RESUMO
Prior to and during the process of human labor, maternal circulating leukocytes infiltrate the maternal-fetal interface (choriodecidua) and become activated resembling choriodecidual leukocytes. Since, there is no evidence comparing maternal circulating and choriodecidual leukocytes, herein, we characterized their transcriptome and explored the biological processes enriched in choriodecidual leukocytes. From women undergoing spontaneous term labor we isolated circulating and choriodecidual leukocytes, performed microarray analysis (n = 5) and qRT-PCR validation (n = 9) and interaction network analysis with up-regulated genes. We found 270 genes up-regulated and only 17 genes down-regulated in choriodecidual leukocytes compared to maternal circulating leukocytes. The most up-regulated genes were CCL18, GPNMB, SEPP1, FN1, RNASE1, SPP1, C1QC, and PLTP. The biological processes enriched in choriodecidual leukocytes were cell migration and regulation of immune response, chemotaxis, and humoral immune responses. Our results show striking differences between the transcriptome of choriodecidual and maternal circulating leukocytes. Choriodecidual leukocytes are enriched in immune mediators implicated in the spontaneous process of labor at term.
Assuntos
Decídua/metabolismo , Trabalho de Parto/genética , Leucócitos/metabolismo , Transcriptoma , Adulto , Decídua/citologia , Feminino , Humanos , Trabalho de Parto/sangue , Trabalho de Parto/metabolismo , GravidezRESUMO
PROBLEM: 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.
Assuntos
Córion/imunologia , Decídua/imunologia , Leucócitos Mononucleares/imunologia , Subpopulações de Linfócitos/imunologia , Placenta/imunologia , Células Cultivadas , Microambiente Celular , Citocinas/metabolismo , Feminino , Humanos , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Gravidez , Nascimento a Termo/imunologiaRESUMO
OBJECTIVE: To evaluate the presence of merocyanine 540 (M540) bodies and their impact on the measurement of apoptotic biomarkers in human spermatozoa. DESIGN: Case-control, prospective study. SETTING: Academic centers. PATIENT(S): Fertile and subfertile subjects. INTERVENTION(S): Semen samples from subfertile and fertile men, 11 per group, were analyzed for basic semen parameters and early (annexin-V binding) and late (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) sperm apoptotic biomarkers by flow cytometry. Samples were also stained with M540 to assess the presence of M540 apoptotic bodies. MAIN OUTCOME MEASURE(S): Presence of M540 apoptotic bodies. RESULT(S): Groups differed significantly in the expression of early and late apoptosis biomarkers. The percentage of M540 bodies between groups was not different. The exclusion of M540 bodies from TUNEL results did not have a significant impact on measurement in either fertile or subfertile groups. CONCLUSION(S): This study confirmed the occurrence of M540 bodies in semen and that male factor infertility is associated with an increased expression of apoptosis biomarkers. Moreover, we demonstrated that the presence of M540 bodies did not affect the quantification of apoptotic biomarkers in either group.
Assuntos
Apoptose/fisiologia , Citometria de Fluxo/métodos , Infertilidade Masculina/patologia , Pirimidinonas , Espermatozoides/patologia , Adulto , Anexina A5/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Estudos Transversais , Fragmentação do DNA , Ejaculação , Corantes Fluorescentes , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Estudos Prospectivos , Espermatozoides/metabolismoRESUMO
PROBLEM: Spontaneous labor at term involves leukocyte recruitment and infiltration into the choriodecidua; yet, characterization of these leukocytes and their immunological mediators is incomplete. The purpose of this study was to characterize the immunophenotype of choriodecidual leukocytes as well as the expression of inflammatory mediators in human spontaneous parturition at term. METHOD OF STUDY: Choriodecidual leukocytes were analyzed by FACS, immunohistochemistry, and RT-PCR in three different groups: (i) preterm gestation delivered for medical indications without labor; (ii) term pregnancy without labor; and (iii) term pregnancy after spontaneous labor. RESULTS: Two T-cell subsets of memory-like T cells (CD3(+) CD4(+) CD45RO(+) and CD3(+) CD4(-) CD8(-) CD45RO(+) cells) were identified in the choriodecidua of women who had spontaneous labor. Evidence for an extensive immune signaling network composed of chemokines (CXCL8 and CXCL10), chemokine receptors (CXCR1-3), cytokines (IL-1ß and TNF-α), cell adhesion molecules, and MMP-9 was identified in these cells during spontaneous labor at term. CONCLUSIONS: The influx of memory-like T cells in the choriodecidua and the evidence that they are active by producing chemokines and cytokines, and expressing chemokine receptors, cell adhesion molecules, and a matrix-degrading enzyme provides support for the participation of the adaptive immune system in the mechanisms of spontaneous parturition at term.
