Specific Lipopolysaccharide Serotypes Induce Differential Maternal and Neonatal Inflammatory Responses in a Murine Model of Preterm Labor

American Journal of Pathology, Volume 185, No. 9, Year 2015

Interpretation

Intrauterine inflammation is recognized as a key mediator of both normal and preterm birth but is also associated with neonatal neurological injury. Lipopolysaccharide (LPS) is often used to stimulate inflammatory pathways in animal models of infection/inflammation-induced preterm labor; however, inconsistencies in maternal and neonatal responses to LPS are frequently reported. We hypothesized that LPS serotype-specific responses may account for a portion of these inconsistencies. Four different Escherichia coli LPS serotypes (O111:B4, O55:B5, O127:B8, and O128:B12) were administered to CD1 mice via intrauterine injection at gestational day 16. Although control animals delivered at term 60 ± 15 hours postinjection (p.i.), those administered with O111:B4 delivered 7 ± 2 hours p.i., O55:B5 delivered 10 ± 3 hours p.i., O127:B8 delivered 16 ± 10 hours p.i., and O128:B12 delivered 17 ± 2 hours p.i. (means ± SD). A correlation between the onset of preterm labor and myometrial activation of the inflammatory transcription factor, activator protein 1, but not NF-κB was observed. Specific LPS serotypes induced differential activation of downstream contractile and inflammatory pathways in myometrium and neonatal pup brain. Our findings demonstrate functional disparity in inflammatory pathway activation in response to differing LPS serotypes. Selective use of LPS serotypes may represent a useful tool for targeting specific inflammatory response mechanisms in these models. Animal studies were performed under UK Home Office License 70/6906, in accordance with the guidance to Animals Scientific Procedures Act of 1986, and with approval of the Imperial College London and University College London (London, UK) Ethical Review Committees. CD1 outbred virgin females were timed mated, and the presence of a copulatory plug was classified as embryonic day 0 (E0) of gestation. Mice were housed in open cages at 21°C ± 1°C with ad libitum access to standard rodent food and water and were exposed to a 12:12 light-dark cycle regimen. Unless otherwise stated, five biological replicates were collected for all experimental groups. Pregnant (E16) female dams were administered a s.c. injection of 2.5 mg/kg morphine 20 minutes before surgery. Animals were anesthetized by isoflurane, and a laparotomy was performed as previously described.9,30,31 Briefly, both uterine horns were exteriorized and the number of live fetuses per horn was recorded. An intrauterine injection of 20 μg [25 μL total volume in phosphate-buffered saline (PBS)] of either E. coli LPS serotype O111:B4, O55:B5, O127:B8, or O128:B12 (Sigma Aldrich, Gillingham, UK) or sterile PBS was injected into the upper right uterine horn between the first and second sacs. Biochemical characteristics of the LPS serotypes were consistent [phenol extracted; protein content, ≤3%; solubility, 4.9 to 5.1 mg/mL; endotoxin level, ≥500,000 endotoxin units (EU)/mg]. Animals were continuously monitored after surgery remotely via an infrared closed circuit TV camera system during recovery until tissue collection or the onset of spontaneous delivery. The onset of labor was defined as delivery of the first pup. For tissue collection, mice were anesthetized and sacrificed by cervical dislocation. A laparotomy was performed, uteri were immediately incised in the longitudinal direction, and pups were isolated and sacrificed by decapitation. Right and left horns of the uterus were snap frozen separately after removal of gestational membranes, placentas, and vasculature. Myometrium samples from the right uterine horns were used for mRNA and protein analyses. After decapitation, whole pup brains were isolated and snap frozen. Tissue was stored at −80°C until extraction. Before culling, pup viability was qualitatively assessed using a scoring system as follows: 3, pups displaying full body spontaneous movement when removed from the myometrium or amniotic sac; 2, pups with partial body movement (ie, lower half or full body movement when gently prodded with forceps); 1, pups exhibiting partial body movement or movement of limbs only in response to forceful prodding or squeezing of a limb with forceps’ and 0, pups with no response when forcefully prodded or limbs squeezed. Myometrial and whole pup brain protein lysates were prepared by grinding tissue under liquid nitrogen in a mortar and pestle, then homogenizing in a modified radioimmunoprecipitation assay buffer (1% Triton X-100, 1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 0.1% SDS, 1% deoxycholic acid, 50 mmol/L NaF, 1 mmol/L orthovanadate, protease inhibitor cocktail, 25 mmol/L Tris [pH 7.4], and 150 mmol/L NaCl) at a ratio of 1 mL buffer:100 mg wet weight tissue. Protein lysates were centrifuged at 13,000 × g, and protein concentration was determined via a Bradford assay (Bio-Rad, Hemel Hempstead, Hertfordshire, UK). Extracted proteins (20 μg per sample) were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (GE