We analyzed the vaginal fluid proteome to identify biomarkers of intraamniotic infection among women in preterm labor.
Proteome analysis was performed on vaginal fluid specimens from women with preterm labor, using multidimensional liquid chromatography, tandem mass spectrometry, and label-free quantification. Enzyme immunoassays were used to quantify candidate proteins. Classification accuracy for intraamniotic infection (positive amniotic fluid bacterial culture and/or interleukin-6>2 ng/mL) was evaluated using receiver-operator characteristic curves obtained by logistic regression.
Of 170 subjects, 30 (18%) had intraamniotic infection. Vaginal fluid proteome analysis revealed 338 unique proteins. Label-free quantification identified 15 proteins differentially expressed in intraamniotic infection, including acute-phase reactants, immune modulators, high-abundance amniotic fluid proteins and extracellular matrix–signaling factors; these findings were confirmed by enzyme immunoassay. A multi-analyte algorithm showed accurate classification of intraamniotic infection.
Vaginal fluid proteome analyses identified proteins capable of discriminating between patients with and without intraamniotic infection.
intraamniotic infection; preterm labor; proteomics; vaginal fluid
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 (Macaca nemestrina) at 118–125 days gestation (term = 172 days) received choriodecidual inoculation of either GBS (n = 5) or saline (n = 5). Cesarean section was performed in the first week after GBS or saline inoculation. RNA extracted from chorioamnion (inoculation site) was profiled by microarray. Single gene, Gene Set, and Ingenuity Pathway Analysis results were validated using qRT-PCR (chorioamnion), Luminex (amniotic fluid, AF), immunohistochemistry, and transmission electron microscopy (TEM). Despite uterine quiescence in most cases, significant elevations of AF cytokines (TNF-α, IL-8, IL-1β, IL-6) were detected in GBS versus controls (p<0.05). Choriodecidual infection resolved by the time of cesarean section in 3 of 5 cases and GBS was undetectable by culture and PCR in the AF. A total of 331 genes were differentially expressed (>2-fold change, p<0.05). Remarkably, GBS exposure was associated with significantly downregulated expression of multiple cytokeratin (CK) and other cytoskeletal genes critical for maintenance of tissue tensile strength. Immunofluorescence revealed highly significant changes in the CK network within amniocytes with dense CK aggregates and retraction from the cell periphery (all p = 0.006). In human pregnancies affected by PPROM, there was further evidence of CK network retraction with significantly shorter amniocyte foot processes (p = 0.002). These results suggest early choriodecidual infection results in decreased cellular membrane integrity and tensile strength via dysfunction of CK networks. Downregulation of CK expression and perturbations in the amniotic epithelial cell intermediate filament network occur after GBS choriodecidual infection, which may contribute to PPROM.
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 birth and stillbirth are complex local and global health problems requiring an interdisciplinary approach and an international commitment. Stakeholders developed recommendations for a Global Action Agenda (GAA) at the 2009 International Conference on Prematurity and Stillbirth. The primary goal of this GAA is to forge a collaborative effort toward achieving common goals to prevent preterm birth and stillbirth, and to improve related maternal, newborn, and child health outcomes.
GAPPS co-convened this four-day conference with the Bill & Melinda Gates Foundation, March of Dimes, PATH, Save the Children, UNICEF and the World Health Organization. Participants included about 200 leading international researchers, policymakers, health care practitioners and philanthropists. A near-final draft of this report was sent three weeks in advance to help co-chairs and participants prepare for workgroup discussions.
Global Action Agenda
Twelve thematic workgroups, composed of interdisciplinary experts, made recommendations on short-, intermediate-, and long-term milestones, and success metrics. Recommendations are based on the following themes: (1) advance discovery of the magnitude, causes and innovative solutions; (2) promote development and delivery of low-cost, proven interventions; (3) improve advocacy efforts to increase awareness that preterm birth and stillbirth are leading contributors to the global health burden; (4) increase resources for research and implementation; and (5) consider ethical and social justice implications throughout all efforts.
