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1.  Choriodecidual Cells from Term Human Pregnancies Show Distinctive Functional Properties Related to the Induction of Labor 
Human parturition is associated with an intrauterine pro-inflammatory environment in the choriodecidua. Evidence that some mediators of this signaling cascade also elicit responses leading to labor prompted us to characterize the cellular sources of these mediators in the human choriodecidua.
Method of study
Leukocyte-enriched preparations from human choriodecidua (ChL) and intervillous placental blood leukocytes (PL) were maintained in culture. Secretions of inflammatory cytokines, chemokines and MMP-9 were documented. Leukocyte phenotype of ChL and PL was determined by flow cytometry using specific fluorochrome-conjugated antibodies.
Results and Conclusions
ChL showed a distinct pro-inflammatory secretion pattern of cytokines and chemokines when compared with PL, including higher amounts of TNF-α and IL-6, and decreased secretions of IL-4 and IL-1ra. ChL also secreted more MIP-1α and MCP-1 and MMP-9 than PL. No significant differences were found in leukocytes subsets between compartments. Based on our findings, we propose that ChL isolated from fetal membranes at term are functionally different from PL and may collaborate to modulate the microenvironment linked to induction and progression of human labor.
PMCID: PMC4000259  PMID: 24286217
amniochorion; choriodecidua; fetal membranes; inflammation; labor
2.  Outdoor Air Pollution, Preterm Birth, and Low Birth Weight: Analysis of the World Health Organization Global Survey on Maternal and Perinatal Health 
Environmental Health Perspectives  2014;122(4):425-430.
Background: Inhaling fine particles (particulate matter with diameter ≤ 2.5 μm; PM2.5) can induce oxidative stress and inflammation, and may contribute to onset of preterm labor and other adverse perinatal outcomes.
Objectives: We examined whether outdoor PM2.5 was associated with adverse birth outcomes among 22 countries in the World Health Organization Global Survey on Maternal and Perinatal Health from 2004 through 2008.
Methods: Long-term average (2001–2006) estimates of outdoor PM2.5 were assigned to 50-km–radius circular buffers around each health clinic where births occurred. We used generalized estimating equations to determine associations between clinic-level PM2.5 levels and preterm birth and low birth weight at the individual level, adjusting for seasonality and potential confounders at individual, clinic, and country levels. Country-specific associations were also investigated.
Results: Across all countries, adjusting for seasonality, PM2.5 was not associated with preterm birth, but was associated with low birth weight [odds ratio (OR) = 1.22; 95% CI: 1.07, 1.39 for fourth quartile of PM2.5 (> 20.2 μg/m3) compared with the first quartile (< 6.3 μg/m3)]. In China, the country with the largest PM2.5 range, preterm birth and low birth weight both were associated with the highest quartile of PM2.5 only, which suggests a possible threshold effect (OR = 2.54; CI: 1.42, 4.55 and OR = 1.99; CI: 1.06, 3.72 for preterm birth and low birth weight, respectively, for PM2.5 ≥ 36.5 μg/m3 compared with PM2.5 < 12.5 μg/m3).
Conclusions: Outdoor PM2.5 concentrations were associated with low birth weight but not preterm birth. In rapidly developing countries, such as China, the highest levels of air pollution may be of concern for both outcomes.
Citation: Fleischer NL, Merialdi M, van Donkelaar A, Vadillo-Ortega F, Martin RV, Betran AP, Souza JP, O´Neill MS. 2014. Outdoor air pollution, preterm birth, and low birth weight: analysis of the World Health Organization Global Survey on Maternal and Perinatal Health. Environ Health Perspect 122:425–430;
PMCID: PMC3984219  PMID: 24508912
3.  Air pollution, inflammation and preterm birth in Mexico City: Study design and methods 
Preterm birth is one of the leading causes of perinatal mortality and is associated with long-term adverse health consequences for surviving infants. Preterm birth rates are rising worldwide, and no effective means for prevention currently exists. Air pollution exposure may be a significant cause of prematurity, but many published studies lack the individual, clinical data needed to elucidate possible biological mechanisms mediating these epidemiological associations. This paper presents the design of a prospective study now underway to evaluate those mechanisms in a cohort of pregnant women residing in Mexico City. We address how air quality may act together with other factors to induce systemic inflammation and influence the duration of pregnancy. Data collection includes: biomarkers relevant to inflammation in cervico-vaginal exudate and peripheral blood, along with full clinical information, pro-inflammatory cytokine gene polymorphisms and air pollution data to evaluate spatial and temporal variability in air pollution exposure. Samples are collected on a monthly basis and participants are followed for the duration of pregnancy. The data will be used to evaluate whether ambient air pollution is associated with preterm birth, controlling for other risk factors. We will evaluate which time windows during pregnancy are most influential in the air pollution and preterm birth association. In addition, the epidemiological study will be complemented with a parallel toxicology invitro study, in which monocytic cells will be exposed to air particle samples to evaluate the expression of biomarkers of inflammation.
