Repeated Measures of Cervicovaginal Cytokines during Healthy Pregnancy: Understanding "Normal" Inflammation to Inform Future Screening.

OBJECTIVE: This study aimed to describe characteristics of cervicovaginal cytokines obtained during pregnancy from women who subsequently delivered at term. STUDY DESIGN: We used repeated measures of 20 cervicovaginal cytokines, collected on average on a monthly basis, from the second to the ninth month of gestation among 181 term pregnancies in the Mexico City Pregnancy Research on Inflammation, Nutrition, & City Environment: Systematic Analyses cohort (2009-2014). Cytokines were quantified using multiplex assay. RESULTS: Cytokine distributions differed more between than within cytokines. Across trimesters, cytokines interleukin (IL)-1Ra, IL-1α, and IL-8 consistently had high concentrations compared with other measured cytokines. Cytokine intraclass correlation coefficients ranged from 0.41 to 0.82. Spearman's correlation coefficients among cytokine pairs varied but correlation directions were stable; 95.3% of the 190 correlation pairs remained either negative or positive across trimesters. Mean longitudinal patterns of log-transformed cytokines from Tobit regression varied across but less within cytokines. CONCLUSION: Although mean concentrations of cervicovaginal cytokines among term pregnancies were high, they were largely stable over time. The high cytokine concentrations corroborate that pregnancy is associated with an active inflammatory state. These characterizations may serve as a baseline for comparison to other obstetric outcomes, which may be helpful in understanding deviations from normal gestational inflammation.

DNA Methylation Variability in a Single Locus of the RXRα Promoter from Umbilical Vein Blood at Term Pregnancy.

DNA methylation status of RXRα gene promoter has been correlated with maternal diet during early pregnancy, and associated with offspring's adiposity and bone mineral content. In adult life, increased methylation of RXRα promoter region is associated with myocardium pathologies. Early growth response proteins (EGR) are zinc finger transcription factors associated with several cellular pathways such as inflammation, apoptosis, and cardiopathies. DNA-binding sequences of EGR proteins have been reported in the RXRα gene promoter using chromatin immunoprecipitation methods. Here, we used correlations between the maternal pre-pregnancy body mass index (p-BMI), gestational weight gain (GWG), and birth weight (BW) as indirect indicators of the maternal nutritional status as modifier of DNA methylation in the offspring. DNA methylation status from newborns' umbilical vein blood in full-term pregnancy was evaluated in a short sequence (116 pb) of the RXRα gene promoter that contains the elements of response sequence for EGR proteins. Fifty-three bisulfite-modified DNA samples were assessed through methyl-sensitive high-resolution melting (MS-HRM) analysis. To validate the results, we directly sequenced MS-HRM samples to confirm the presence of CpG-methylated positions. In addition, the RXRα protein levels in extracts of umbilical vein blood were evaluated by western blot. We found differential methylation in a specific locus of the RXRα promoter surrounding the EGR-binding sequence; however, no correlation was found with the level of RXRα protein expression. Variability in the methylation status of the RXRα promoter near the EGR transcription factor binding site in newborn cord blood provides controversial epigenetic insights into RXRα regulation via EGR proteins.

[Adverse perinatal outcomes in Mexican women with twin pregnancy achieved by assisted reproduction techniques vs. spontaneous twin pregnancies]. / Resultados perinatales adversos en mujeres mexicanas con embarazos gemelares por reproducción asistida vs. gemelares espontáneos.