Healthcare, Little Chalfont, UK) at 100 V (constant voltage) at 4°C. Membranes were blocked with 5% skim milk in Tris-buffered saline (TBS) supplemented with 0.01% Tween 20 (TBST) for 1 hour and then incubated overnight at 4°C with primary antibodies raised against phosphorylated (p) NF-κB: p65 Ser536 (1:1000 in TBS; catalog number 3031; Cell Signaling Technology, Hitchin, Herts, UK), p-c-Jun Ser73 (1:1000 in TBST containing 1% milk; catalog number 9164; Cell Signaling Technology); CX43 (1:1000 in TBST containing 1% milk; catalog number 3512; Cell Signaling Technology); p-C/EBPβ Thr235 (1:1000 in 1% bovine serum albumin; catalog number 3084; Cell Signaling Technology); COX-2 (1:2000 in TBST containing 5% milk; catalog number sc-1745; Santa Cruz Biotechnology, Dallas, TX); and IL-1β (1:1000 in TBST containing 1% milk; catalog number AF-401-NA; R&D Systems, Abingdon, UK). After primary antibody incubation, membranes were washed six times (5 minutes each) with TBST and subsequently incubated with the appropriate horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature (1:2000; Cell Signaling Technology) before being washed again with TBST. Detection of immunoreactive bands was performed by enhanced chemiluminescence (ECL; GE Healthcare) using the ImageQuant LAS 4000 Imager (GE Healthcare). Membranes were stripped with 0.2 mol/L NaOH for 5 minutes at room temperature, washed in TBST, and reprobed with β-actin (1:40,000; Sigma Aldrich) as a loading control. Densitometric analysis was conducted using ImageQuant TL (GE Healthcare). mRNA was isolated from ground myometrial and whole pup brain tissue using the Nucleospin miRNA kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions. RNA concentration and integrity were measured with a NanoDrop 1000 spectrophotometer (ThermoScientific, Waltham, MA) and an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). cDNA synthesis was performed on 2 μg RNA using M-MLV Reverse Transcriptase (Sigma-Aldrich). RNA was incubated with 1 μL of 0.5 μg/μL Oligo(dT)23 Primers Anchored (Sigma-Aldrich) and 1 μL of 10 mmol/L dNTP mix (Sigma-Aldrich) for 10 minutes at 70°C and then transferred onto ice. Master mix (10 μL) containing 6.5 μL of nuclease-free water, 2 μL of M-MLV Reverse Transcriptase Buffer, 0.5 μL of human ribonuclease inhibitor (40,000 U/mL; Sigma-Aldrich), and 1 μL of 200 U/μL M-MLV Reverse Transcriptase was added to each RNA sample and incubated at room temperature for 15 minutes, then at 37°C for 50 minutes, and finally at 85°C for 10 minutes to inactivate the enzyme. Isolated cDNA was stored at −20°C. For each target gene, a gene-specific primer set of 8 to 22 bp was designed using the National Center for Biotechnology Information Database (http://www.ncbi.nlm.nih.gov/tools/primer-blast, last accessed April 29, 2015) with optimal Tm of 60°C (Table 1). Both forward and reverse primers were designed to span exon-exon junctions. Primer Sequences Used for Real-Time Quantitative RT-PCR Real-time quantitative RT-PCR was performed in 384-well plates in duplicate using a total volume of 10 μL containing 2.6-μL nuclease-free water, 0.1 μL ROX reference dye (Sigma-Aldrich), 0.15 μL forward primer (20 μmol/L), 0.15 μL reverse primer (20 μmol/L), 5 μL SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich), and 2 μL of cDNA. Reactions were run on an ABI 7900HT (Applied Biosystems Foster City, CA) as follows: 2 minutes at 94°C and 40 cycles of 15 seconds at 94°C, 60 seconds at 60°C, and 60 seconds at 72°C; the dissociation curve was 15 seconds at 95°C, 1 minute at 60°C, and 15 seconds at 95°C. Data were processed using ABI 7900HT SDS version 2.4 software and analyzed with Excel (Office 2010; Microsoft, Redmond, WA). Gene expression levels for each sample were normalized to the endogenous reference β-actin mRNA for each well (ΔCT). Relative efficiencies of gene target and endogenous control amplification were assessed using standard curves generated for each gene by 10-fold serial dilutions of the same sample run in duplicate. The comparative CT method was used to determine differences between LPS- and PBS-treated control samples. Dissociation curve analysis, confirmed by 2% agarose gel electrophoresis, was used to verify that a single gene-specific product was produced (data not shown). Each experiment consisted of five biological replicates, and results are presented as means ± SD unless otherwise specified. Densitometric values were normalized to β-actin before undertaking statistical analyses using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). The statistical significance between LPS- and PBS-treated control samples was assessed using a two-tailed t-test or one-way analysis of variance (with Tukey’s post hoc test) where appropriate. P < 0.05 was considered statistically significant. For linear regression, mean levels of p-c-Jun normalized to β-actin for each serotype at 6 hours after injection were calculated and regressed against mean time of delivery for each serotype. The coefficient of determination (r2) of the linear regression was calculated and reported as a measure of goodness of fit.

