixel size of 0.069 µm/pixel. Images at each magnification were collected using the same laser and detector settings for all labeled specimens and negative controls.
Image analysis for CK6 content was performed with ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,
The high-resolution images were coded by random number generator and scored by a two blinded reviewers (KAW, RMM) for qualitative assessment of CK retraction and speckling (yes/no). CK retraction was defined as a “blunted or faded” CK staining pattern along the highly folded basement membrane of most cells. Speckling was defined as “bright spots or areas of immunofluorescence” associated with CK aggregation in some or all cells. Five images per slide were taken from random areas of the amniotic epithelium. Each image was scored with one point for retraction and one point for speckling from each reviewer. We also created a binary predictor test variable for retraction and speckling combined so that cases with both retraction and speckling were coded as 0 and other combinations coded as a 1.
Formalin-fixed and paraffin-embedded tissues from macaque (GBS-1 and -2, Saline-3 and -5, Table S1) and human chorioamnion (Table S5) were used for these experiments. First, the tissues were deparaffinized and fixed in 4% paraformaldehyde and 2% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.4) for at least 2 hours to overnight. Tissues were cut (1×4 mm2) and immersed in 0.1M sodium cacodylate buffer (3 changes, 10 min each or overnight). After fixation, tissues were then immersed in 1% osmium tetroxide in buffered saline. Tissues were then dehydrated with 50% ethanol (10 minutes) followed by en bloc staining with 2% uranyl acetate in70% ethanol (2 hours). Further dehydration was done in a series of alcohol dehydration steps (95% for 10 minutes, 100% for 10 minutes ×2) and 100% propylene oxide (10 minutes ×2). Embedding was performed by a 1∶1 mixture of EMBed 812 and Propylene Oxide for 1–2 hr, then 100% EMBed 812 for 2–4 hours followed by drying in an oven at 60°C for 24 hours. Embedded samples were then cut semi-thick (Leica Reichert Ultracot S, Wein, Austria) with toluidine blue stain (1–3 min). Semi-thick sections were observed under microscope for precise location for ultrathin sections. Ultrathin sections at 70 nm were collected onto grids. Grids were stained with lead citrate. Tissue samples were processed at two different sites (University of Washington, Seattle Children's Hospital) and imaged with two different electron microscopes (JEM-1230, Jeol, Peabody, MA, USA or Zeiss EM910, Carl Zeiss AG, Oberkochen, Germany).
Quantitative analysis of TEM images was performed on the human controls and PPROM cases. Amniocyte foot processes were identified along the basement membrane folds were measured for maximum length by Olympus ITEM Soft Imaging System (Münster, Germany) by an author (RPK) blinded to case-control status (
Histopathology of the fetal chorioamnion and umbilical cord are shown for a saline control (A,C) and GBS-exposed with chorioamnionitis (B,D,E). In exposed animals, neutrophilic infiltration (circles) is present in the chorioamnion (B), placental disc (D), and wall of the umbilical vein (E, inset).
(TIF)
Heatmap of 603 out of 52,779 differentially expressed probesets with >2.0 fold change, p<0.05 in GBS vs Saline exposed animals. When probesets were matched to genes and duplicates removed, 331 out of 19448 genes were differentially expressed (>2.0 fold change, p<0.05). The black arrows indicate the GBS cases with chorioamnionitis.
(JPG)
Principal component (PCA) analysis of GBS cases and saline controls. PCA is a tool in exploratory data analysis and allows interpretation of variance in the data. In this figure, the PCA analysis reveals that the saline group is separated from the 5 GBS samples, but the GBS cases without chorioamnionitis are mixed in with the GBS cases with chorioamnionitis. This would indicate that the GBS cases with chorioamnionitis are too confounded with the GBS cases without chorioamnionitis to benefit from a stratified analysis.
(TIF)
The histogram depicts the length of foot processes at the basal amniotic epithelial surface obtained from transmission electron microscopy images from human PPROM cases (blue, n = 7) and human controls (red, n = 6) stratified by site. Human amniocyte foot processes were on average 594 nm shorter in PPROM cases (p = 0.002 after adjustment for processing site).
(TIF)
An example of a measurement (white bar) of an amniocyte foot process length is shown here. The black arrow indicates CK aggregation (aggresome) indicative of CK network dysfunction.
(TIF)
We are grateful for the technical assistance of Louis Paolella, Mike Gough, Keith Vogel, Cliff Astley, Bita Maghsoodi, Jaclyn Bogue, and Glen MacDonald. We also gratefully acknowledge Jennifer Summers and Jan Hamanishi for their help in preparing the figures.