PMCID: PMC3594336  PMID: 23177781
air pollution; epidemiology; toxicology; preterm birth; inflammation; Mexico City
4.  Low Glycemic Index Carbohydrates versus All Types of Carbohydrates for Treating Diabetes in Pregnancy: A Randomized Clinical Trial to Evaluate the Effect of Glycemic Control 
Background. Due to the higher prevalence of obesity and diabetes mellitus (DM), more pregnant women complicated with diabetes are in need of clinical care. Purpose. Compare the effect of including only low glycemic index (GI) carbohydrates (CHO) against all types of CHO on maternal glycemic control and on the maternal and newborn's nutritional status of women with type 2 DM and gestational diabetes mellitus (GDM). Methods. Women (n = 107, ≤29 weeks of gestation) were randomly assigned to one of two nutrition intervention groups: moderate energy and CHO restriction (Group 1: all types of CHO, Group 2: low GI foods). Results. No baseline differences in clinical data were observed. Capillary glucose concentrations throughout pregnancy were similar between groups. Fewer women in Group 2 exceeded weight gain recommendations. Higher risk of prematurity was observed in women in Group 2. No differences in glycemic control were observed between women with type 2 DM and those with GDM. Conclusions. Inclusion of low GI CHO as part of a comprehensive nutrition intervention is equally effective in improving glycemic control as compared to all types of CHO. This strategy had a positive effect in preventing excessive maternal weight gain but increased the risk of prematurity.
PMCID: PMC3517846  PMID: 23251152
5.  Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis 
PLoS ONE  2011;6(12):e28771.
Chemotaxis has been studied by classical methods that measure chemotactic and random motility responses in vitro, but these methods do not evaluate the total number and phenotype of migrating leukocytes simultaneously. Our objective was to develop and validate a novel assay, combined Boyden-flow cytometry chemotaxis assay (CBFCA), for simultaneous quantification and phenotypification of migrating leukocytes. CBFCA exhibited several important advantages in comparison to the classic Boyden chemotaxis assay (CBCA): 1) improved precision (intra-assay coefficients of variation (CVs): CBFCA-4.7 and 4.8% vs. CBCA-30.1 and 17.3%; inter-observer CVs: CBFCA-3.6% vs. CBCA 30.1%); 2) increased recovery of cells, which increased assay to provide increased sensitivity; 3) high specificity for determining the phenotype of migrating/attracted leukocytes; and 4) reduced performance time (CBFCA 120 min vs. CBCA 265 min). Other advantages of CBFCA are: 5) robustness, 6) linearity, 7) eliminated requirement for albumin and, importantly, 8) enabled recovery of migrating leukocytes for subsequent studies. This latter feature is of great benefit in the study of migrating leukocyte subsets. We conclude that the CBFCA is a novel and improved technique for experiments focused on understanding leukocyte trafficking during the inflammatory response.
PMCID: PMC3235166  PMID: 22174892
6.  Effect of supplementation during pregnancy with L-arginine and antioxidant vitamins in medical food on pre-eclampsia in high risk population: randomised controlled trial 
Objective To test the hypothesis that a relative deficiency in L-arginine, the substrate for synthesis of the vasodilatory gas nitric oxide, may be associated with the development of pre-eclampsia in a population at high risk.
Design Randomised, blinded, placebo controlled clinical trial.
Setting Tertiary public hospital in Mexico City.
Participants Pregnant women with a history of a previous pregnancy complicated by pre-eclampsia, or pre-eclampsia in a first degree relative, and deemed to be at increased risk of recurrence of the disease were studied from week 14-32 of gestation and followed until delivery.
Interventions Supplementation with a medical food—bars containing L-arginine plus antioxidant vitamins, antioxidant vitamins alone, or placebo—during pregnancy.