Assuntos
Imunidade Adaptativa , Decídua/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Antígenos CD/metabolismo , Separação Celular , Células Cultivadas , Quimiocina CXCL10/metabolismo , Citocinas/metabolismo , Membranas Extraembrionárias/imunologia , Feminino , Citometria de Fluxo , Humanos , Memória Imunológica , Mediadores da Inflamação/metabolismo , Interleucina-8/metabolismo , Parto/imunologia , Gravidez , Receptores de Citocinas/metabolismo , Transdução de SinaisRESUMO
A gradient of immunological mediators exists in the fetal membranes from the periplacental zone (PZ) to the rupture zone (RZ) at term delivery (rupture of fetal membranes [ROM]). However, it is unknown if this gradient is different in premature rupture of these tissues (premature rupture of fetal membranes [PROM]). We therefore analyzed leukocyte chemotactic activity and chemokine/cytokine production in fetal membrane zones in ROM and PROM. In ROM, leukocyte chemotactic activity increased from the PZ to the RZ; however, this did not occur in PROM. This was due to consistently elevated leukocyte chemotactic activity in PROM compared to ROM tissues. In the RZ, ROM was characterized by increased T-cell attraction and high levels of chemokine (C-X-C motif) ligand 8 (CXCL-8)/interleukin 8, and PROM by increased granulocyte attraction and high levels of granulocyte-macrophage colony-stimulating factor and CXCL-10/interferon gamma-induced protein 10. We conclude that normal and premature rupture of fetal membranes differ in regional chemotactic activity and related chemokine/cytokine production, which may represent evidence for differential mechanisms of rupture at term delivery.
Assuntos
Quimiocinas/metabolismo , Quimiotaxia/fisiologia , Citocinas/metabolismo , Parto Obstétrico , Membranas Extraembrionárias/metabolismo , Ruptura Prematura de Membranas Fetais/metabolismo , Feminino , Ruptura Prematura de Membranas Fetais/diagnóstico , Humanos , Técnicas de Cultura de Órgãos , GravidezRESUMO
Pre-eclampsia is a syndrome characterized by inadequate placentation, which is due to deficient trophoblastic invasion of the uterine spiral arteries. This deficiency can lead to placental hypoxia, secretion of proinflammatory cytokines, and release of angiogenic and antiangiogenic factors. Hypoxic conditions in the placenta can promote oxidative stress and the production of angiogenic factors that are antagonized by soluble receptors, which are also elevated in this syndrome. In addition to these factors, the development of hypertension in women with pre-eclampsia may be associated with the renin-angiotensin system and endothelial dysfunction. The presence of antiangiotensin II type 1 receptor autoantibodies is relevant in pre-eclampsia because it has been related to the secretion of antiangiogenic factors through cytokine pathways, indicating that autoimmune mechanisms may participate in the pathophysiology of this syndrome.
Assuntos
Proteínas Angiogênicas/metabolismo , Autoanticorpos/metabolismo , Placenta/fisiopatologia , Pré-Eclâmpsia/fisiopatologia , Linfócitos B/imunologia , Feminino , Humanos , Hipóxia/fisiopatologia , Pré-Eclâmpsia/imunologia , Pré-Eclâmpsia/metabolismo , Gravidez , Receptor Tipo 1 de Angiotensina/imunologia , Sistema Renina-Angiotensina , Proteínas ras/imunologia , Proteínas ras/metabolismoRESUMO
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.