BACKGROUND: Twin pregnancies (TP) have shown a greater risk of adverse perinatal outcomes than singletons have. However, there is still no consensus about whether the TP achieved by assisted reproduction techniques (ART) have worse perinatal outcomes compared with TP achieved spontaneously. OBJECTIVE: Compare the incidence of adverse perinatal outcomes (preterm birth, premature rupture of membranes, gestational diabetes, pregnancy-induced hypertension, anemia, weight and destination of the newborn) in Mexican women with TP achieved spontaneously vs those with TP achieved by ART. METHODS: Historical cohort study with two sample groups: group 1, women with TP achieved spontaneously, and group 2, women with TP achieved by ART. Women with TP achieved by TRA were matched 1:1 with women with EG achieved spontaneously for age, weeks of gestation, chorionicity and body mass index at admission to prenatal care. Adverse perinatal outcomes were compared between the two groups and the odds ratio (OR) had a 95% confidence interval. RESULTS: There were 57 women per group. Baseline characteristics were similar in both groups except for nulliparity (38.6% in group 1 vs 82.5% in group 2 (p < 0.0001)). In group 2 there was a higher incidence of pregnancy-induced hypertension (group 1 (19.3%) vs. group 2 (42.1%), OR 2.5 (95% CI 1.07-5.8)). Newborns in group 2 were admitted to nursery more often than those in group 1 (49.1% vs. 35.1% OR 1.7 IC 95% 1.04-3.04). There was no difference in other adverse perinatal outcomes. CONCLUSIONS: Mexican women with TP achieved by ART had higher risk of pregnancy-induced hypertension compared to women with TP achieved spontaneously.

Association between adiposity and inflammatory markers in maternal and fetal blood in a group of Mexican pregnant women.

In the present pilot study, we evaluated the effect of maternal adiposity on the plasma concentration of adipocytokines in pregnant women and their newborns. Twenty patients with term gestations without labour were initially selected by pregestational BMI and then classified into two study groups (n 10 each), according to their median value of adiposity (total body fat). Concentrations of TNF-α, IL-1ß, IL-6, leptin and adiponectin in plasma of maternal peripheral blood and fetal cord blood were measured and correlated to maternal adiposity. Maternal adiposity showed a significant negative correlation with fetal adiponectin (r - 0·587, P = 0·01) and IL-6 (r - 0·466, P = 0·05), a significant positive correlation with maternal leptin (r 0·527, P = 0·02) and no correlation with TNF-α or IL-1ß. Adiponectin was higher in fetal plasma than in maternal plasma (P = 0·043), but significantly lower in newborns from women with high adiposity than in newborns from women with low adiposity (P = 0·040). Our results suggest that fetuses from obese women may be less able to control inflammation, due to lower circulating anti-inflammatory adipocytokines, which could limit their optimal development or even increase the risk of abortion or preterm labour.

[Expression of cell adhesion molecules in the maternal fetal period of human gestation]. / Expresión de moléculas de adhesión celular en la interfase materno fetal al término de la gestación humana.

BACKGROUND: The human labor is an inflammatory process invading leukocytes modulated by gestational tissues. The local increase of cell adhesion molecules (CAMs) promotes the permanence of these leukocytes in the coriodecidua. Gestational tissues express ICAM-1, while circulating leukocytes expressing its ligand Mac-1. OBJECTIVE: To analyze, first, if the expression of CAMs in the fetal membranes is associated with progress of gestational age, and second, the expression of CAMs on circulating leukocytes in the uterus (placenta). MATERIAL AND METHOD: original and closed study conducted at the Instituto Nacional de Perinatologia Isidro Espinosa de los Reyes (Mexico City). We included samples from healthy women between 15 and 44 years of age with term pregnancies (> or =37 weeks gestation). RESULTS: Real time PCR analysis showed that the expression of CAMs in the fetal membranes remained constant before labor. ICAM3 and ICAM1 tended to increase during labor, while ICAM2, VCAM1, SELE and SELP decrease with advancing gestational age. Placental leukocytes showed a clear increase in the expression of ITGAM (Mac-1) during labor. CONCLUSIONS: These results show that the maternal-fetal interface expresses a specific combination of CAMs during labor, including ICAM1, ICAM-3 and Mac-1. The expression of these molecules could promote the retention of leukocytes in the local tissues to modulate the local inflammatory microenvironment during human labor.