The conference provided an unprecedented opportunity for maternal, newborn and child health stakeholders to create a collaborative strategy for addressing preterm birth and stillbirth globally. Participants and others have already completed or launched work on key milestones identified in the GAA. Updates will be provided at www.gapps.org.
Normal and abnormal processes of pregnancy and childbirth are poorly understood. This second article in a global report explains what is known about the etiologies of preterm births and stillbirths and identifies critical gaps in knowledge. Two important concepts emerge: the continuum of pregnancy, beginning at implantation and ending with uterine involution following birth; and the multifactorial etiologies of preterm birth and stillbirth. Improved tools and data will enable discovery scientists to identify causal pathways and cost-effective interventions.
Pregnancy and parturition continuum
The biological process of pregnancy and childbirth begins with implantation and, after birth, ends with the return of the uterus to its previous state. The majority of pregnancy is characterized by rapid uterine and fetal growth without contractions. Yet most research has addressed only uterine stimulation (labor) that accounts for <0.5% of pregnancy.
The etiologies of preterm birth and stillbirth differ by gestational age, genetics, and environmental factors. Approximately 30% of all preterm births are indicated for either maternal or fetal complications, such as maternal illness or fetal growth restriction. Commonly recognized pathways leading to preterm birth occur most often during the gestational ages indicated: (1) inflammation caused by infection (22-32 weeks); (2) decidual hemorrhage caused by uteroplacental thrombosis (early or late preterm birth); (3) stress (32-36 weeks); and (4) uterine overdistention, often caused by multiple fetuses (32-36 weeks). Other contributors include cervical insufficiency, smoking, and systemic infections. Many stillbirths have similar causes and mechanisms. About two-thirds of late fetal deaths occur during the antepartum period; the other third occur during childbirth. Intrapartum asphyxia is a leading cause of stillbirths in low- and middle-income countries.
Utilizing new systems biology tools, opportunities now exist for researchers to investigate various pathways important to normal and abnormal pregnancies. Improved access to quality data and biological specimens are critical to advancing discovery science. Phenotypes, standardized definitions, and uniform criteria for assessing preterm birth and stillbirth outcomes are other immediate research needs.
Preterm birth and stillbirth have multifactorial etiologies. More resources must be directed toward accelerating our understanding of these complex processes, and identifying upstream and cost-effective solutions that will improve these pregnancy outcomes.
This is the first of seven articles from a preterm birth and stillbirth report. Presented here is an overview of the burden, an assessment of the quality of current estimates, review of trends, and recommendations to improve data.
Few countries have reliable national preterm birth prevalence data. Globally, an estimated 13 million babies are born before 37 completed weeks of gestation annually. Rates are generally highest in low- and middle-income countries, and increasing in some middle- and high-income countries, particularly the Americas. Preterm birth is the leading direct cause of neonatal death (27%); more than one million preterm newborns die annually. Preterm birth is also the dominant risk factor for neonatal mortality, particularly for deaths due to infections. Long-term impairment is an increasing issue.
Stillbirths are currently not included in Millennium Development Goal tracking and remain invisible in global policies. For international comparisons, stillbirths include late fetal deaths weighing more than 1000g or occurring after 28 weeks gestation. Only about 2% of all stillbirths are counted through vital registration and global estimates are based on household surveys or modelling. Two global estimation exercises reached a similar estimate of around three million annually; 99% occur in low- and middle-income countries. One million stillbirths occur during birth. Global stillbirth cause-of-death estimates are impeded by multiple, complex classification systems.
Recommendations to improve data
(1) increase the capture and quality of pregnancy outcome data through household surveys, the main data source for countries with 75% of the global burden; (2) increase compliance with standard definitions of gestational age and stillbirth in routine data collection systems; (3) strengthen existing data collection mechanisms—especially vital registration and facility data—by instituting a standard death certificate for stillbirth and neonatal death linked to revised International Classification of Diseases coding; (4) validate a simple, standardized classification system for stillbirth cause-of-death; and (5) improve systems and tools to capture acute morbidity and long-term impairment outcomes following preterm birth.