Assuntos
Bioensaio/métodos , Quimiotaxia de Leucócito/fisiologia , Citometria de Fluxo/métodos , Adulto , Quimiotaxia de Leucócito/efeitos dos fármacos , Feminino , Humanos , Variações Dependentes do Observador , Fenótipo , Reprodutibilidade dos Testes , Albumina Sérica/farmacologiaRESUMO
OBJECTIVE: The purpose of this study was to examine the histologic and immunologic differences between fetal membrane zones after membrane rupture at term delivery. STUDY DESIGN: Fetal membrane explants from postrupture zones (periplacental, middle, rupture) were obtained from women following spontaneous vaginal delivery at term (n = 5). Tissues for histology, protein extracts, and RNA were isolated. RESULTS: The collagen distribution decreased and the leukocyte density increased from the periplacental zone to the rupture zone. T cells were mainly present in the rupture zone and granulocytes in the middle zone. CXCL10, CXCR1, ICAM-1, -2, PSEL, tumor necrosis factor alpha, and matrix metalloproteinase-9 levels were higher in the middle zone than in the rupture zone and periplacental zone (P < .021). Interleukin-1beta and CXCL8 levels were higher in the rupture zone than in the middle zone and periplacental zone (P = .018 and P < .0001). CONCLUSION: During labor specific immunologic microenvironments are created in the zones of the fetal membrane that may be involved in their rupture at the end of gestation.
Assuntos
Microambiente Celular/fisiologia , Membranas Extraembrionárias/metabolismo , Inflamação/metabolismo , Adulto , Parto Obstétrico , Feminino , Humanos , Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Gravidez , Nascimento a TermoRESUMO
Procedural learning refers to the acquisition of motor skills and the practice that refines their performance. The striatum participates in this learning through a function regulated by endocannabinoid signaling and other systems. This study relates the efficiency in learning a procedural task with the AATn polymorphism of the CNR1 gene, which encodes for the CB1 receptor. The mirror-drawing star task was solved by 99 healthy young subjects in three trials. The sample was divided into high- and low-performance groups based on performance efficiency. AAT12/14 carriers were more frequent in the former group, while there were more AAT12/13 carriers in the latter, which also made more errors/min. Therefore, we characterized two efficiency phenotypes: high- vs. low-performers associated with the two AATn genotypes, AAT12/14 vs. AAT12/13. The findings suggest that AATn polymorphism modifies CNR1 translation, indicating a different modulation of CB1.
Assuntos
Corpo Estriado/fisiologia , Aprendizagem/fisiologia , Polimorfismo Genético , Receptor CB1 de Canabinoide/genética , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Memória/fisiologia , Destreza Motora/fisiologia , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Adulto JovemRESUMO
OBJECTIVE: The purpose of this study was to compare the chemotactic activity of the choriodecidua and amnion, and to identify the phenotype of the leukocytes chemoattracted by each tissue. STUDY DESIGN: Amnion, choriodecidua and whole fetal membranes extracts were obtained from women at term (>37 weeks of gestation) with or without labor (n = 5 each). Extracts were assayed for leukocyte chemotactic activity, and the number and phenotype of the chemoattracted leukocytes were characterized by flow cytometry. RESULTS: Although all of the extracts exhibited chemotactic activity, more leukocytes were chemoattracted by the choriodecidua and the whole fetal membranes during labor compared with no labor (P = .010, .008). During labor the choriodecidua is responsible for granulocyte, T-lymphocyte, monocyte, and natural killer-cell chemoattraction, and the amnion is responsible for B-lymphocyte chemoattraction. CONCLUSION: Choriodecidua and amnion exhibit chemotactic activity for selective leukocytes and thus, each fetal membrane differentially regulates leukocyte chemotactic activity during labor.
Assuntos
Âmnio/metabolismo , Quimiotaxia de Leucócito , Córion/metabolismo , Decídua/metabolismo , Trabalho de Parto , Leucócitos/metabolismo , Membranas Extraembrionárias/metabolismo , Feminino , Citometria de Fluxo , Humanos , GravidezRESUMO
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-1ß (IL-1ß) and prostaglandin E(2) (PGE(2)) 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 PGE(2), probably via IL-1ß, 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.
