Specific bacteria in cervix of a pregnant woman may lead to preterm birth


https://speciality.medicaldialogues.in/specific-bacteria-in-cervix-of-a-pregnant-woman-may-lead-to-preterm-birth/

Bacterial membrane vesicles can cause preterm birth


Bacterial membrane vesicles can cause preterm birth
As a novel mechanism contributing to streptococcal pathogenesis, Surve et al. identified membrane bound vesicles (MVs) produced by Streptococcus agalactiae. The scanning electron micrograph depicts MVs (red hue) dispersed around S. agalactiae cells (gray). See Anirban Banerjee and colleagues. 

Approximately 20-30% of women carry bacteria called group B streptococcus (GBS) in their vagina or rectum. In most cases, these bacteria cause no problems, but GBS has been linked to complications during pregnancy, including pre-term delivery.

A study published on September 1st in PLOS Pathogens reports that GBS produces membrane-bound vesicles containing bacterial factors that can attack the host tissue. In mice, the study shows, these vesicles can move from the vagina to the uterus and cause inflammation of the membranes surrounding the fetus. When injected directly into the amniotic cavity of mice, these vesicles can induce preterm and still births.

Membrane-bound vesicles (MVs) loaded with toxins, immune-modulators, and other bacterial factors, contribute to the survival and virulence (the ability to cause disease) of many pathogenic bacteria. Whether GBS produces MVs was not known. However, because in pregnant women who carry GBS and deliver prematurely, bacterial infection is rarely found in the womb, Anirban Banarjee, from the Indian Institute of Technology Bombay, and colleagues hypothesized that if GBS produces MVs, they might move up to the womb during pregnancy and cause tissue damage at interface between mother and fetus.

To test this, the researchers started by growing GBS in liquid media. When they removed the bacteria and examined the remaining liquid by electron microscopy, they found numerous spherical structures. Zooming in on the surface of growing bacteria, they detected vesicles that were just budding off the bacterial cell, confirming that GBS produces MVs. They next examined the protein content of the MVs and identified 8 bacterial proteins, all with predicted properties of virulence factors that can attack the human host and cause disease.

When the researchers mixed MVs and cells of human origin, they found that the MVs can invade and kill these cells, suggesting that GBS MVs are toxic to the human host. The researchers then deposited the MVs without the bacteria into mouse vagina and hours later found them throughout the uterus and in the developing fetus, indicating that MVs can indeed travel up the birth canal. Adding MVs to mouse chorio-decidual membrane (which is found at the interface between mother and fetus) caused collagen degradation, reducing the elasticity and weakening the mechanical properties of the membrane.

When the researchers injected MVs directly into the amniotic sac (the fluid-filled cavity surrounding the embryo) of pregnant mice, they observed that 24 hours later the tissue of the interface between mother and fetus was severely disrupted, with broken collagen fibers, hallmarks of inflammation, and signs of extensive cell death. To test whether these changes could lead to pre-term birth, the researchers carefully monitored females whose amniotic sacs had been injected with MVs at day 14.5 of pregnancy (a full-term mouse pregnancy lasts 19 days). Approximately 60% of the fetuses were born prematurely (by day 18 of pregnancy), compared with only 10% of pups following control injection with saline. Along with , the researchers observed an increased frequency of fetal death in utero, and that the pups born to MV injected mothers were too small and some had abnormal morphology. Collectively, these results suggest that GBS MVs can cause preterm birth and fetal injury.

Discussing their results, the researchers emphasize the finding that MVs alone could induce features resembling clinical chorio-amnionitis in the mice. “Clinically”, they say, “this observation is highly relevant as 50-80% women with chorio-amnionitis do not have bacteria in their amniotic fluid or the decidual tissue”. Based on their study, they hypothesize that “MVs secreted by the pathogens residing in lower genital tract may be responsible for cases with unexplained chorio-amnionitis”.

Acknowledging the gap between experimental results in mice following direct injection of MVs into the amnion sac and human pathogenesis, the researchers nevertheless suggest that their findings “provide a novel insight into how GBS while simply sitting in the vagina can orchestrate events at the fetal membrane leading to premature birth”. Because MVS are not susceptible to antibiotics, the researchers speculate that instead “new drugs that prevent vesicle production may […] be a viable therapeutic option to prevent GBS mediated preterm birth”.

 

Abstract

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.

Discussion

Membrane vesicles are bilayered structures, found to be secreted almost ubiquitously by gram negative bacteria and a large number of gram positive bacteria [24,25,28,29,3134]. In the current study, we demonstrate for the first time that GBS (a gram positive bacteria) is capable of secreting MVs in a strain independent manner. Employing different microscopic techniques we could not only detect the presence of vesicles in GBS culture supernatant, but also demonstrate the budding of vesicular structures from GBS surface. Interestingly, similar budding structures from GBS surface resembling MVs have been reported in GBS colonized in the murine genital tract, implying that GBS could also produce MVs in vivo [35]. We isolated and characterized these MVs both physically and biochemically and examined their pattern of interaction with host cells in order to explore their potential contribution in GBS pathogenesis. Intriguingly, we could extract DNA and amplify the cfb gene, an important virulence factor of GBS, using the MV DNA as template. Occasionally, DNA has been found to be associated with MVs from other microorganisms such as P. aeruginosa [36], S. vesicuolsa [37] and C.perfringens [26]. Though DNA is detected in GBS MVs, we believe this DNA is fragmented as we could only detect the cfb gene, but genes representing other virulence factors (cylE, pepB,zooA) or housekeeping gene (gapN) could not be amplified. This implies that the DNA packaged in MVs is perhaps not a random phenomenon but may involve a regulated mechanism. While the functional significance of the DNA packaging in MVs is yet unknown, since the MVs fuse with the host or other bacterial cells, these might act as carriers of DNA from one cell to another [38]. It will be of interest to determine if the MV DNA specifically enters the cells to induce a pathological activation of host nuclease machinery.