Influence of pre-pregnancy body mass index (p-BMI) and gestational weight gain (GWG) on DNA methylation and protein expression of obesogenic genes in umbilical vein.

Understanding the regulatory mechanisms that affect obesogenic genes expression in newborns is essential for early prevention efforts, but they remain unclear. Our study aimed to explore whether the maternal p-BMI and GWG were associated with regulatory single-locus DNA methylation in selected obesogenic genes. For this purpose, DNA methylation was assayed by Methylation-Sensitive High Resolution Melting (MS-HRM) technique and Sanger allele-bisulfite sequencing in fifty samples of umbilical vein to evaluate glucosamine-6-phosphate deaminase 2 (GNPDA2), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), and leptin receptor (LEPR) genes. Correlations between DNA methylation levels and indicators of maternal nutritional status were carried out. Western blotting was used to evaluate protein expression in extracts of the same samples. Results indicated that GNPDA2 and PGC1α genes have the same level of DNA methylation in all samples; however, a differential DNA methylation of LEPR gene promoter was found, correlating it with GWG and this correlation is unaffected by maternal age or unhealthy habits. Furthermore, leptin receptor (Lep-Rb) was upregulated in samples that showed the lowest levels of DNA methylation. This study highlights the association between poor GWG and adjustments on obesogenic genes expression in newborn tissues with potential consequences for development of obesity in the future.

[Spontaneous uterine rupture in a patient with polyhydramnios as only risk factor. Case report, literature review and institutional experience]. / Rotura uterina espontánea en una paciente con polihidramnios como único factor de riesgo. Comunicación de un caso, revisión de la bibliografía y experiencia institucional.

The uterine rupture is a catastrophic obstetric complication. The main risk factor is an antecedent of uterine surgery, usually caesarean. It is reported the case of a 39-years-old patient with 37 week-pregnancy and polyhydramnios, without surgical antecedents, whose was not in labor and developed complete rupture of the lateral face of the uterus, which was spontaneous, without previous uterine scar and with a unusual outcome.

[Evidence that periplacentary leukocytes subpopulations of term pregnancy have functional properties associated with labor induction]. / Evidencia de que las subpoblaciones de leucocitos periplacentarios al término de la gestación poseen propiedades funcionales relacionadas con la inducción del trabajo de parto.

BACKGROUND: The rise of cholagenolitic activity in fetal membranes, associated to premature rupture, have been related to abnormal activity of the extracellular matrix metalloproteinases discharged to the extracellular space as inactive enzymes that have to be activated to selectively degrade its components. OBJECTIVE: To analyze the functional properties of leukocytes subpopulations coming from the placental circulation. MATERIAL AND METHODS: Biomedical experimental study in which placental and outlying blood leukocytes were cultivated during 96 hours, from women with pregnancy to term without labor. Leukocytes subpopulations were stained by flow cytometry. Culture media were analyzed with zymography and enzymatic activity profile was evaluated in presence of proteases inhibitors. RESULTS: Placental leukocytes are composed of T-, NK- and B-lymphocytes, and monocytes; it was documented a progressive increase of inactive MMP-9 secretion (92 kDa), accompanied by an 82 kDa form MMP-9 activation since 48 hours. Enzymatic profile mainly showed metalloproteasas. CONCLUSIONS: Placental blood leukocytes showed functional capacities different from those that circulate in pregnant women's outlying circulation. Placental leukocytes, mainly T-lymphocytes, are characterized by the specific capacity to secrete and activate MMP-9; an enzyme that participates in fetal membranes degradation. It suggests that in placental surroundings are recruited cells specialized in labor changes induction.

[Interleukin-6 levels changes in cervicovaginal exudates in labor evolution]. / Cambios en la concentración de la interleucina 6 en exudados cervicovaginales en trabajo de parto.