Assuntos
Âmnio/imunologia , Córion/imunologia , Leucócitos/imunologia , Trabalho de Parto Prematuro/imunologia , Streptococcus agalactiae/fisiologia , Ureaplasma urealyticum/fisiologia , Âmnio/metabolismo , Córion/metabolismo , Dinoprostona/metabolismo , Feminino , Humanos , Inflamação/imunologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Técnicas de Cultura de Órgãos , Placenta/citologia , Placenta/imunologia , Gravidez , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To evaluate the impact of age on the expression of apoptotic biomarkers in human spermatozoa. DESIGN: Cross sectional, prospective study. SETTING: Academic centers. PATIENT(S): Healthy volunteers with proven fertility, stratified by age (n = 25, range: 20-68 years). INTERVENTION(S): Examination of serum hormone levels and basic semen parameters, and assessment of early (plasma membrane translocation of phosphatidylserine) and late (DNA fragmentation) sperm apoptotic markers by flow cytometry (using Annexin-V binding and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). MAIN OUTCOME MEASURE(S): Apoptosis markers. RESULT(S): Advancing male age was significantly and positively correlated with Annexin-V binding results. Although not significant, there was a clear trend for increased DNA fragmentation in the older groups. The age threshold for these observations appears to be 40 years. Advancing male age was positively correlated with FSH and sex hormone-binding globulin (SHBG) levels, and negatively correlated with sperm concentration. CONCLUSION(S): Advancing male age is associated with the expression of early apoptotic markers as evidenced by significantly increased plasma membrane translocation of phosphatidylserine, as well as with a more subtle proportion of sperm carrying DNA fragmentation. This study confirmed that male age is also associated with a decline in sperm concentration.
Assuntos
Envelhecimento/metabolismo , Apoptose , Biomarcadores/metabolismo , Idade Paterna , Espermatozoides/metabolismo , Adulto , Idoso , Envelhecimento/sangue , Envelhecimento/genética , Anexina A5/metabolismo , Apoptose/genética , Biomarcadores/análise , Biomarcadores/sangue , Fragmentação do DNA , Humanos , Marcação In Situ das Extremidades Cortadas , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/metabolismo , Masculino , Pessoa de Meia-Idade , Análise do Sêmen , Espermatozoides/química , Adulto JovemRESUMO
BACKGROUND: Pre-eclampsia is a syndrome of heterogeneous origin characterized by deficient placentation due to the inability of the cytotrophoblast to acquire an invasive phenotype and to remodel the uterine spiral arteries. One of the main problems observed early in pre-eclampsia is an altered regulation of the immune system, where the shift toward a Th2 cytokine profile observed in normal pregnancies, does not occur. In pre-eclampsia, high interferon (IFN)-gamma concentrations are present, along with transforming growth factor-beta cytokines, which retard migration of cytotrophoblasts. METHODS: A review of the scientific literature was performed on the immunological factors associated with the origins of pre-eclampsia. The various components of the immune system that may be participating in the aberrant immune activation that pathologically affect early pregnancy events and inhibit cytotrophoblast invasion were identified. RESULTS AND CONCLUSIONS: Cells and their signaling and regulatory molecules have been implicated in the immunological alterations found in the placental microenvironment of patients who develop pre-eclampsia. One of the main differences found in pre-eclampsia is a shift toward Th1 responses and the production of IFN-gamma. The origin of IFN-gamma is not clearly identified and could be the uterine natural killer cells, the placental dendritic cells modulating Th responses, alterations in synthesis of or response to regulatory molecules, or changes in the function of regulatory T cells in pregnancy. Aberrant immune responses promoting pre-eclampsia may also be due to an altered fetal allorecognition or to inflammatory triggers. Understanding the immunological basis for pre-eclampsia will expand knowledge regarding other adverse pregnancy outcomes.