Proteomic analysis of the GBS MV proteins revealed presence of numerous ECM degrading enzymes. The preferential packaging of ECM degrading enzymes compared to other abundant membrane proteins implies presence of a selective sorting mechanism. Such a sorting mechanism have been identified in P. gingivalis which not only facilitates preferential packaging of important virulence factors but also enables it to exclude other abundant outer membrane proteins from the cargo [39]. Similar enrichment of acidic glycosidases and proteases were observed in Bacteroides species suggesting presence of a species specific machinery devoted to selectively pack proteins into the vesicles to do specific jobs, in this case securing nutrients for the benefit of the whole bacterial community present in the microbiota [40].

The presence of various ECM degrading enzymes in GBS MVs therefore points towards its significant role in GBS pathogenesis. Indeed, we observed that the MVs could not only interact with HeLa cells extracellularly but also internalize and cause cell death. These observations prompted us to hypothesize that the presence of various virulence factors and ECM degrading proteins in GBS MVs might lead to tissue degradation and cell death that may contribute to PPROM and preterm births. We next performed a series of experiments to investigate this hypothesis.

The fetal membranes (amnion and chorion) rest upon a collagenous basement membrane of type II and IV collagen and beneath this layer lays a fibrous layer that contains collagen types I, III, V, and VI. Collagen, therefore provides major structural strength for the membranes and degradation of it leads to loss of membrane integrity [4143]. The enrichment of ECM degrading proteases, coupled with the presence of gelatinolytic activity in GBS MVs suggested that these might cause loss of fetal membrane integrity by ECM degradation. Indeed, ex vivo treatment of chorio-decidual membrane with GBS MVs led to collagen fragmentation. This collagen fragmentation had profound effect on fetal membrane integrity as the stiffness of the membranes challenged with MVs were significantly reduced. It is likely that during pregnancy, GBS via its MVs lead to collagen degradation and reduction in tissue stiffness which together would resist further expansion of the amniotic sac, a prerequisite to accommodate the growing fetus. Thus our findings suggest that GBS MVs lead to loss of ECM and weaken the amniotic membrane making it susceptible to rupture upon pressure from the growing fetus.

Epidemiological data suggest that colonization of vagina and cervix with GBS increases the probability of chorio-amnionitis. We suspected that the MVs produced by GBS while colonizing can move upwards in the female reproductive tract and affect cells and tissue at distant sites. Budding structures resembling MVs have been observed in GBS colonized in the murine genital tract [35]. Extending this data, herein we show that GBS MVs when vaginally instilled could traverse anterogradely upto anterior most segment of the uterus. Recently, it has been shown that GBS mutant for hyaluronidase is not capable of ascending infection in a mouse model [44]. Since, hyaluronidase is also enriched in our GBS MV preparations; it is possible that it could aid ascend of MVs into the female tract. While it needs to be demonstrated that like GBS, MVs can also anterogradely transport into the gravid uterus; it is tempting to hypothesize that GBS colonization at a distant site such as vagina can affect the feto-maternal tissues in the uterus by the virtue of secreting MVs.

We next asked if GBS MVs have any pathogenic effects in vivo. Corroborating our ex-vivoobservations; we observed that instillation of GBS MVs intra-amniotically led to extensive disruption of the chorio-decidual structure. This disruption is also associated with collagen fragmentation which could be due to the proteases present in the MVs. Beyond collagen, extensive alterations in the expression of genes encoding for extracellular matrix proteins has been reported in chorio-decidua of rhesus monkeys infected with GBS [16]. While it will be of interest to see the alterations in other ECM molecules in response to MVs, we believe that the changes in extracellular matrix and collagen degradation would contribute to the mechanical weakening of the tissue resulting in the loss of membrane stiffness. Indeed we did see a reduction on stiffness of the chorio-decidua tissue challenged with MVs ex vivo. It will be of interest to test the effects of MVs derived from GBS mutants for various ECM degrading enzymes and determine the mechanistic basis of this phenomenon. Along with loss of collagen, we also detected extensive apoptosis in the chorio-decidua derived from sacs injected with MVs. These observations suggested that coupled with mechanical weakening, cell death might further contribute to loosening of the membrane. Such tissue damage by MVs could make the membrane prone to rupture leading to PPROM.

A leading feature of PPROM and preterm births due to inflammation is chorio-amnionitis. Chorio-amnionitis is acute inflammation of the fetal membrane and chorion which is typically caused due to ascending microbial infection especially in case of infection with genital mycoplasmas [45]. Histologically, chorio-amnionitis is characterized by leukocyte infiltration in the chorion/amnion and the decidua. In the context of GBS, in vivo administration of the live bacteria in pregnant mice led to histopathologic characteristics resembling chorio-amnionitis [16]. Herein for the first time we demonstrated that chorio-amnionitis can be caused by MVs even in absence of live bacteria. We observed that intra-amniotic administration of GBS MVs caused extensive leukocytic infiltration in the subchorion layer and in decidua resembling chorio-amnionitis. Beyond leukocytic infiltration, the decidua of mice injected with GBS MVs also had macrophage infiltration. Moreover, these vesicles also induced inflammatory cytokines such as, Kc, Il-1β, Il-6, and Tnf-α in the decidua. Levels of these pro-inflammatory cytokines are also known to be elevated in the amniotic fluid of patients in independent contexts of GBS infections and PPROM [16,46]. Since HeLa cells also showed similar response upon treatment with MVs (S3B Fig), we believe that the elevation in levels of these cytokines in the decidua may not be exclusively due to higher numbers of immune cells present in these tissues, but because of the ability of MVs to generate an inflammatory response in the decidual cells. We therefore conclude that GBS by the virtue of production of MVs activates the host immune system which might trigger the immune cell homing and activation leading to chorio-amnionitis. These observations are novel as for the first time we have shown that even in absence of active infection in the chorio-decidua, features resembling clinical chorio-amnionitis could be mimicked by MVs. Clinically this observation is highly relevant as 50–80% women with chorio-amnionitis do not have bacteria in their amniotic fluid or the decidual tissue [4750]. Based on our findings we can hypothesize that MVs secreted by the pathogens residing in lower genital tract may be responsible for cases with unexplained chorio-amnionitis. It would be imperative to study the presence of MVs in amniotic fluid and chorio-decidual tissues of woman with culture negative chorio-amnionitis.