AI Digest

Study Justification:

This study aimed to investigate the differential maternal and neonatal inflammatory responses to different lipopolysaccharide (LPS) serotypes in a murine model of preterm labor. Intrauterine inflammation is known to play a role in both normal and preterm birth, as well as neonatal neurological injury. However, inconsistencies in the responses to LPS in animal models have been reported. The researchers hypothesized that the specific LPS serotypes used may account for these inconsistencies. Understanding the specific inflammatory response mechanisms can help in targeting interventions and treatments for preterm labor and associated complications.

Highlights:

– Four different LPS serotypes (O111:B4, O55:B5, O127:B8, and O128:B12) were administered to CD1 mice via intrauterine injection.
– Control animals delivered at term, while those administered with LPS serotypes delivered earlier.
– The onset of preterm labor was correlated with the activation of the inflammatory transcription factor, activator protein 1.
– Different LPS serotypes induced differential activation of contractile and inflammatory pathways in the myometrium and neonatal pup brain.
– These findings suggest that selective use of LPS serotypes can be a useful tool for targeting specific inflammatory response mechanisms in preterm labor models.

Recommendations:

Based on the findings of this study, the following recommendations can be made:

1. Further investigate the specific mechanisms by which different LPS serotypes induce differential inflammatory responses.
2. Explore the potential of targeting specific inflammatory pathways for interventions and treatments in preterm labor.
3. Conduct clinical studies to validate the findings in animal models and assess the relevance to human preterm labor.

Key Role Players:

To address the recommendations, the following key role players are needed:

1. Researchers and scientists specializing in reproductive biology, obstetrics, and neonatology.
2. Animal care and ethics committees to ensure compliance with regulations and ethical considerations.
3. Funding agencies to provide financial support for further research and clinical studies.
4. Collaborative partnerships between research institutions, hospitals, and pharmaceutical companies to facilitate the translation of findings into clinical practice.

Cost Items:

While the actual costs may vary, the following cost items should be considered in planning the recommendations:

1. Research personnel salaries and benefits.
2. Animal care and housing facilities.
3. Laboratory supplies and equipment for sample collection and analysis.
4. Animal models and LPS serotypes.
5. Ethical review and regulatory compliance fees.
6. Data analysis and statistical software.
7. Publication and dissemination of research findings.
8. Clinical study expenses, including patient recruitment, medical procedures, and data collection.
Please note that the provided cost items are general and may not cover all specific expenses associated with implementing the recommendations.