Lack of adequate data hampers visibility, effective policies, and research. Immediate opportunities exist to improve data tracking and reduce the burden of preterm birth and stillbirth.
To determine if treatment with ampicillin together with dexamethasone and indomethacin delays preterm birth induced by intra-amniotic Group B Streptococcus (GBS) in a nonhuman primate model.
After contraction onset induced by GBS (106 cfu/ml), chronically instrumented rhesus macaques received either: no treatment (controls, n=6); ampicillin (n=4); or ampicillin + dexamethasone + indomethacin (n=5). Outcomes included the interval from contraction onset until delivery and concentrations of amniotic fluid (AF) inflammatory mediators.
Mean interval (hours) from contraction onset until delivery was 33 ± 8.7 in controls, 82 ± 28.0 with ampicillin (p=0.18 vs. controls); and 213 ± 50.8 with ampicillin + dexamethasone + indomethacin (p=0.004 vs. controls). Ampicillin eradicated GBS, but uterine activity, AF cytokines, prostaglandins, and MMP-9 remained elevated. AMP/DEX/INDO suppressed IL-1β, TNF-α, PGE2 and PGF2α, but did not alter MMP expression or chorioamnionitis.
The combination of AMP/DEX/INDO suppressed inflammation and significantly prolonged gestation.
Intra-amniotic infection; Preterm labor; Cytokines; Prostaglandins; Rhesus macaques
Intra-amniotic infection (IAI) is associated with preterm birth and perinatal mortality. To identify potential biomarkers, we performed a comprehensive survey of cervical-vaginal fluid (CVF) proteome from a primate IAI model utilizing Multidimensional protein identification technology (LC/LC-MS/MS) and MALDI-TOF-MS analyses. Analyses of CVF proteome identified 205 unique proteins and differential expression of 27 proteins in controls and IAI samples. Protein expression signatures and immunodetection of specific biomarkers identified can be employed for non-invasive detection of IAI.
The rhesus monkey is an important animal model to study human vaginal health to which lactic acid bacteria play a significant role. However, the vaginal lactic acid bacterial species richness and relative abundance in rhesus monkeys is largely unknown.
Vaginal swab samples were aseptically obtained from 200 reproductive aged female rhesus monkeys. Following Rogosa agar plating, single bacterial colonies representing different morphotypes were isolated and analyzed for whole-cell protein profile, species-specifc PCR, and 16S rRNA gene sequence.
A total of 510 Lactobacillus strains of 17 species and one Pediococcus acidilactici were identified. The most abundant species was L. reuteri, which colonized the vaginas of 86% monkeys. L. johnsonii was the second most abundant species, which colonized 36% of monkeys. The majority of monkeys were colonized by multiple Lactobacillus species.
The vaginas of rhesus monkeys are frequently colonized by multiple Lactobacillus species, dominated by L. reuteri.
Vaginal microbiota; commensal bacteria; 16S rRNA; phylogenetic tree
Michael Gravett and colleagues review the burden of pregnancy-related infections, especially in low- and middle-income countries, and offer suggestions for a more effective intervention strategy.
Intrauterine exposure to amniotic fluid (AF) cytokines is thought to predispose to bronchopulmonary dysplasia (BPD). We evaluated the effects of GBS exposure on RNA expression in fetal lung tissue to determine early molecular pathways associated with fetal lung injury that may progress to BPD.
Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118–125 days gestation (term = 172 days) received choriodecidual inoculation of either: 1) Group B Streptococcus (n = 5) or 2) saline (n = 5). Cesarean section and fetal necropsy was performed in the first week after GBS or saline inoculation regardless of labor. RNA was extracted from fetal lungs and profiled by microarray. Results were analyzed using single gene, Gene Set, and Ingenuity Pathway Analysis. Validation was by RT-PCR and immunohistochemistry.