Since, GBS MVs lead to tissue damage, inflammation and chorio-amnionitis, resulting in mechanical weakening of the fetal membrane, we finally asked if GBS MVs can lead to premature birth. Indeed, we observed that intra-amniotic injection of GBS MVs lead to preterm delivery. More than 50% of mouse pups were delivered preterm (almost 2 days prior to their expected day of delivery) when challenged with MVs. Finally, we also observed that like live GBS [17], the MVs are also highly pathogenic to the fetus. Almost 30–40% of fetuses died in utero amounting to resorption and some fetuses were still born. The fetuses that were stillborn or recovered from uterine sacs after GBS MV challenge, were smaller and had major damage to its organs. These results together imply that GBS MVs can cause IUFD or preterm births. To our knowledge this is the first report describing the direct role of membrane vesicles produced by any pathogenic bacteria in disease pathogenesis. In the light of the fact that intrauterine infections can lead to autism like changes in the brain [51], it is possible that GBS MVs might have a similar effect. It will be of interest to study the effects of sub-lethal dose of GBS MVs on fetal development and physiology.

In summary, the results of the present study have shown that GBS MVs cause host cell death, membrane weakening and inflammation of the feto-maternal interface which causes preterm birth and IUFD. Coupling this with the fact that pathogen derived vesicles can function as vehicles for long distance delivery of virulence factors [52], our results imply that the production of extracellular membrane vesicles serves not only as a tool for secretion but also arms GBS with an additional weapon by which while colonizing it can orchestrate events at distant sites including the fetal membrane. Our findings provide a plausible explanation for the occurrence of PPROM and premature delivery in woman with chorio-amnionitis without detectable bacteria in their amniotic fluid or the decidual tissue. We conclude that GBS utilizes MVs as a surrogate to spread its virulence factors in the host which are responsible for the clinical features of GBS infection during pregnancy. Prevention of vesicle biogenesis may therefore be a viable therapeutic option to prevent GBS mediated preterm birth.

Materials and Methods

Ethics statement

All the experimental work on animals was done as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The study protocol has been reviewed and approved by the Institutional Animal Ethics Committee of National Institute for Research in Reproductive Health (NIRRH) under the project number 10/15.

Bacterial strains and isolation of MVs

S. agalactiae strains A909 (serotype IA), COH1 (serotype III), NEM316 (serotype III) and 2603V/R (serotype V) (kindly provided by Dr. Lakshmi Rajagopal, Department of Pediatric Infectious Diseases, University of Washington School of Medicine, Univ. of Washington, Seattle, USA) were cultured in Todd Hewitt broth (THB) at 37C. Unless otherwise stated MVs from GBS strain A909 was used for further experiments. MVs were purified from growing GBS cultures as per the standard protocol used for many other bacteria [25,27,32,53,54]. Briefly, GBS cells grown upto optical density (OD600nm) 1.2, were harvested by centrifugation (12,000 x g, 30 min, 4C) and the supernatant was passed through 0.22μm filter. The filtrate was concentrated using Amicon ultrafiltration system (10 kDa) and ultracentrifuged (150000 x g, 3 h, 4C) to pellet down MVs. The MV pellet was resuspended in PBS (pH 7.2) and protein content was determined by Bradford assay following lysis of MVs by 0.05% Triton X-100.

To quantitate the number of MVs produced from GBS culture, MVs were labeled with 20 μM of Vybrant DiO cell labeling solution (Molecular Probes) and quantified by flow cytometry (FACS Aria II, BD Biosciences, USA) using fluorescent counting beads (CountBright Absolute counting beads; Invitrogen) as standards as described earlier [25]. The varying diameters and size distribution of MV preparations were measured using a Goniometer (Brookhaven Instrument Co., USA) as described earlier [55].

Electron microscopy and atomic force microscopy for GBS MVs

For Scanning Electron Microscopy (SEM), GBS cells were air dried, desiccated overnight and visualized with Field Emission Gun Scanning Electron Microscope (JEOL, USA) at an accelerating voltage of 5 kV. For Transmission Electron Microscopy (TEM), MV samples were applied to Formvar/Carbon film coated 200-mesh copper grids (Pacific Grid-Tech) and negatively stained with 2.5% uranyl acetate followed by visualization under Transmission Electron Microscope (FEI Technai, USA) (120 kV). For Atomic Force Microscope (AFM), bacterial suspension was loaded onto poly-L-lysine coated coverslips and visualized under an atomic force microscope (Asylum Research, USA) under contact mode at a scanning rate of 1 Hz using silicon nitride cantilevers.

Fatty acid analysis

Lipids were extracted from intact GBS cells and MVs using protocol described earlier [25]. Following extraction, lipids were silylated using 1:1 ratio of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine at 75C for 30 min. Silylated lipids were then analyzed using a GC-MS (Agilent, USA) fitted with a HP-5MS fused silica capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) with Helium as carrier gas (flow rate 1ml/min). Identification of compounds was based on mass spectra including comparison to a library (NIST).