Strength of Evidence

The strength of evidence for this abstract is 7 out of 10.
The evidence in the abstract is rated 7 because the study provides detailed information about the experimental design, methods, and results. However, the abstract does not mention the sample size or statistical analysis used, which could be improved by including this information.

Abstract

Animal studies were performed under UK Home Office License 70/6906, in accordance with the guidance to Animals Scientific Procedures Act of 1986, and with approval of the Imperial College London and University College London (London, UK) Ethical Review Committees. CD1 outbred virgin females were timed mated, and the presence of a copulatory plug was classified as embryonic day 0 (E0) of gestation. Mice were housed in open cages at 21°C ± 1°C with ad libitum access to standard rodent food and water and were exposed to a 12:12 light-dark cycle regimen. Unless otherwise stated, five biological replicates were collected for all experimental groups. Pregnant (E16) female dams were administered a s.c. injection of 2.5 mg/kg morphine 20 minutes before surgery. Animals were anesthetized by isoflurane, and a laparotomy was performed as previously described.9,30,31 Briefly, both uterine horns were exteriorized and the number of live fetuses per horn was recorded. An intrauterine injection of 20 μg [25 μL total volume in phosphate-buffered saline (PBS)] of either E. coli LPS serotype O111:B4, O55:B5, O127:B8, or O128:B12 (Sigma Aldrich, Gillingham, UK) or sterile PBS was injected into the upper right uterine horn between the first and second sacs. Biochemical characteristics of the LPS serotypes were consistent [phenol extracted; protein content, ≤3%; solubility, 4.9 to 5.1 mg/mL; endotoxin level, ≥500,000 endotoxin units (EU)/mg]. Animals were continuously monitored after surgery remotely via an infrared closed circuit TV camera system during recovery until tissue collection or the onset of spontaneous delivery. The onset of labor was defined as delivery of the first pup. For tissue collection, mice were anesthetized and sacrificed by cervical dislocation. A laparotomy was performed, uteri were immediately incised in the longitudinal direction, and pups were isolated and sacrificed by decapitation. Right and left horns of the uterus were snap frozen separately after removal of gestational membranes, placentas, and vasculature. Myometrium samples from the right uterine horns were used for mRNA and protein analyses. After decapitation, whole pup brains were isolated and snap frozen. Tissue was stored at −80°C until extraction. Before culling, pup viability was qualitatively assessed using a scoring system as follows: 3, pups displaying full body spontaneous movement when removed from the myometrium or amniotic sac; 2, pups with partial body movement (ie, lower half or full body movement when gently prodded with forceps); 1, pups exhibiting partial body movement or movement of limbs only in response to forceful prodding or squeezing of a limb with forceps’ and 0, pups with no response when forcefully prodded or limbs squeezed. Myometrial and whole pup brain protein lysates were prepared by grinding tissue under liquid nitrogen in a mortar and pestle, then homogenizing in a modified radioimmunoprecipitation assay buffer (1% Triton X-100, 1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 0.1% SDS, 1% deoxycholic acid, 50 mmol/L NaF, 1 mmol/L orthovanadate, protease inhibitor cocktail, 25 mmol/L Tris [pH 7.4], and 150 mmol/L NaCl) at a ratio of 1 mL buffer:100 mg wet weight tissue. Protein lysates were centrifuged at 13,000 × g, and protein concentration was determined via a Bradford assay (Bio-Rad, Hemel Hempstead, Hertfordshire, UK). Extracted proteins (20 μg per sample) were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (GE Healthcare, Little Chalfont, UK) at 100 V (constant voltage) at 4°C. Membranes were blocked with 5% skim milk in Tris-buffered saline (TBS) supplemented with 0.01% Tween 20 (TBST) for 1 hour and then incubated overnight at 4°C with primary antibodies raised against phosphorylated (p) NF-κB: p65 Ser536 (1:1000 in TBS; catalog number 3031; Cell Signaling Technology, Hitchin, Herts, UK), p-c-Jun Ser73 (1:1000 in TBST containing 1% milk; catalog number 9164; Cell Signaling Technology); CX43 (1:1000 in TBST containing 1% milk; catalog number 3512; Cell Signaling Technology); p-C/EBPβ Thr235 (1:1000 in 1% bovine serum albumin; catalog