Despite uterine quiescence in most cases, fetal lung injury occurred in four GBS cases (intra-alveolar neutrophils, interstitial thickening) and one control (peri-mortem hemorrhage). Significant elevations of AF cytokines (TNF-α, IL-8, IL-1β, IL-6) were detected in GBS versus controls (p<0.05). Lung injury was not directly caused by GBS, because GBS was undetectable by culture and PCR in the AF and fetal lungs. A total of 335 genes were differentially expressed greater than 1.5 fold (p<0.05) with GBS exposure associated with a striking upregulation of genes in innate and adaptive immunity and downregulation of pathways for angiogenesis, morphogenesis, and cellular growth and development.
A transient choriodecidual infection may induce fetal lung injury with profound alterations in the genetic program of the fetal lung before signs of preterm labor. Our results provide a window for the first time into early molecular pathways disrupting fetal lung angiogenesis and morphogenesis before preterm labor occurs, which may set the stage for BPD. A strategy to prevent BPD should target the fetus in utero to attenuate alterations in the fetal lung genetic program.
Preterm birth is the most important direct cause of neonatal mortality and remains a major challenge for obstetrics and global health. Intrauterine infection causes approximately 50% of early preterm births. Animal models using pregnant mice, rabbits, or sheep, demonstrate the key link between infection and premature birth, but differ in mechanisms of parturition and placental structure from humans. The nonhuman primate (NHP) is a powerful model which emulates many features of human placentation and parturition. The contributions of the NHP model to preterm birth research are reviewed emphasizing the role of infections, and potential development of preventative and therapeutic strategies.
Early events leading to intrauterine infection and fetal lung injury remain poorly defined, but may hold the key to preventing neonatal and adult chronic lung disease. Our objective was to establish a nonhuman primate model of an early stage of chorioamnionitis in order to determine the time course and mechanisms of fetal lung injury in utero.
Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118–125 days gestation (term = 172 days) received one of two treatments: 1) choriodecidual and intra-amniotic saline (n = 5), or 2) choriodecidual inoculation of Group B Streptococcus (GBS) 1×106 colony forming units (n = 5). Cesarean section was performed regardless of labor 4 days after GBS or 7 days after saline infusion to collect fetal and placental tissues. Only two GBS animals developed early labor with no cervical change in the remaining animals. Despite uterine quiescence in most cases, blinded review found histopathological evidence of fetal lung injury in four GBS animals characterized by intra-alveolar neutrophils and interstitial thickening, which was absent in controls. Significant elevations of cytokines in amniotic fluid (TNF-α, IL-8, IL-1β, IL-6) and fetal plasma (IL-8) were detected in GBS animals and correlated with lung injury (p<0.05). Lung injury was not directly caused by GBS, because GBS was undetectable in amniotic fluid (∼10 samples tested/animal), maternal and fetal blood by culture and polymerase chain reaction. In only two cases was GBS cultured from the inoculation site in low numbers. Chorioamnionitis occurred in two GBS animals with lung injury, but two others with lung injury had normal placental histology.
A transient choriodecidual infection can induce cytokine production, which is associated with fetal lung injury without overt infection of amniotic fluid, chorioamnionitis or preterm labor. Fetal lung injury may, thus, occur silently without symptoms and before the onset of the fetal systemic inflammatory response syndrome.
There have been very few systematic reviews looking at maternal infections in the developing world, even though cutting maternal mortality by three quarters is United Nation’s Millennium Development Goal number five. This systematic review has two aims. The first is to present the prevalence of parasitic infections in the developing world over the last 30 years and the second is to evaluate the quality and distribution of research in this field.
A systematic review of Medline, EMBASE and Global Health databases was undertaken using pre-determined search criteria. Three levels of quality criteria for exclusion of inadequate studies identified 115 out of initial 8580 titles. The data were extracted for 5 domains: worldwide pathogen prevalence, year of study, study setting, sample size and diagnostic test for each pathogen.
The initial search retrieved 8580 results. From these titles, 43 studies on malaria, 12 studies on helminths, 49 studies on Toxoplasma gondii, 7 studies on Chagas disease, 5 studies on Trichomonas, 1 leishmaniasis study and 1 study on trichinellosis were extracted for analysis. High prevalence of malaria was found in Gabon (up to 57%) India (55%), Cameroon (50%), Yemen (55%), Nigeria (up to 64%) and Ghana (54%). High prevalence of hookworm infections was found in Nepal at 78.8% and high values of Ascaris lumbricoides were found in Nepal, (56.2%), Kenya (52.3%) and Gabon (45.5%). High levels of Schistosoma mansoni were found in Zimbabwe (50%) and Tanzania (63.5%). The prevalence of active Toxoplasma gondii infection was found to be highest in India (27.7%).