Assuntos
Placentação/imunologia , Pré-Eclâmpsia/imunologia , Citocinas/fisiologia , Células Dendríticas/fisiologia , Feminino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/fisiologia , Interferon gama/fisiologia , Células Matadoras Naturais/fisiologia , Complexo Principal de Histocompatibilidade/fisiologia , Gravidez , Receptores Imunológicos/fisiologia , Linfócitos T Reguladores/fisiologia , Células Th1/fisiologia , Células Th2/fisiologiaRESUMO
Several studies indicate that at the choriodecidual interface, where maternal and fetal tissues make contact, a network of signals is established during labor that includes infiltration of leukocytes and secretion of pro-inflammatory cytokines. In this review, we provide an overview of the inflammatory milieu present in the choriodecidua during membrane rupture, describe the recruitment and homing of leukocytes to the reproductive tissues, and detail specific actions of the key chemokines released by the choriodecidual cells. These data lend further support to the hypothesis that labor is an inflammatory response, wherein the infiltrated leukocytes in the choriodecidua interface could be contributing to the creation of a microenvironment leading to collagenolysis, which would promote the rupture of these tissues during labor. In addition to the available information describing biological actions of chemokines during various pathological conditions such as infection, preterm labor and preterm rupture of membranes suggest that these compounds play important roles in other gestational events such as cervical dilation and myometrial contractions. Even though we do not know the totality of biochemical signals that integrate the molecular dialogue between leukocytes and the various gestational tissues, it is becoming increasingly evident that this microenvironment is characterized, at least in part, by the differential expression and secretion of chemokines that induce selective trafficking of leukocyte subsets to the fetal membranes. Therefore, chemokines should be considered as important regulatory molecules with the ability to initiate the events that characterize normal and pathological labor.
Assuntos
Quimiocinas/metabolismo , Membranas Extraembrionárias/metabolismo , Ruptura Prematura de Membranas Fetais/metabolismo , Trabalho de Parto/metabolismo , Quimiotaxia/fisiologia , Colágeno/metabolismo , Feminino , Humanos , Subpopulações de Linfócitos/fisiologia , GravidezRESUMO
Rupture of the fetal membranes during human labor is associated with an inflammatory process localized to the maternal-fetal interface. There is evidence that specific leukocytes subsets are attracted to the choriodecidua, and that after homing they condition a local inflammatory microenvironment, possibly being directly involved in rupture of the membranes. In this study our aim was to compare the phenotypes and function of leukocytes located in the placental intervillous blood with peripheral leukocytes obtained before or after labor, including expression of modulators of inflammation in these cells. Flow cytometry revealed that the proportion of CD14(+) cells is increased in intervillous blood, suggesting the participation of monocytes/macrophages during labor. Real time qRT-PCR showed that at term gestation and particularly during labor, placental blood leukocytes adopt a different expression pattern of pro-inflammatory cytokines than leukocytes in peripheral blood, including IL-1beta and IL-1RA. During labor, both placental and peripheral leukocytes increase their secretion of matrix metalloproteinase-9. Moreover, we showed that placental leukocytes respond differently than peripheral leukocytes to bacterial lipopolysaccharide, secreting differential amounts of TNF-alpha, IL-1beta and IL-6. Finally, a preliminary proteomic characterization of placental leukocytes revealed a significantly higher number of individual proteins than in peripheral leukocytes. Our results support the existence of selective subsets of leukocytes recruited to the maternal-fetal interface that may participate in the triggering of parturition.
Assuntos
Trabalho de Parto/imunologia , Leucócitos/imunologia , Placenta/imunologia , Membranas Extraembrionárias/imunologia , Feminino , Humanos , Inflamação/imunologia , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Receptores de Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Metaloproteinase 9 da Matriz/imunologia , Monócitos/imunologia , Polissacarídeos Bacterianos/imunologia , Gravidez , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: biomolecular processes associated with aging and programmed cell death during spermatogenesis are well known, but not its biological significance in ejaculated sperm, because it ignores the behavior of these apoptotic markers in relation to the age of man. OBJECTIVE: To evaluate the effect of aging on the functional capacity of sperm and their relationship to programmed cell death processes. MATERIAL AND METHODS: Prospective, cross-sectional and analytical performed with semen samples from 25 healthy subjects 20 to 70 years old, were divided into two groups [(A: under 40 years) and (B: over 40 years age)]. Semen parameters were evaluated WHO (1999) and transformation processes biomolecular membrane and the expression of oligonucleosomes in the terminal cascade of apoptosis. Were measured by flow cytometry with an argon laser as a source of reading at 480 nm, the degree of cellularity discriminate negative and positive for each of the indicators. RESULTS: The percentage of live cells with phosphatidylserine translocation in the membrane (annexin-V / PI) was significantly higher in men older than 40 years (p <0.05). These findings are enriched with a significant positive correlation (r = 0.50, P <0.008) between early biomarker and age of the subjects. With regard to DNA fragmentation, although no statistically significant differences were found, it is a clear trend of increase as older the subjects (r = 0.51). CONCLUSIONS: The increasing age of the man is associated with increased expression of apoptosis, as demonstrated by the increased expression of phosphatidylserine translocation at the Membrane. Thus, this study confirms that the subject's age is associated with a decline in some of the seminal parameters.