number 3084; Cell Signaling Technology); COX-2 (1:2000 in TBST containing 5% milk; catalog number sc-1745; Santa Cruz Biotechnology, Dallas, TX); and IL-1β (1:1000 in TBST containing 1% milk; catalog number AF-401-NA; R&D Systems, Abingdon, UK). After primary antibody incubation, membranes were washed six times (5 minutes each) with TBST and subsequently incubated with the appropriate horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature (1:2000; Cell Signaling Technology) before being washed again with TBST. Detection of immunoreactive bands was performed by enhanced chemiluminescence (ECL; GE Healthcare) using the ImageQuant LAS 4000 Imager (GE Healthcare). Membranes were stripped with 0.2 mol/L NaOH for 5 minutes at room temperature, washed in TBST, and reprobed with β-actin (1:40,000; Sigma Aldrich) as a loading control. Densitometric analysis was conducted using ImageQuant TL (GE Healthcare). mRNA was isolated from ground myometrial and whole pup brain tissue using the Nucleospin miRNA kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions. RNA concentration and integrity were measured with a NanoDrop 1000 spectrophotometer (ThermoScientific, Waltham, MA) and an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). cDNA synthesis was performed on 2 μg RNA using M-MLV Reverse Transcriptase (Sigma-Aldrich). RNA was incubated with 1 μL of 0.5 μg/μL Oligo(dT)23 Primers Anchored (Sigma-Aldrich) and 1 μL of 10 mmol/L dNTP mix (Sigma-Aldrich) for 10 minutes at 70°C and then transferred onto ice. Master mix (10 μL) containing 6.5 μL of nuclease-free water, 2 μL of M-MLV Reverse Transcriptase Buffer, 0.5 μL of human ribonuclease inhibitor (40,000 U/mL; Sigma-Aldrich), and 1 μL of 200 U/μL M-MLV Reverse Transcriptase was added to each RNA sample and incubated at room temperature for 15 minutes, then at 37°C for 50 minutes, and finally at 85°C for 10 minutes to inactivate the enzyme. Isolated cDNA was stored at −20°C. For each target gene, a gene-specific primer set of 8 to 22 bp was designed using the National Center for Biotechnology Information Database (http://www.ncbi.nlm.nih.gov/tools/primer-blast, last accessed April 29, 2015) with optimal Tm of 60°C (Table 1). Both forward and reverse primers were designed to span exon-exon junctions. Primer Sequences Used for Real-Time Quantitative RT-PCR Real-time quantitative RT-PCR was performed in 384-well plates in duplicate using a total volume of 10 μL containing 2.6-μL nuclease-free water, 0.1 μL ROX reference dye (Sigma-Aldrich), 0.15 μL forward primer (20 μmol/L), 0.15 μL reverse primer (20 μmol/L), 5 μL SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich), and 2 μL of cDNA. Reactions were run on an ABI 7900HT (Applied Biosystems Foster City, CA) as follows: 2 minutes at 94°C and 40 cycles of 15 seconds at 94°C, 60 seconds at 60°C, and 60 seconds at 72°C; the dissociation curve was 15 seconds at 95°C, 1 minute at 60°C, and 15 seconds at 95°C. Data were processed using ABI 7900HT SDS version 2.4 software and analyzed with Excel (Office 2010; Microsoft, Redmond, WA). Gene expression levels for each sample were normalized to the endogenous reference β-actin mRNA for each well (ΔCT). Relative efficiencies of gene target and endogenous control amplification were assessed using standard curves generated for each gene by 10-fold serial dilutions of the same sample run in duplicate. The comparative CT method was used to determine differences between LPS- and PBS-treated control samples. Dissociation curve analysis, confirmed by 2% agarose gel electrophoresis, was used to verify that a single gene-specific product was produced (data not shown). Each experiment consisted of five biological replicates, and results are presented as means ± SD unless otherwise specified. Densitometric values were normalized to β-actin before undertaking statistical analyses using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). The statistical significance between LPS- and PBS-treated control samples was assessed using a two-tailed t-test or one-way analysis of variance (with Tukey’s post hoc test) where appropriate. P < 0.05 was considered statistically significant. For linear regression, mean levels of p-c-Jun normalized to β-actin for each serotype at 6 hours after injection were calculated and regressed against mean time of delivery for each serotype. The coefficient of determination (r2) of the linear regression was calculated and reported as a measure of goodness of fit.