OBJECTIVE: To evaluate the secretion of interleukin 6 (IL-6) in cervicovaginal fluid in a pseudocohort that emulates the evolution of the labor. MATERIAL AND METHOD: Samples of cervicovaginal fluid of patients with 20 to 40 weeks of gestation were taken, patients were classified in 5 strata of the development of the labor. Each stratum reflects the progressive activation of the uterine activity and the cervical changes. To each sample was made determination of IL-6 by means of the system Multiplex. The samples of patient with infection data were eliminated. RESULTS: 173 samples were included distributed in each one of the five strata. Basal secretion exists of IL-6 to the cervicovaginal fluid along the gestation, that doesn't modify until the active labor appears. Only when the uterine activity is manifested in form to regulate and effective, very significant increase is documented in the concentration of the IL-6 in the cervicovaginal fluid. CONCLUSIONS: The IL-6 it is a proinflammatory cytokine that increases in a specific way in the moment in that the effective uterine activity begins, thus, it is an excellent candidate to be evaluated as marker presage of the event of normal labor and preterm.

[Extracellular matrix metalloproteases profile identification in chorioamniotic membranes of term and preterm pregnancies through soluble microarrays]. / Identificación del perfil de metaloproteasas de matriz extracelular en membranas corioamnióticas de embarazos a término y pretérmino mediante microarreglos solubles.

BACKGROUND: Physiological and pathological membrane rupture is a complex phenomenon with different biochemical processes; it is known that collagenolitic activity rises and collagen content diminishes within term tissue membranes in comparison to preterm membranes. Identification of these processes within rupture mechanism allows to suggest that fetal membranes and decidua can respond to biochemical and mechanical stimulus alike, and to produce mediators that degrade matrix of intracellular membranes. OBJECTIVE: To identify simultaneously, whit a soluble microarray, different matrix metalloproteinases in extracts from amniochorion of pregnancies at term and preterm. PATIENTS AND METHODS: Biomedical experimental study where amniochorion explants were obtained from four women groups. Group 1: at term with spontaneous labor; group 2: at term without labor; group 3: at term with premature rupture of membranes, and group 4: preterm labor. Explants were cultured for 24 h and then homogenated in their own culture media to obtain cell free extracts. MMP were identified in these extracts using a soluble microarray for MMPs that included: MMP-1, -2, -3, -7, -8, -9, -12 and -13. RESULTS: MMP-8 and -2 were the enzymes most abundant in all the extracts of amniochorion. However, the concentration of MMP-8 in the extracts of group 3 (PROM) was significantly greater in comparison with the extracts of groups 1 and 2 (p = 0.01). The MMP-8 also was in greater concentration in the extracts of group 4 (preterm labor) in comparison with in the extracts of group 1 (p = 0.01). CONCLUSION: Activation of cellular processes that lead to the degradation of connective tissue in the MCA under physiological conditions seems to defer in originating tissues from cases with PROM or preterm labor, and this activation is characterized by an increase in the concentration of MMP-8.

[Three-dimensional analysis of human chorioamniotic membranes structure in term pregnancy]. / Análisis tridimensional de la estructura de las membranas corioamnióticas humanas al término de la gestación.

BACKGROUND: Physical and structural chorioamniotic membranes integrity is due to a precise process of synthesis and degradation of collagen; surrounding collagenolitic activity raises during labor, what leads to a structural loss and mechanical resistance weakening, the main cause of its rupture under physiological and pathological conditions. Understanding of its three-dimensional structure is essential to characterize normal and pathological labor. OBJECTIVE: To analyze three-dimensional structure of human chorioamniotic membranes at gestational term. MATERIAL AND METHODS: Descriptive study to analyze the distribution of collagens type I, II and IV in human chorioamniotic membranes at term (37 to 40 gestational weeks) without labor by means of confocal and electronic scan microscopy. RESULTS: Cells' amnios shapes a homogeneous epithelium without a close intercellular contact (classic epithelium) what may contribute to transmembranal diffusion molecules' transport. Amnios connective tissue is too a complex fibrilar net of type I collagen, structurally supported by type IV collagen. On the contrary, corion has a great amount of cells in close contact, with a few fibers of type I and II collagen, and almost none of type IV collagen cells. CONCLUSION: Three-dimensional analysis of chorioamniotic membranes connective tissue, particularly amnios, allows to understand the main role of type IV collagen on supporting its structure, as well as collagenolitic enzymes in its degradation and rupture under normal and pathological conditions.