Protein analysis

150 μg of MV proteins were separated on 12% SDS-PAGE. After staining with Coomassie Brilliant Blue R-250 (CBB), bands were excised and processed for in-gel trypsin digestion [56]. The eluted oligopeptides were co-crystallized with CHCA (5 mg/ml) and spectra were acquired using MALDI-ToF mass spectrometer (UltraFlex III, Bruker Daltonics). Mascot (Version 2.2.04, Matrix Science) searches were conducted using the NCBI non-redundant database with the following settings: 1 missed cleavage; Carbamidomethyl on cysteine as fixed modifications, methionine oxidation as variable modification and 100 ppm error (150 ppm error for band No. 4). A match with S. agalactiae protein with the best score in each Mascot search was accepted as successful identification (p< 0.05). Subcellular locations were predicted by LocateP database and confirmed by pSORT and TMHMM algorithms.

Isolation and analysis of nucleic acid from MVs

MVs were initially treated with 50 μg/ml DNaseI in presence of 10 mM MgCl2 at 37°C for 1 h to remove any surface bound DNA. Following heat inactivation (10 min at 80°C) and lysis of MVs using Triton X-100 (0.05%) at 37⁰C for 30 min, DNA was extracted by phenol-chloroform-isoamyl alcohol, precipitated using ammonium acetate as described earlier [26]. The purified DNA was quantified and used for PCR using primers specific for cylE, cfb, pepB, zooA andgapN genes (S1 Table). Genomic DNA isolated from GBS strain A909 served as control template.

Cell culture, internalization of MVs and cytotoxicity

HeLa cells (procured from National Center for Cell Science, Pune, India) were grown in DMEM media (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) at 37C in 5% CO2. HeLa cells were incubated with increasing protein concentrations of MVs (10 μg/ml to 300 μg/ml) for 24 h and the viability was assessed using MTT assay kit (HiMedia, India).

To study internalization, MVs were labeled with FITC (Sigma) in 0.1 M sodium carbonate buffer (pH 9.0) for 1 h at room temperature and washed to remove unbound FITC [57]. FITC labeled MVs (30 μg MV protein) were then allowed to interact with HeLa cells for 6 h. After washing to remove unbound MVs, cells were fixed and stained with mouse polyclonal anti-FITC antibody (Invitrogen) followed by an anti-mouse secondary antibody conjugated to AlexaFluor 555 (Invitrogen). Images were acquired with an oil immersion Plan-Apochromat 63X/1.4 NA objective using a confocal laser scanning microscope (Zeiss).

Gelatin zymography

10 μg of MV proteins were separated (150 V, 5 h, 4°C) on 12% SDS-PAGE with 0.1% co-polymerized gelatin. After treatment in 1% Triton X-100, gelatinolysis was promoted by further incubation for 16 h at 37°C in developing buffer (50 mM Tris-HCl, 50 mM Tris base, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij 35, pH 7.6). The gel was then stained with Coomassie Brilliant Blue R-250 and destained to intensify the digestion halos.

Preparation of liposome

BSA entrapped liposomes were prepared by thin film hydration method [58]. Briefly, phosphatidylcholine (10 mg) was dissolved in a mixture of chloroform and methanol (2:1) and the solvent was evaporated under vacuum using a rotary evaporator to form a thin layer of lipid film in a round bottom flask. The dried lipid film was hydrated using 1 ml of PBS containing BSA (10 mg/ml) at 45°C for 1 h to form multilamellar vesicles (MLV). The liposome suspension was then sonicated using a probe sonicator at 40% amplitude to obtain unilamellar vesicles.

Atomic force microscopy for tissues

Chorio-decidual membrane was collected from pregnant mice on E14.5. Immediately after dissection, the membranes were incubated in DMEM (Invitrogen) containing 10% FBS (Invitrogen) at 37°C and treated for 24 h with either PBS, or BSA entrapped liposomes (100 μg/ml) or MVs (100 μg/ml) in presence or absence of protease inhibitor cocktail (Sigma). Collagenase (10 μg/ml) was used as positive control. Following incubation, the membranes were washed, carefully spread on a slide layered with double adhesive tape, allowed to air dry for 5 min. Subsequently, the membranes were hydrated with PBS, and their mechanical properties were probed with an Atomic Force Microscope (AFM). A 5 μm diameter spherical probe with a nominal spring constant of 32.66 pN/nm was used. Using a custom-written code, force-indentation curves were fitted with Hertz model to estimate the Young’s modulus of elasticity of each of the membranes. Each sample was probed randomly multiple number of times (greater than 50) at multiple different positions to estimate average stiffness of the membranes.

Anterograde transport of MVs

The vagina of mice in estrus phase were flushed thrice with 40 μl of 0.2% Triton X-100 in 0.9% saline followed by 40 μl 0.9% saline. MVs were labeled with FITC as described earlier [57] and 100 μg of FITC labeled GBS MVs in 100 μl PBS were vaginally instilled in 25–30 μl aliquots using a micropipette. Control mice received PBS only. After 6 h mice were euthanized and the reproductive tract (cervix to uterus) was collected. Tissues were briefly fixed in 4% paraformaldehyde and mounted on slides with Vectashield containing DAPI (Vector Laboratories). Images of different parts of the tissue such as utero-cervical junction, distal uterus and proximal uterus were captured with an oil immersion Plan-Apochromat 40X/1.3 NA objective of a confocal laser scanning microscope.