Materials

Innovations

Based on the provided description, it is difficult to determine specific innovations for improving access to maternal health. The description focuses on a study involving the use of lipopolysaccharide (LPS) serotypes in a murine model of preterm labor. It does not provide information on innovations or recommendations for improving access to maternal health.

AI Innovations Description

The recommendation to improve access to maternal health based on the provided description is to further investigate the specific lipopolysaccharide (LPS) serotypes that induce differential maternal and neonatal inflammatory responses in a murine model of preterm labor. This research suggests that the use of selective LPS serotypes may be a useful tool for targeting specific inflammatory response mechanisms in animal models. By understanding the specific serotypes that elicit different responses, researchers can develop targeted interventions and treatments to improve maternal and neonatal health outcomes. Further studies can be conducted to explore the potential of using specific LPS serotypes as a basis for developing innovative approaches to improve access to maternal health.

AI Innovations Methodology

Based on the provided description, here are some potential recommendations for improving access to maternal health:

1. Strengthening healthcare infrastructure: Investing in healthcare facilities, equipment, and trained healthcare professionals in areas with limited access to maternal health services can help improve access and quality of care.

2. Mobile health (mHealth) interventions: Utilizing mobile technology to provide maternal health information, reminders for prenatal care appointments, and access to telemedicine consultations can help overcome geographical barriers and improve access to care.

3. Community-based interventions: Implementing community-based programs that provide education, support, and resources for pregnant women can help increase awareness about maternal health and encourage early and regular prenatal care.

4. Transportation support: Providing transportation services or subsidies for pregnant women in remote areas can help overcome transportation barriers and ensure timely access to maternal health services.

5. Financial incentives: Offering financial incentives, such as cash transfers or vouchers, to pregnant women who seek prenatal care and deliver at healthcare facilities can help reduce financial barriers and increase utilization of maternal health services.

To simulate the impact of these recommendations on improving access to maternal health, a methodology could include the following steps:

1. Define the target population: Identify the specific population or region where the recommendations will be implemented.

2. Collect baseline data: Gather data on the current access to maternal health services, including the number of healthcare facilities, healthcare professionals, utilization rates, and health outcomes.

3. Develop a simulation model: Create a mathematical or computational model that represents the target population and the healthcare system. The model should include variables such as population demographics, healthcare infrastructure, transportation availability, and financial factors.

4. Input data and parameters: Input the baseline data and parameters related to the recommendations being simulated, such as the number of healthcare facilities to be built, the cost of transportation subsidies, or the amount of financial incentives.

5. Run simulations: Run the simulation model multiple times, varying the input parameters to simulate different scenarios. For example, simulate the impact of building different numbers of healthcare facilities or providing different levels of financial incentives.

6. Analyze results: Analyze the simulation results to determine the impact of the recommendations on access to maternal health services. This could include measures such as the number of additional women accessing prenatal care, the reduction in travel time to healthcare facilities, or the increase in facility-based deliveries.

7. Validate and refine the model: Validate the simulation results by comparing them to real-world data, if available. Refine the model based on feedback and additional data to improve its accuracy and reliability.

8. Communicate findings: Present the findings of the simulation study, including the potential impact of the recommendations on improving access to maternal health services, to stakeholders and policymakers. This can help inform decision-making and resource allocation for implementing the recommendations.

Author

Dima Health

Statistics:

Citations: 52

Identifiers:

DOI: 10.1016/j.ajpath.2015.05.015

ISSN: 00029440

Research Areas:

Environmental, Food Security, Genetics and Genomics, Health System and Policy, Maternal and Child Health, Social Determinants, Technology and Innovations, Violence and Injury

Study Countries:

Mali

Study Design:

Cohort Study

Study Approach:

Quantitative

Participants Gender:

Female