[Development and validation of an in vitro culture model for the study of the differentiation of human trophoblast]. / Desarrollo y validación de un modelo de cultivo in vitro para el estudio de la diferenciación del trofoblasto humano.

OBJECTIVES: To develop a method to isolate cells of human citotrophoblast and to assess its invading and differentiation capacity. TYPE OF STUDY: Experimental biomedical. MATERIALS AND METHOD: Citotrophoblasts of healthy placentas of full-term pregnancies were isolated by digestion with dispase and purification in a density gradient. The purity by immunoreactivity to citokeratin 7 and the invasiveness of the cells of citotrophoblast in Matrigel were evaluated. The enzymatic activity was determined through zimography and hCG secreted was quantified by means of ELISA. The expression of alpha 1 and alpha 5 integrins was evaluated by immunohistochemistry. RESULTS: Citotrophoblasts with a purity of 97% were obtained; they differentiated themselves, in a spontaneous way, to a syncytium after four days. There was a growing production of hCG. Maximum invasiveness of citotrophoblasts ocurred the first two days, when their phenotype was mononuclear and coincided with the secretion of pro-MMP-9, and then it diminished according with the culture time. Immunoreactivity to the alpha 1 and alpha 5 integrins was observed in citotrophoblast cells with mononuclear phenotype. This immunoreactivity was lower in cells with phenotype of syncytium. CONCLUSIONS: It was created an in vitro model that replicates events of the early development of the placenta. These events resemble the invasion and differentiation phase of the citotrophoblast. This model has potential utility in the study of the mechanisms of damage in preeclampsia.

[Identification of extracellular matrix metalloprotease 3 in the fetal membrane of the rat and its possible implication in the rupture of chorioamniotic membranes]. / Identificación de la metaloproteasa de matriz extracelular-3 en la membrana fetal de la rata y su posible implicación en la rotura de las membranas corioamnióticas.

OBJECTIVE: Human corioamniotic membranes, or their equivalent in the rat, function as selective barrier during gestation and their rupture is part of the mechanisms implied in the labor. Molecular mechanisms carried out in this process are unknown. TYPE OF STUDY: Experimental animal model. MATERIAL AND METHODS: Corioamniotic membranes (obtained at the beginning of the labor) of rats with programmed and synchronous pregnancies were analized. The coexistence and distribution of metalloproteinase of extracellular-3 matrix (estromelisine) in these tissues were determined. RESULTS: Secretion and tissue location of metalloproteinase of extracellular-3 matrix in fetal membranes were identified for the first time. Metalloproteinase of extracellular-3 matrix was immunolocated in the compact layer of the amnion and its secretion (by the membranes) was confirmed through electrophoresis, zimography and Western blot. By confocal microscopy it was verified that metalloproteinase of extracellular-3 matrix is located in the same places of that of extracellular-9 matrix. CONCLUSIONS: Rupture of corioamniotic membranes relates to the expression and local activity of the metalloproteinases of extracellular matrix. The coexistence of metalloproteinase of extracellular-3 matrix in the amnion of the rat has been identified; this element is added to the biochemical process of rupture, since metalloproteinase of extracelular-3 matrix is an activator of that of extracellular-9 matrix. It is possible that the physiological function of this enzyme is implied, of a main way, in the process of rupture of corioamniotic membranes during the childbirth.

[Assessment of a microarray of solid-phase antibodies to the study of chemokines secreted by the chorioamniotic membrane]. / Evaluación de un microarreglo de anticuerpos en fase sólida para el estudio de quimiocinas secretadas por la membrana corioamniótica.