Intra-amniotic administration of MVs

For pregnancy-outcome experiments, C57BL6/J- were bred and maintained at the Experimental Animal Facility of NIRRH under constant temperature and 12 h light and dark cycles were used. Female mice in estrus were impregnated naturally by a male of proven fertility and mating was confirmed by the presence of a vaginal plug (E0.5). Intra-amniotic injections were performed on E14.5 of a 19–20 d gestation. Briefly, animals were anesthetized and a 1.5-cm midline incision was made in the lower abdomen. The mouse uterus is a bicornuate where the fetuses are arranged in a “beads-on-a-string” pattern. Both the horns were exposed and individual fetal sacs were injected with 100 μl of MVs (5 or 10 μg protein) or equivalent amount of PBS or BSA-liposomes. After injection the uterus was returned to the abdomen, muscle and skin layers were sutured and dams were returned to their cages and monitored on regular intervals. Surgical procedures lasted ∼10 min and post-operative care was taken as per standard protocols at NIRRH. Delivery of one or more pups in the cage or lower vagina within 48 h was considered preterm.

For collection of tissues, animals were euthanized 24 h after surgery. The inoculated horns were incised longitudinally along the anti-mesenteric border. Gestational tissues (full-thickness biopsies from the middle region) and fetal membranes were harvested and frozen in Trizol reagent (Invitrogen) and stored at −80°C for RNA extraction or fixed in 4% paraformaldehyde for histopathology. Fixed tissues were paraffin embedded and 5 μm thick paraffin sections were collected on poly-lysine coated glass slides and processed for routine hematoxylin and eosin staining.

RNA extraction, cDNA synthesis and qRT-PCR

RNA from HeLa cells or mouse tissue was isolated using Trizol reagent as detailed previously [59]. RNA was treated for 30 min with DNaseI to remove any DNA contamination and processed for reverse transcription. The details of qRT-PCR have been described previously [59]. Briefly, 1 μg of RNA was reverse-transcribed using SuperScriptTM First-Strand Synthesis System (Invitrogen). The cDNA was further used for quantitative RT-PCR (qRT-PCR) for various cytokines. β-actin (for HeLa cells) and 18S (for mouse tissues) were used as the house keeping genes. The sequences of the primers are as mentioned in S1 Table. Care was taken to design primers that spanned an intron to eliminate any amplification due to genomic DNA contamination. The specificity of amplicons was confirmed by performing dissociation / melt curve analysis. Only those primer pairs that resulted in a single sharp melt peak with a consistent melt temperature were included in the study. The relative changes in the expression of above genes was analyzed by 2-ΔΔCt method [60].

Quantification of leukocytes

Hematoxylin and Eosin stained slides of the chorio-decidua were examined under 40X objective for neutrophils and lymphocytes. The cells were identified based on their morphology. The number of neutrophils and lymphocytes per field were counted in 8–10 random fields per section. Five random sections from each fetus were analyzed. The analysis was done in three biological replicates.

Immunohistochemistry

Immunohistochemistry on tissue sections was performed as detailed previously [61]. For detection of macrophages, paraffin embedded 5 μm sections of the chorio-decidual membrane (with or without MV treatment) were stained with rabbit polyclonal anti-F4/80 Ab (1:100; Santa Cruz Biotechnology) and detected using HRP conjugated goat anti-rabbit secondary antibody (1:100; Dako) and 3.3’ Diaminobenzidine with H2O2. Sections were counterstained with hematoxylin and mounted in DPX.

Collagen staining

Total collagen staining in chorio-decidual membrane sections were performed using Picrosirius red [62]. Paraffin sections were deparaffinized, rehydrated in graded methanol series and stained with hematoxylin followed by 0.5% Direct Red 80 (Sigma) prepared in picric acid. Following dehydration with 100% methanol and clearing in xylene, the slides were mounted with DPX.

TUNEL

Induction of apoptosis by GBS MVs was analyzed by In Situ Cell Death Determination Kit (Roche) according to manufacturer’s protocol. Briefly, following deparaffinization, tissue sections were rehydrated and digested using proteinase K. After permeabilization with 0.25% Triton X 100, the sections were incubated with terminal deoxynucleotidyl transferase and fluorescein-dUTPs for 1 h at 37°C. Following washing to remove unbound dUTPs, the sections were mounted with Vectashield containing DAPI (Vector Laboratories). Images were acquired with an oil immersion Plan-Apochromat 40X/1.3 NA objective of confocal laser scanning microscope (Zeiss).

 

Mom’s Bacteria During Pregnancy Linked with Preterm Birth


The bacteria in a pregnant woman’s body may provide clues to her risk of going into labor early, according to a new study.

Researchers found that the pregnant women in the study with lower levels of bacteria calledLactobacillus in the vagina had an increased risk of preterm labor, compared with women whose vaginal bacterial communities were rich in this type of bacteria.

Moreover, among the women with lower levels of Lactobacillus, a higher abundance of two other bacterial species — Gardnerella and Ureaplasma — was tied to an even more pronounced risk of preterm labor, the investigators found.

 Pregnant Woman

If the results are confirmed in other populations of women, then measuring vaginal bacteria could become a useful way to determine who is at risk of preterm labor, said study author Dr. David Relman, a professor at Stanford University in California. “The first possible application is to use high-risk features in the vaginal microbiome as a marker, as a diagnostic tool to identify women early in pregnancy who are destined to have a higher risk of preterm labor,” Relman told Live Science.

Births that occur before 37 weeks of pregnancy are considered preterm. About 11 percent of pregnancies worldwide involve preterm labor, the researchers said.

It is not clear whether the differences in vaginal bacteria contribute in any way to causing a woman to go into labor early, the researchers said. It is also possible, for example, that other factors that cause premature birth also cause changes in the vaginal bacterial community, Relman said.