Main outcome measure Development of pre-eclampsia/eclampsia.
Results 222 women were allocated to the placebo group, 228 received L-arginine plus antioxidant vitamins, and 222 received antioxidant vitamins alone. Women had 4-8 prenatal visits while receiving the bars. The incidence of pre-eclampsia was reduced significantly (χ2=19.41; P<0.001) in women randomised to L-arginine plus antioxidant vitamins compared with placebo (absolute risk reduction 0.17 (95% confidence interval 0.12 to 0.21). Antioxidant vitamins alone showed an observed benefit, but this effect was not statistically significant compared with placebo (χ2=3.76; P=0.052; absolute risk reduction 0.07, 0.005 to 0.15). L-arginine plus antioxidant vitamins compared with antioxidant vitamins alone resulted in a significant effect (P=0.004; absolute risk reduction 0.09, 0.05 to 0.14).
Conclusions Supplementation during pregnancy with a medical food containing L-arginine and antioxidant vitamins reduced the incidence of pre-eclampsia in a population at high risk of the condition. Antioxidant vitamins alone did not have a protective effect for prevention of pre-eclampsia. Supplementation with L-arginine plus antioxidant vitamins needs to be evaluated in a low risk population to determine the generalisability of the protective effect, and the relative contributions of L-arginine and antioxidant vitamins to the observed effects of the combined treatment need to be determined.
Trial registration Clinical trials NCT00469846.
PMCID: PMC3100912  PMID: 21596735
7.  Interaction between Pathogenic Bacteria and Intrauterine Leukocytes Triggers Alternative Molecular Signaling Cascades Leading to Labor in Women▿  
Infection and Immunity  2010;78(11):4792-4799.
Increased risk of preterm labor has been linked to cervicovaginal infection with Ureaplasma urealyticum and group B streptococci. Although various experimental models have been developed to study the role of amniochorion infection in preterm labor, they typically exclude the initial interaction between intrauterine leukocytes (recruited from decidual vessels into the avascular fetal membranes) and infecting bacteria. In this work, we ascertained whether inflammatory molecules secreted by bacterium-activated intrauterine leukocytes stimulate the amniochorion production of mediators involved in human labor. Using a two-step process beginning with placental circulating leukocytes as a proxy for intrauterine leukocytes, we found that coincubation of amniochorion explants with plasma from placental whole blood preincubated with group B streptococci resulted in a significant increase in tumor necrosis factor alpha (TNF-α) and matrix metalloproteinase 9 (MMP-9) levels in tissue. Extensive changes in the connective tissue arrangement and a decrease in collagen content demonstrated the degradation of the extracellular matrix following this treatment. In contrast, plasma from blood preconditioned with U. urealyticum induced a highly significant secretion of interleukin-1β (IL-1β) and prostaglandin E2 (PGE2) by the amniochorion without changes in the extracellular matrix organization or content. These data demonstrate that group B streptococci induce degradation of the amniochorion as a result of MMP-9 production, probably via TNF-α, whereas U. urealyticum stimulates the secretion of PGE2, probably via IL-1β, potentially stimulating myometrial contraction. Our study provides novel evidence that the immunological cells circulating within the uterine microenvironment respond differentially to an infectious agent, triggering alternative molecular signaling pathways leading to human labor.
PMCID: PMC2976360  PMID: 20805331
8.  In vitro secretion and activity profiles of matrix metalloproteinases, MMP-9 and MMP-2, in human term extra-placental membranes after exposure to Escherichia coli 
Premature rupture of fetal membranes (PROM) complicated with intrauterine infection has been associated to alterations of the extracellular matrix (ECM) homeostasis. The aim of this work was to evaluate the integral/functional response of the amnion (AMN) and choriodecidua (CHD) to synthesis, secretion, and activity of MMP-2 and MMP-9 and of their inhibitors TIMP-1, -2, and -4, after stimulation with Escherichia coli.
Full-thickness membranes were mounted on a Transwell device, constituting two independent chambers, Escherichia coli (1×10 (6) CFU/mL) were added to either the amniotic or the choriodecidual face or to both. Secretion profiles of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-4 were quantified by ELISA and gelatinolytic activity by zymography. Immunoreactivity for MMP-2 and MMP-9 was revealed by immunohistochemistry and the collagen content was assessed by the hydroxyproline assay.