OBJECTIVE: At the end of pregnancy, a complex signaling network of cytokines, chemokines and proteic effector molecules is started. Due to such complexity, this network is very difficult to analyze. The aim of the present study was to evaluate the effectiveness of an antibody-based proteomic analysis method to distinguish the simultaneous presence of different molecules in biological samples. MATERIAL AND METHOD: Amniochorion explants were obtained from women at term with spontaneous labor (n = 4) and subjected to cesarean section without labor (n = 4). Explants were cultured for 24 h and then homogenated in their own culture media to obtain cell free extracts. Chemokines were identified in these extracts using a commercial array for chemokines that included: eotaxin, eotaxin 2, 1-309, IL-8, IP-10, MCP-1, MCP-2, MIG, MIP-1alpha, MIP-1beta, MIP-1delta and RANTES. RESULTS: All the included chemokines were found in amniochorion extracts, being IL-8 the most abundant. However, once labor is present, all chemokines tend to appear in greater concentrations than those from non-in-labor tissues, except for RANTES, which disappeared when labor began. CONCLUSION: This methodological approach shows that amniochorion from term pregnancies secrete chemokines with a characteristic qualitative profile during labor. This approach would allow the fast evaluation of potential markers of this phenomenon in physiologic or pathologic conditions.

Initial characterization of the microenvironment that regulates connective tissue degradation in amniochorion during normal human labor.

Extracellular matrix degradation in fetal membranes leading to its rupture is coupled to myometrial activity and cervical ripening during human normal labor. Mechanisms which modulate collagen degradation in amniochorion during labor have not been elucidated. Initial characterization of the effect of different blood compartments on connective tissue degradation in amniochorion during human labor was explored. Amniochorion explants were stimulated with plasma of maternal venous blood, umbilical cord blood or placental blood, obtained from women with pregnancies at term, with or without labor. MMP-2 and MMP-9 activities were quantified in conditioned media by gelatin-zymography as an index of connective tissue degradation. Collagen content was measured in tissue explants and collagen fibrils distribution was examined by electron microscopy. Placental plasma from term pregnancies, with or without labor, is enriched with soluble signals that enhance the in vitro MMP-9 production by amniochorion. Accompanying ultrastructural distortion of collagen fibers and demonstration of collagen degradation fragments confirmed induction of extracellular matrix degradation. Control experiments in which MMP-9 activity was blocked with TIMP-1 resulted in inhibition of all the above mentioned changes. These results suggest that placental intervillous space is a functional compartment in which mediators capable to induce collagen degradation in amniochorion are selectively expressed during human labor.

[Connective tissue degradation in human amniochorion after stimulation with choriodecidual lymphocytes infected with group B streptococci]. / La interacción de las células inmunológicas coriodeciduales con estreptococo del grupo B condiciona la degradación del tejido conectivo del corioamnios.

OBJECTIVE: To identify whether soluble products from choriodecidual blood cells stimulated with group B streptococci (GBS) induce connective tissue degradation in human amniochorion. MATERIAL AND METHODS: Blood samples from choriodecidual compartment were collected by direct aspiration from placental cotyledons draining blood and represent local circulating cells. Samples were divided into two aliquots: one was stimulated with GBS (1 X 10(6) CFU/mL) and the other was kept free of bacteria as negative control. After overnight incubation, plasmas were separated. Chorioamnion explants were stimulated with 10% plasma for 12h at 37 degrees C in 5% CO2. MMP-9 proteolytic activity was measured in the supernatants by gelatin-zymography and IL-1beta and TNF-alpha were quantified by ELISA. Distribution of the collagenous fibrils in explants was examined by electron microscopy. STATISTICAL ANALYSIS: three independent experiments on duplicate were carried out and the statistical significance of experimental differences between groups was assessed with ANOVA test. RESULTS: MMP-9, IL-1beta and TNF-alpha production was significantly higher in supernatants from explants co-cultured with choriodecidual plasma from blood previously infected with GBS, compared with control plasma. Accompanying extensive changes of connective tissue arrangement confirm induction of extracellular matrix degradation. CONCLUSIONS: Choriodecidual plasma from blood stimulated with GBS is enriched with biochemical signals that enhance the MMP-9, IL-1alpha and TNF-beta production by amniochorion. These findings suggest that local circulating cells are capable to act in response to GBS choriodecidual infection through extracellular matrix degradation and the consequent rupture of membranes.