In the study, the researchers examined bacterial communities weekly in 49 women, ages 18 and older, while the women were pregnant and after they gave birth. The researchers examined bacteria from the women’s vaginas, gums, stool and saliva. Of all the women in the study, 15 delivered preterm babies.

The investigators also found that the composition of vaginal bacteria in most women in the study changed drastically right at the time of delivery. Specifically, the levels of Lactobacillus decreased after delivery and were replaced by a diverse array of bacteria. This change persisted for up to one year later, according to the study.

When the researchers compared bacterial samples from the vagina to samples from the other body sites, they found that the vaginal bacterial communities became more similar shortly after delivery to the bacterial communities in the gut.

It is not clear how exactly this shift in bacteria occurs, or how it may affect a woman’s health, the researchers said. However, it is possible that an altered community of vaginal bacteria after delivery might affect a woman’s risk of preterm birth in a subsequent pregnancy, if another conception occurs soon after delivery. However, more research is needed to examine whether this is the case, the researchers said.

MRI May Predict Preterm Birth


Using MR imaging to evaluate apparent diffusion coefficient (ADC) of the cervix among pregnant women may help detect impending preterm birth, according to a study published in Radiology.
Researchers from Italy and Switzerland performed a prospective hypothesis-generating study to determine the utility of using MRI to assess ADC among asymptomatic patients with a sonographic cervical length of 15 mm or less and positive fetal fibronectin test results between 23 and 28 weeks of gestation.

Thirty pregnant women (mean gestational age, 26 weeks) were included in the study. The women had a parity of 0 or 1, with no history of preterm birth, cervical procedures, or congenital abnormalities of the uterus. All underwent pelvic 1.5-T MR imaging the same day as undergoing vaginal ultrasound. Oblique sagittal diffusion-weighted images were obtained with b values of 0, 400, and 800 sec/mm2. ADC values at MR imaging of the subglandular and stromal cervix and the difference between both were correlated to the interval to delivery. The images were reviewed by three radiologists.

The results showed that eight (27%) of the 30 patients delivered within six or seven days of MR imaging (impending delivery group), and 22 (73%) of 30 patients delivered between 18 and 89 days after imaging, a mean of 55 days (late delivery group). The women in the impending delivery group had higher mean subglandular ADC and mean ADC difference than those with late delivery:

Impending delivery Late delivery
Mean subglandular ADC (2,406.3 ± 166.0) × 10−6 mm2/sec (1,708.9 ± 108.1) × 10−6 mm2/sec
Mean ADC (657.3 ± 129.9) × 10−6 mm2/sec (69.2 ± 70.2) × 10−6 mm2/sec

The receiver operating characteristic curve analysis of subglandular ADC revealed 100% sensitivity and specificity in association with impending delivery with a 1921 × 10−6 mm2/sec threshold. The radiologists did not find significant differences between the two groups in cervical length or incidence of US visualization of the cervical glandular with ultrasound examination.
Figure 1. Image in a 29-year-old woman at 28 weeks of gestation with a sonographic cervical length of 6 mm who was referred for evaluation of suspected corpus callosum dysgenesis. Oblique sagittal T2 half-Fourier acquisition single-shot fast spin-echo MR image shows a short cervix (white line) with funneling.

Smoking ban ‘cuts premature births’.


The theory that public smoking bans cut the number of children born prematurely has been strengthened by new research.

Baby exposed to cigarette smoke

The study of 600,000 births found three successive drops in babies born before 37 weeks – each occurring after a phase of a public smoking ban was introduced.

There was no such trend in the period before the bans were put in place, the British Medical Journal reported.

The study, by Hasselt University in Belgium, comes after Scottish research in 2012 found a similar pattern.

But experts could not fully state the smoking ban was the cause of the change because pre-term births had started to drop before the ban.

It is already well established that smoking leads to reduced birth weight and an increased risk of premature birth.

Successive drops

In the latest study researchers were able to look at the rate of premature births after each phase of a smoking ban came into force in Belgium.

Public places and most workplaces were first to introduce smoke-free rules in 2006, followed by restaurants in 2007 and bars serving food in 2010.

The rate of premature births was found to fall after each phase of the ban with the biggest impact seen after the second two bans with restaurants and bars introducing no smoking rules.

After the bans in 2007 and 2010, the premature birth rate dropped by around 3% each time.

Overall it corresponds to a fall of six premature babies in every 1,000 births.

The changes could not be explained by other factors – such as mother’s age and socioeconomic status or population effects such as changes in air pollution and influenza epidemics.

There was no link found with birth weight.

Study leader Dr Tim Nawrot from Hasselt University said that even a mild reduction in gestational age has been linked in other studies to adverse health outcomes in early and later life.

“Because the ban happened at three different moments, we could show there was a consistent pattern of reduction in the risk of preterm delivery.”

He added: “It supports the notion that smoking bans have public health benefits even from early life.”

Patrick O’Brien, spokesperson for the Royal College of Obstetricians and Gynaecologists said: “It is very gratifying to see further strong evidence that smoking bans have had a beneficial impact on pregnant women and their babies.”

Scans show premature-baby brain arrested development.


Premature birth may interrupt vital brain development processes, medical scans reveal.

Researchers at King’s College London scanned 55 premature infants and 10 babies born at full term, using a novel type of MRI scan.

Premature baby in hospital

The brain scans showed arrested development in the premature babies at a key stage of maturation.

Experts say the work, in PNAS, could further understanding, but that parents should not be alarmed by the findings.

In recent decades there have been big advances in caring for premature babies, which mean most can go on to live a healthy life.