Levels of MMP-9 in CHD and AMN increased 4- and 8-fold, respectively, after simultaneous infection. MMP-2 secreted to the medium by CHD increased a mean of 3 times after direct stimulation. Secretion profiles of TIMP-1, TIMP-2, and TIMP-4 remained without significant changes. Collagen content was significantly decreased (4-fold) in infected membranes, and was associated with loss of structural continuity and co-localization with immunoreactive forms of MMP-2 and MMP-9.
Infection of chorioamniotic membranes with E. coli induces an increase in the secretion of inactive forms and an association to ECM of active forms of MMP-2 and MMP-9 without changes in TIMP-1, -2, and -4. These changes could explain the significant decrease of collagen content and loss of structural continuity.
PMCID: PMC3036613  PMID: 21266053
9.  Glycosylated VCAM-1 isoforms revealed in 2D western blots of HUVECs treated with tumoral soluble factors of breast cancer cells 
Several common aspects of endothelial phenotype, such as the expression of cell adhesion molecules, are shared between metastasis and inflammation. Here, we analyzed VCAM-1 variants as biological markers of these two types of endothelial cell activation. With the combination of 2-DE and western blot techniques and the aid of tunicamycin, we analyzed N-glycosylation variants of VCAM-1 in primary human endothelial cells stimulated with either TNF or tumoral soluble factors (TSF's) derived from the human breast cancer cell line ZR75.30.
Treatments induced a pro-adhesive endothelial phenotype. 2D western blots analysis of cells subjected to both treatments revealed the expression of the two known VCAM-1 isoforms and of previously unknown isoforms. In particular TSFZR75.30 induced an isoform with a relative molecular mass (Mr) and isoelectric point (pI) of 75-77 kDa and 5.0, respectively.
The unknown isoforms of VCAM-1 that were found to be overexpressed after treatment with TSF's compared with TNF, could serve as biomarkers to discriminate between inflammation and metastasis. 2D western blots revealed three new VCAM-1 isoforms expressed in primary human endothelial cells in response to TSF stimulation. Each of these isoforms varies in Mr and pI and could be the result of differential glycosylation states.
PMCID: PMC2787495  PMID: 19930605
10.  In vitro secretion profiles of interleukin (IL)-1beta, IL-6, IL-8, IL-10, and TNF alpha after selective infection with Escherichia coli in human fetal membranes 
Chorioamniotic membranes infection is a pathologic condition in which an abnormal secretion of proinflammatory cytokines halts fetal immune tolerance. The aim of the present study was to evaluate the functional response of human chorioamniotic membranes, as well as the individual contribution of the amnion and choriodecidua after stimulation with Escherichia coli, a pathogen associated with preterm labor.
Explants of chorioamniotic membranes from 10 women (37–40 weeks of gestation) were mounted and cultured in a Transwell system, which allowed us to test the amnion and choriodecidua compartments independently. Escherichia coli (1 × 10 6 CFU/mL) was added to either the amniotic or the choriodecidual regions or both; after a 24-h incubation, the secretion of IL-1beta, IL-6, TNFalpha, IL-8, and IL-10 in both compartments was measured using a specific ELISA. Data were analyzed by Kruskal-Wallis one-way analysis of variance.
After stimulation with Escherichia coli, the choriodecidua compartment showed an increase in the secretion of IL-1beta (21-fold), IL-6 (2-fold), IL-8 (6-fold), and IL-10 (37-fold), regardless of which side of the membrane was stimulated; TNFalpha secretion augmented (22-fold) also but only when the stimulus was applied simultaneously to both sides. When the amnion was stimulated directly, the level of IL-1beta (13-fold) rose significantly; however, the increase in IL-8 secretion was larger (20-fold), regardless of the primary site of infection. TNFalpha secretion in the amnion compartment rose markedly only when Escherichia coli was simultaneously applied to both sides.
Selective stimulation of fetal membranes with Escherichia coli results in a differential production of IL-1beta, IL-6, TNFalpha, IL-8, and IL-10. These tissues were less responsive when the amnion side was stimulated. This is in agreement with the hypothesis that the choriodecidua may play a primary role during an ascending intrauterine infection, being the main barrier to progression of the infection into the amniotic cavity. Therefore, the tissue-specific capacities for the secretion of these immune modulators could be a determining factor for the degree of severity of the inflammation process in fetal membranes.
PMCID: PMC2175507  PMID: 18078521

Results 1-10 (10)