Matrix Metalloproteinase-3 (MMP-3) Is an Endogenous Activator of the MMP-9 Secreted by Placental Leukocytes: Implication in Human Labor.

BACKGROUND: The activity of matrix degrading enzymes plays a leading role in the rupture of the fetal membranes under normal and pathological human labor, and matrix metalloproteinase-9 (MMP-9) it is considered a biomarker of this event. To gain further insight into local MMP-9 origin and activation, in this study we analyzed the contribution of human placental leukocytes to MMP-9 secretion and explored the local mechanisms of the pro-enzyme activation. METHODS: Placental blood leukocytes were obtained from women at term gestation without labor and maintained in culture up to 72 h. MMP-9 activity in the culture supernatants was determined by zymography and using a specific substrate. The presence of a potential pro-MMP-9 activator in the culture supernatants was monitored using a recombinant biotin-labeled human pro-MMP-9. To characterize the endogenous pro-MMP-9 activator, MMP-1, -3, -7 and -9 were measured by multiplex assay in the supernatants, and an inhibition assay of MMP-9 activation was performed using an anti-human MMP-3 and a specific MMP-3 inhibitor. Finally, production of MMP-9 and MMP-3 in placental leukocytes obtained from term pregnancies with and without labor was assessed by immunofluorescence. RESULTS: Placental leukocytes spontaneously secreted pro-MMP-9 after 24 h of culture, increasing significantly at 48 h (P≤0.05), when the active form of MMP-9 was detected. Culture supernatants activated the recombinant pro-MMP-9 showing that placental leukocytes secrete the activator. A significant increase in MMP-3 secretion by placental leukocytes was observed since 48 h in culture (P≤0.05) and up to 72 h (P≤0.001), when concentration reached its maximum value. Specific activity of MMP-9 decreased significantly (P≤0.005) when an anti-MMP-3 antibody or a specific MMP-3 inhibitor were added to the culture media. Placental leukocytes from term labor produced more MMP-9 and MMP-3 compared to term non-labor cells. CONCLUSIONS: In this work we confirm that placental leukocytes from human term pregnancies are able to secrete large amounts of MMP-9, and that the production of the enzyme it is enhanced by labor. We also demonstrate for the first time that endogenous MMP-3 plays a major role in MMP-9 activation process. These findings support the contribution of placental leukocytes to create the collagenolytic microenvironment that induces the rupture of the fetal membranes during human labor.

[Characterization of choriodecidual space as an effector molecule-rich environment that induces rupture of fetal membranes during labor]. / Caracterización del espacio coriodecidual como un microambiente rico en moléculas efectoras que inducen la rotura de las membranas corioamnióticas durante el trabajo de parto.

OBJECTIVE: To identify a specific microenvironment in direct contact with fetal membranes where effector molecules acumulate, aiming to degrade the components of its extracellular matrix during labor. TYPE OF STUDY: Experimental, analytic, longitudinal and prospective. MATERIALS AND METHODS: Blood samples were collected from maternal, fetal and choriodecidual compartments, and mononuclear cells were isolated. Part of these cells was stained with antibodies to determine leukocyte subpopulations by flow cytometry. The other part was cocultured for 12 h with amniochorion explants. After coculture, MMP-9 was identified on the mononuclear cells by immunofluorescence. IL-1beta and TNF-alpha were determined in the supernatants by ELISA. Three independent experiments were carried out with duplicates and analyzed with Mann-Whitney's U test. RESULTS: Although there were no significant differences in the mononuclear cell subpopulations from the three compartments, MMP-9 production was higher in choriodecidual cells than in those of the maternal and fetal compartments. Furthermore, IL-1beta and TNF-alpha were significantly more abundant in cocultures with choriodecidual cells compared with the other two compartments. CONCLUSIONS: During labor, choriodecidual cell subpopulations are not phenotypically different from those of the maternal or fetal compartments, but they are regarding MMP-9 production, which suggests that the environment surrounding chorioamniotic membranes enhances the synthesis of this enzyme, thus promoting degradation of connective tissue.