Assuntos
Envelhecimento/fisiologia , Lipídeos de Membrana/análise , Nucleossomos/ultraestrutura , Fosfatidilserinas/análise , Espermatozoides/fisiologia , Adulto , Idoso , Apoptose , Distinções e Prêmios , Estudos Transversais , Fragmentação do DNA , Ginecologia , Humanos , Infertilidade Masculina/epidemiologia , Masculino , México , Pessoa de Meia-Idade , Obstetrícia , Estudos Prospectivos , Maturação do Esperma , Motilidade dos Espermatozoides , Espermatozoides/química , Espermatozoides/ultraestrutura , Coloração e Rotulagem , Adulto JovemRESUMO
BACKGROUND: The packaging of heterochromatin during spermatogenesis has been correlated with the expression of residual apoptotic bodies (which stain with merocyanine A) that will impact on sperm function in the fertilization process; as well as the joint expression of the transmembrane translocation phosphatidyl serine and oligonucleosomes. OBJECTIVE: To evaluate the expression of bodies stained with merocyanine in the functional processes of sperm and their level of agreement with apoptotic Annexin V and TUNEL biomarkers. MATERIAL AND METHOD: We performed a prospective, cross, including 11,000 cells belonging to semen samples from infertile men, were evaluated according to WHO criteria (1999), bounded by the lines of Tygerberg. The biomolecular transformation processing of the membrane and the expression of oligonucleosomes in the terminal cascade of apoptosis were quantified by cytometry flow, using an argon lasser as a reading source of 480 nm, discriminating the degree of cellularity, both negative and positive for each indicators. RESULTS: Because of the study design was found low quantification in semen parameters, motility, morphology and sperm concentration. The average expression of cells [DNA-PI(+) / dUTP-FITC(+)] (quantification of TUNEL) and [Annexin-V(+) / PI(-)] was 36.5 +/- 17.4% and 31.2 +/- 17.4%, respectively. By comparing the expression of TUNEL without the effect of M540 bodies (36.3 +/- 1.7% vs. 36 +/- 1.7%) a significant difference was not determined. CONCLUSIONS: This study shows that there is a remnant of the primary processes of spermiation, which can take an important role in apoptotic and functional processes of the sperm. However, its expression does not affect measurement of biomarkers of apoptosis seminal, whose determination changed the diagnosis and functional perception of reproductive parameters in the sperm.
Assuntos
Apoptose , Artefatos , Fragmentação do DNA , Fertilização/efeitos dos fármacos , Corantes Fluorescentes/farmacologia , Fluorometria , Marcação In Situ das Extremidades Cortadas/métodos , Infertilidade Masculina/metabolismo , Nucleossomos/efeitos dos fármacos , Pirimidinonas/farmacologia , Espermatozoides/efeitos dos fármacos , Coloração e Rotulagem , Anexina A5/metabolismo , Distinções e Prêmios , Estudos Transversais , Reações Falso-Positivas , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/farmacologia , Corantes Fluorescentes/análise , Ginecologia , Heterocromatina/metabolismo , Humanos , Infertilidade Masculina/patologia , Masculino , Lipídeos de Membrana/metabolismo , México , Nucleossomos/ultraestrutura , Obstetrícia , Fosfatidilserinas/metabolismo , Estudos Prospectivos , Pirimidinonas/análise , Motilidade dos Espermatozoides , Espermatozoides/química , Espermatozoides/ultraestruturaRESUMO
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.
Assuntos
Âmnio/química , Córion/química , Trabalho de Parto/metabolismo , Metaloproteinases da Matriz Secretadas/análise , Trabalho de Parto Prematuro/enzimologia , Feminino , Humanos , Análise em Microsséries , GravidezRESUMO
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.
Assuntos
Âmnio/ultraestrutura , Córion/ultraestrutura , Colágeno/ultraestrutura , Feminino , Humanos , Trabalho de Parto , Microscopia Confocal , Microscopia Eletrônica de Varredura , GravidezRESUMO
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.