“Start Quote

Most premature babies are unaffected by their early arrival and families of these babies should not be unduly concerned”

Andy Cole Bliss

The researchers say the new type of imaging – which tracks the movement of water in the brain – will enable them to explore how the disruption of key processes might cause conditions such as autism.

It could also be used to monitor possible treatments to prevent brain damage.

Interrupted development

The scans showed cortical development was reduced in the preterm babies compared with those born at full term, with the greatest effect in the most premature infants – those born at about 27 weeks.

The brain regions affected govern social and emotional processing, as well as memory.

The same children were assessed at two years of age and those who had been born prematurely performed less well on neurodevelopmental tests, which the researchers say suggests the weeks a baby loses in the womb may matter.

Lead investigator Prof David Edwards said: “The number of babies born prematurely is increasing, so it has never been more important to improve our understanding of how preterm birth affects brain development and causes brain damage.

“We know that prematurity is extremely stressful for an infant, but by using a new technique we are able to track brain maturation in babies to pinpoint the exact processes that might be affected by premature birth.”

Andy Cole, chief executive of the premature baby charity Bliss, said: “It is very exciting that this ground-breaking research is being driven forward here in the UK.

“A better understanding of the way that preterm babies’ brains develop is an important step for doctors to help identify improvements in care that will benefit the 60,000 preterm children born every year.

“It is important to mention that most premature babies are unaffected by their early arrival and families of these babies should not be unduly concerned.”

‘Sugar gel’ helps premature babies.


A dose of sugar given as a gel rubbed into the inside of the cheek is a cheap and effective way to protect premature babies against brain damage, say experts.

premature baby

Dangerously low blood sugar affects about one in 10 babies born too early. Untreated, it can cause permanent harm.

Researchers from New Zealand tested the gel therapy in 242 babies under their care and, based on the results, say it should now be a first-line treatment.

Their work is published in The Lancet.

Sugar dose

Dextrose gel treatment costs just over £1 per baby and is simpler to administer than glucose via a drip, say Prof Jane Harding and her team at the University of Auckland.

“Start Quote

This is a cost effective treatment and could reduce admissions to intensive care services which are already working at high capacity levels”

Andy Cole Bliss

Current treatment typically involves extra feeding and repeated blood tests to measure blood sugar levels.

But many babies are admitted to intensive care and given intravenous glucose because their blood sugar remains low – a condition doctors call hypoglycaemia.

The study assessed whether treatment with dextrose gel was more effective than feeding alone at reversing hypoglycaemia.

Neil Marlow, from the Institute for Women’s Health at University College London, said that although dextrose gel had fallen into disuse, these findings suggested it should be resurrected as a treatment.

We now had high-quality evidence that it was of value, he said.

Andy Cole, chief executive of premature baby charity Bliss, said: “This is a very interesting piece of new research and we always welcome anything that has the potential to improve outcomes for babies born premature or sick.

“This is a cost-effective treatment and could reduce admissions to intensive care services, which are already working at high capacity levels.

“While the early results of this research show benefits to babies born with low blood sugars, it is clear there is more research to be done to implement this treatment.”

More than one in 10 babies worldwide born prematurely.


Fifteen million babies, one in 10 births, are born prematurely every year, a global project suggests.

Baby in mother's arms in Nepal

One million of these babies die soon after birth. The joint report, led by the WHO, says three quarters of deaths could be prevented with basic care.

For the first time premature birth rates have been estimated by country, with the highest risk being in Africa.

In the UK about 8% of babies are born too soon and this rate is rising partly due to obesity and later motherhood.

There are nearly 60,000 premature births every year in the UK.

Andy Cole, from premature baby charity Bliss, said “it is worrying that the UK’s preterm birth rate is significantly higher than countries such as Sweden, Norway and Ireland, and highlights the need for well-co-ordinated and high-quality antenatal care for all women identified as high risk”.

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Being born too soon is an unrecognised killer”

Dr Joy Lawn Co-editor of the report

Worldwide problem

A premature or preterm baby is one that is born before 37 weeks after the first day of the mother’s last period. A full term baby is when pregnancy lasts 40 weeks.

Forty-four organisations contributed to The Born Too Soon report, which estimated premature birth rates – the number of babies born too soon, out of the total of number of live births – for 184 individual countries, in the first study of its kind.

Of the 11 countries where over 15% of babies are born too early, all but two are in sub-Saharan Africa. However, the report highlights it is a problem worldwide.

“It is very striking to see that preterm births have a similar burden all around the world – but due to different reasons,” Dr Lale Say from the WHO said.

“In developing countries it is due to things like infections, HIV, malaria and poor nutrition.

Baby in arms
Premature babies are likely to have developmental problems as they grow up

“In developed countries there are totally different risk factors – an older delivery age, diabetes, obesity and multiple births due to IVF.”

The report also mentions caesarean sections before full term, which are not always medically needed, as a reason for increasing premature baby rates.

A leading cause of death

Dr Joy Lawn, co-editor of the report and Director for Save the Children said: “This report shows the problem is much bigger than expected or realised. Being born too soon is an unrecognised killer.”

In children under five, prematurity is the leading cause of death worldwide, after pneumonia. Many premature babies that do survive develop learning difficulties and visual and hearing problems.

“This has been a shocker to those who work in child survival programmes – people were almost falling off their chairs when I reported back our findings,” said Dr Lawn.

“We have been working on problems identified 20 years ago. There has been progress in pneumonia, and diarrhoea as a cause of death has seen a major drop, but preterm birth has not been on anyone’s ‘to do’ list.”

“There is no excuse for 80% of babies, who are less than eight weeks early, to die – it’s lack of food and warmth, not lack of intensive care.”