[Connective tissue degradation in human amniochorion after stimulation with choriodecidual lymphocytes infected with group B streptococci]. / La interacción de los linfocitos coriodeciduales con los estreptococos del grupo B degrada el tejido conectivo del corioamnios.

OBJECTIVE: To identify whether soluble products from choriodecidual blood cells stimulated with group B streptococci (GBS) induce connective tissue degradation in human amniochorion. MATERIAL AND METHODS: Blood samples from choriodecidual compartment were collected by direct aspiration from placental cotyledons draining blood and represent local circulating cells. Samples were divided into two aliquots: one was stimulated with GBS (1 x 10(6) CFU/mL) and the other was kept free of bacteria as negative control. After overnight incubation, plasmas were separated. Chorioamnion explants were stimulated with 10% plasma for 12h at 37 degrees C in 5% CO2. MMP-9 proteolytic activity was measured in the supernatants by gelatin-zymography and IL-1beta and TNF-alpha were quantified by ELISA. Distribution of the collagenous fibrils in explants was examined by electron microscopy. STATISTICAL ANALYSIS: three independent experiments on duplicate were carried out and the statistical significance of experimental differences between groups was assessed with ANOVA test. RESULTS: MMP-9, IL-1beta and TNF-alpha production was significantly higher in supernatants from explants co-cultured with choriodecidual plasma from blood previously infected with GBS, compared with control plasma. Accompanying extensive changes of connective tissue arrangement confirm induction of extracellular matrix degradation. CONCLUSIONS: Choriodecidual plasma from blood stimulated with GBS is enriched with biochemical signals that enhance the MMP-9, IL-1beta and TNF-alpha production by amniochorion. These findings suggest that local circulating cells are capable to act in response to GBS choriodecidual infection through extracellular matrix degradation and the consequent rupture of membranes.

[Changes in the systemic immunologic response in association with endometriosis using an animal model]. / Alteración de la respuesta inmunológica sistémica en asociación con endometriosis, empleando un modelo de estudio animal.

INTRODUCTION: Endometriosis constitutes the growth of endometrial tissue in a place other than the uterine cavity. Its etiopathogenesis is unknown, although there is some evidence associating it with the decrease of cytotoxic activity in the immunological system. OBJECTIVE: Evaluating the relationship between the development of ectopic endometrial tissue and the immunological status, and enumerating lymphocyte subpopulations and cytokine synthesis in T lymphocytes, using a murine endometriosis model. METHODOLOGY: Spleen lymphocytes isolated from two study groups of 10 female mice of the Balb/c strain that had been submitted to the surgical implantation of autologous endometrial tissue in the peritoneal cavity, and sacrificed after 5 (group I) and 8 (group II) weeks, were incubated--or not--with PMA/lonomicine. Lymphocyte T numbers and their cytokine production were determined by flow cytometry. RESULTS: A lower dispersion of the ectopic tissue growth value was observed in group II (24% vs. 42%). A smaller population of cytotoxic T lymphocytes and a greater IL-4 production in the stimulated cells of the study group (p < 0.05) were observed, as compared to the control group. CONCLUSIONS: The presence of endometrial tissue in the uterine cavity decreases the amount of cytotoxic T lymphocytes and increases IL-4 production in total T lymphocytes, suggesting a modulation of the systemic immunological response to TH-2.