This study highlights the large burden of maternal parasitic infections globally. It may serve as a useful starting point for health policy development and research prioritization in this area.
More than 500 000 children die each year in low resource settings due to serious neonatal infections. Better diagnostics that can be utilized in these settings to identify infected infants have the potential to significantly reduce neonatal deaths and the associated morbidity. A systematic review was performed and identified more than 250 potential new biomarkers for the diagnosis of serious neonatal infections. Eight of these biomarkers were both high-performance and high-abundance (antithrombin, inter-α inhibitor proteins, interferon-γ inducible protein-10, interleukin-1 receptor antagonist, LPS binding protein, mannose binding lectin, serum amyloid A, resistin, visfatin), and are promising for the diagnosis of serious neonatal infections in low resource settings. Future clinical trials comparing these biomarkers with more traditional biomarkers seem warranted.
Maternal morbidity and mortality in low- and middle-income countries has remained exceedingly high. However, information on bacterial and viral maternal infections, which are important contributors to poor pregnancy outcomes, is sparse and poorly characterised. This review aims to describe the epidemiology and aetiology of bacterial and viral maternal infections in low- and middle-income countries.
A systematic search of published literature was conducted and data on aetiology and epidemiology of maternal infections was extracted from relevant studies for analysis. Searches were conducted in parallel by two reviewers (using OVID) in the following databases: Medline (1950 to 2010), EMBASE (1980 to 2010) and Global Health (1973 to 2010).
Data from 158 relevant studies was used to characterise the epidemiology of the 10 most extensively reported maternal infections with the following median prevalence rates: Treponema pallidum (2.6%), Neisseria gonorrhoeae (1.5%), Chlamydia trachomatis (5.8%), Group B Streptococcus (8.6%), bacterial vaginosis (20.9%), hepatitis B virus (4.3%), hepatitis C virus (1.4%), Cytomegalovirus (95.7% past infection), Rubella (8.9% susceptible) and Herpes simplex (20.7%). Large variations in the prevalence of these infections between countries and regions were noted.
This review confirms the suspected high prevalence of maternal bacterial and viral infections and identifies particular diseases and regions requiring urgent attention in public health policy planning, setting research priorities and donor funding towards reducing maternal morbidity and mortality in low- and middle-income countries.
Microbial-specific factors are likely critical in determining whether bacteria trigger preterm labor. Structural variations in lipopolysaccharide (LPS), a component of gram-negative bacteria, can determine whether LPS has an inflammatory (agonist) or anti-inflammatory (antagonist) effect through Toll-like receptor 4 (TLR4). Our objective was to determine whether amniochorion can discriminate between LPS variants in a nonhuman primate model. We also cloned Macaca nemestrina TLR4 and MD-2 and compared this complex functionally to the human homologue to establish whether nonhuman primates could be used to study TLR4 signaling in preterm birth.
Amniochorion explants from M. nemestrina were stimulated with a panel of LPS variants for 24 hours. Supernatants were analyzed for IL-1β, TNF-α, IL-6, IL-8 and prostaglandins E2 and F2α. Tissue expression of TLR1, 2, 4, 6, MyD88 and NF-kB was studied by RT-PCR. M. nemestrina TLR4 and MD2 genes were cloned and compared with their human counterparts in a recombinant TLR4 signaling system to determine LPS sensitivity.
LPS variants differentially stimulated cytokines and prostaglandins, which was not related to transcriptional changes of TLR4 or other TLRs. Nearly all elements of LPS binding and TLR4 leucine-rich repeats were conserved between humans and M. nemestrina. TLR4/MD-2 signaling complexes from both species were equally sensitive to LPS variants.