Easy care

Experts at the UN say simple and inexpensive care, like antiseptic cream to prevent cord infection, one US dollar (60 pence) steroid injections given to mothers to help foetal lung development, and antibiotics to fight infection, can help keep premature babies alive.

They also advocate the use of kangaroo care – where the baby is tied, skin to skin, on the mothers’ front – which reduces infection, keeps the baby warm and makes it easy to breastfeed. This has been proven to dramatically reduce newborn death.

Not surprisingly, there are big inequalities in survival rates around the world.

Countries with the highest premature birth numbers (2010)

Source: World Health Organization

This is highlighted in the US – as, while it is makes the top ten for the highest number of premature births in the world, it is only number 37 for deaths – because of very effective and expensive intensive care.

Dr Christopher Howson, from March of Dimes, a baby charity which collaborated on the study said: “In low income countries, more than 90% of extremely preterm babies die within the first few days of life, while less than 10% die in high income countries.”

“However, this is a solvable problem. A number of countries, for example, Ecuador, Botswana, Turkey, Oman and Sri Lanka have halved their neonatal deaths from preterm birth through improving [care for] serious complications like infections and respiratory distress.”

It is hoped the report will spotlight premature births, especially in sub-Saharan Africa, and make it an urgent priority to help reach the UN Millennium Development Goal 4 set in 2000 – which calls for the reduction of young child deaths by two-thirds in 2015.

Map of premature birth rates across the world Premature birth rates across the world (2010)

‘Kangaroo care’ key for prem babies.


Mothers carrying babies skin-to-skin could significantly cut global death and disability rates from premature birth, a leading expert has said.

Prof Joy Lawn says “kangaroo care“, not expensive intensive care, is the key.

Premature baby in an incubator

The 15 million babies every year born at or before 37 weeks gestation account for about 10% of the global burden of disease, and one million of them die.

Of those who survive, just under 3% have moderate or severe impairments and 4.4% have mild impairments.

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Unless there are those breathing problems, kangaroo care is actually better ”

Prof Joy Lawn London School of Hygiene & Tropical Medicine

Prof Lawn, from the London School of Hygiene and Tropical Medicine (LSHTM), said: “The perception is you need intensive care for pre-term babies,

“But 85% of babies born premature are six weeks early or less. They need help feeding, with temperature control and they are more prone to infection.

“It’s really only before 32 weeks that their lungs are immature and they need help breathing,

She added: “Unless there are those breathing problems, kangaroo care is actually better because it promotes breastfeeding and reduces infection.”

Speaking ahead of World Prematurity Day on Friday, UN Secretary General Ban Ki-moon, who leads the Every Woman Every Child movement, which promotes improvements to healthcare for women and children, said: “Three-quarters of the one million babies who die each year from complications associated with prematurity could have been saved with cost-effective interventions, even without intensive care facilities.”

Duncan Wilbur, from the UK charity Bliss, said, “While kangaroo care saves lives in countries such as Africa, it is also incredibly important for babies born too soon all over the world.

“Here in the UK our medical technology is extremely advanced but simply giving a baby kangaroo care or skin-to-skin can help make a baby’s breathing and heart rate more regular, it can help a baby’s discomfort during certain medical procedures and importantly can benefit breastfeeding and bonding between the baby and parents.”

Pregnancy risks

Studies to be published this weekend in the Pediatric Research journal show boys are 14% more likely to be born prematurely – and boys who are premature are more likely to die or experience disability than girls.

Common disabilities include learning disorders and cerebral palsy.

Prof Lawn said: “One partial explanation for more preterm births among boys is that women pregnant with a boy are more likely to have placental problems, pre-eclampsia, and high blood pressure, all associated with preterm births.”

She added: “Baby boys have a higher likelihood of infections, jaundice, birth complications, and congenital conditions, but the biggest risk for baby boys is due to preterm birth.

“For two babies born at the same degree of prematurity, a boy will have a higher risk of death and disability compared to a girl.

“Even in the womb, girls mature more rapidly than boys, which provides an advantage, because the lungs and other organs are more developed.

‘Sugar gel’ helps premature babies


A dose of sugar given as a gel rubbed into the inside of the cheek is a cheap and effective way to protect premature babies against brain damage, say experts.

Dangerously low blood sugar affects about one in 10 babies born too early. Untreated, it can cause permanent harm.

Researchers from New Zealand tested the gel therapy in 242 babies under their care and, based on the results, say it should now be a first-line treatment.

Their work is published in The Lancet.

Sugar dose

Dextrose gel treatment costs just over £1 per baby and is simpler to administer than glucose via a drip, say Prof Jane Harding and her team at the University of Auckland.

 “Start Quote

This is a cost effective treatment and could reduce admissions to intensive care services which are already working at high capacity levels”

Andy ColeBliss

Current treatment typically involves extra feeding and repeated blood tests to measure blood sugar levels.

But many babies are admitted to intensive care and given intravenous glucose because their blood sugar remains low – a condition doctors call hypoglycaemia.

The study assessed whether treatment with dextrose gel was more effective than feeding alone at reversing hypoglycaemia.

_70094382_premature_baby-spl-2

Neil Marlow, from the Institute for Women’s Health at University College London, said that although dextrose gel had fallen into disuse, these findings suggested it should be resurrected as a treatment.

We now had high-quality evidence that it was of value, he said.

Andy Cole, chief executive of premature baby charity Bliss, said: “This is a very interesting piece of new research and we always welcome anything that has the potential to improve outcomes for babies born premature or sick.

“This is a cost-effective treatment and could reduce admissions to intensive care services, which are already working at high capacity levels.

“While the early results of this research show benefits to babies born with low blood sugars, it is clear there is more research to be done to implement this treatment.”

Source: BBC