LPS variants elicit a hierarchical inflammatory response within amniochorion that may contribute to preterm birth. LPS sensitivity is similar between M. nemestrina and humans, validating M. nemestrina as an appropriate model to study TLR4 signaling in preterm birth.
amniochorion; LPS; TLR4; TLR4 antagonist; Macaca nemestrina; nonhuman primate
Objective. An outbreak of 20 peripartum Clostridium difficile infections (CDI) occurred on the obstetrical service at the University of Washington Medical Center (UWMC) between April 2006 and June 2007. In this report, we characterize the clinical manifestations, describe interventions that appeared to reduce CDI, and determine potential risk factors for peripartum CDI. Methods. An investigation was initiated after the first three peripartum CDI cases. Based on the findings, enhanced infection control measures and a modified antibiotic regimen were implemented. We conducted a case-control study of peripartum cases and unmatched controls. Results. During the outbreak, there was an overall incidence of 7.5 CDI cases per 1000 deliveries. Peripartum CDI infection compared to controls was significantly associated with cesarean delivery (70% versus 34%; P = 0.03
), antibiotic use (95% versus 56%; P = 0.001), chorioamnionitis (35% versus 5%; P = 0.001), and the use of the combination of ampicillin, gentamicin, and clindamycin (50% versus 3%; P < 0.001
). Use of combination antibiotics remained a significant independent risk factor for CDI in the multivariate analysis. Conclusions. The outbreak was reduced after the implementation of multiple infection control measures and modification of antibiotic use. However, sporadic CDI continued for 8 months after these measures slowed the outbreak. Peripartum women appear to be another population susceptible to CDI.
Intra-uterine infection, which occurs in the majority of early preterm births, triggers an immune response culminating in preterm labor. We hypothesized that blockade of lipopolysaccharide (LPS)-induced immune responses by a Toll-like receptor 4 antagonist (TLR4A) would prevent elevations in amniotic fluid (AF) cytokines, prostaglandins, and uterine contractility. Chronically catheterized rhesus monkeys at 128-147 days gestation received intra-amniotic infusions of either: 1) saline (n=6), 2) LPS (0.15-10μg; n=4), or 3) TLR4A pre-treatment with LPS (10 μg) one hour later (n=4). AF cytokines, prostaglandins, and uterine contractility were compared using oneway ANOVA with Bonferroni-adjusted pairwise comparisons. Compared to saline controls, LPS induced significant elevations in AF IL-8, TNF-α, PGE2, PGF2α, and uterine contractility (p<0.05). In contrast, TLR4A pre-treatment inhibited LPS-induced uterine activity and was associated with significantly lower AF IL-8, TNF-α, PGE2, and PGF2α versus LPS alone (p<0.05). Toll-like receptor antagonists, together with antibiotics, may delay or prevent infection-associated preterm birth.
TLR4; intrauterine infection/inflammation; preterm labor; rhesus monkey; animal model
Preterm birth remains the most common cause of perinatal mortality. Although the causes of preterm labor are multifactorial and vary according to gestational age, preterm labor and term labor share common cellular and molecular mechanisms, including stimulation of the fetal hypothalamic-pituitary-adrenal (HPA) axis and endocrine/immune system interactions. We have developed a non-human primate experimental model for intrauterine infection and preterm labor using chronically instrumented rhesus monkeys (Macaca mulatta) with timed gestations. We have documented the temporal and quantitative relationships among intrauterine infection, the synthesis and release of proinflammatory cytokines, prostaglandins, and fetal-placental steroid biosynthesis in this model. Infection-induced preterm parturition is characterized by significant elevations in amniotic fluid proinflammatory cytokines and by increases in fetal adrenal steroid biosynthesis, but not by corresponding increases in placental estrogen biosynthesis characteristic of spontaneous parturition. This suggests that activation of the fetal HPA axis by the stress of infection is accompanied by placental dysfunction and also that infection-induced preterm parturition is not dependent upon the increased estrogen biosynthesis observed in spontaneous parturition. These different endocrine and immune responses have important diagnostic and therapeutic implications in the management of preterm labor.