Exosome-like small-extracellular vesicles (sEVs) are extracellular vesicles that act in intercellular communication and are involved in several biologic and pathologic processes. While sEVs increase the stability of t...Exosome-like small-extracellular vesicles (sEVs) are extracellular vesicles that act in intercellular communication and are involved in several biologic and pathologic processes. While sEVs increase the stability of their cargo molecules, there is still a need for standardization of sampling and isolation of these microvesicles. We aimed to determine the best sampling method for isolation of sEVs from peripheral blood from reproductive-aged women. Material and Methods: We included samples of plasma from our biobank collected in 2014 by venipuncture in heparin tubes and stored at -80°C. We also included blood samples collected in heparin tubes and Ethylenediamine tetraacetic acid (EDTA) tubes and stored at -80°C for one to two weeks prior processing. All blood samples were collected from the same nine reproductive-aged female volunteers. sEVs were isolated from plasma by ultracentrifugation and filtration and indirectly quantified using Pierce BCA Protein Assay kit. Transmission electron microscopy (TEM) and Nano Tracking Analysis (NTA) were performed to confirm the isolation of sEVs. Results and Discussion: TEM and NTA confirmed the isolation of sEVs. Protein concentration of short-time stored heparin samples was not statistically different from long-time stored heparin samples (1847.2 ± 651.4 vs. 2363.2 ± 1025.1, p = 0.14). There was no difference between heparin and EDTA plasma samples recently collected (2363.2 ± 1025.1 vs. 2044.8 ± 653.2, p = 0.44). In conclusion, blood samples may be collected using heparin or EDTA for isolation of sEVs. Long-time stored plasma samples maintain sEVs integrity and may be used, especially in comparative studies.展开更多
Background: Adolescents are disproportionally affected by sexually transmitted infections (STI). Chlamydia trachomatis (CT) and Trichomonas vaginalis (TV) are the most frequent curable STI in adolescents, causing seri...Background: Adolescents are disproportionally affected by sexually transmitted infections (STI). Chlamydia trachomatis (CT) and Trichomonas vaginalis (TV) are the most frequent curable STI in adolescents, causing serious consequences for their reproductive health. Therefore, we aimed to determine the prevalence and incidence of CT and TV, as well as their risk factors in pregnant adolescents from Belém, northern Brazilian Amazon. Methods: This prospective study enrolled 199 adolescents up to 20 weeks of pregnancy. They were scheduled for follow-up visit between 28 and 29 weeks of pregnancy. Sociodemographic and behavioral data were obtained by interview. Cervicovaginal samples were taken to test for TV, CT, Neisseria gonorrhoeae and bacterial vaginosis. Univariate and multivariate analyses were performed to test the association of prevalent/incident CT and TV with the variables. Results: Prevalence of cervical CT infection was 33.7% (n = 67/ 199), and for trichomoniasis it was 4.0% (n = 8/199). Cervical ectopy increased the risk for prevalent CT (OR, 1.93;95% CI, 1.01 - 3.70), while having treated vaginal discharge in the past (OR, 0.51;95% CI, 0.26 - 0.98) and being married (OR, 0.10;95% CI, 0.01 - 0.83) were protective against current CT and TV, respectively. Among the 95 (47.7%) adolescents who completed follow-up, 15 cases of incident CT were identified. Incident CT was associated with having a formal or informal job (OR, 28.4;95% CI, 2.1 - 391.6) and bacterial vaginosis treatment at the baseline (OR, 0.08;95% CI, 0.01 - 0.69). Conclusion: Prevalence and incidence rates of TV and CT are high in this population devoid of STI routine screening. Treatment of bacterial vaginosis may benefit this population by reducing risk for CT acquisition.展开更多
Amniochorion membranes were collected from 25 pregnant women at preterm labor, in the presence or not of Preterm Premature Rupture of Membranes (PPROM) and 27 pregnant women at term in the presence at labor, in order ...Amniochorion membranes were collected from 25 pregnant women at preterm labor, in the presence or not of Preterm Premature Rupture of Membranes (PPROM) and 27 pregnant women at term in the presence at labor, in order to quantify the expression and to evaluate the immunoreactivity of human beta defensins (HBD)1, HBD2, HBD3 and HBD4 in amniochorion membranes from pregnancies complicated by spontaneous prematurity. The HBDs were evaluated by immunohistochemistry, real time quantitative PCR and ELISA. Statistical analyses were performed using Chi-squared and Mann Whitney tests. There was no significant difference in HBDs expression between study and control groups: HBD1 (Md = 0.62 (0.0 - 105.0) vs Md = 0.80 (0.02 - 25.0);p = 0.85), HBD2 (Md = 0.17 (0.0 - 5.2) vs Md = 0.0 (0.0 - 43.2);p = 0.16), HBD3 (Md = 0.11 (0.0 - 140.5) vs Md = 0.06 (0.0 - 972.1);p = 0.91). Also, HBD1, HBD2 and HBD3 protein expression was not significant different between the groups: HBD1 (1.32 pg/mL (0.0 - 1.85) vs 1.08 pg/mL (0.04 - 2.22);p = 0.67), HBD2 (0.00 pg/mL (0.0 - 1.74) vs 0.02 pg/mL (0.0 - 1.24);p = 0.69), HBD3 (0.04 pg/mL (0.0 - 1.05) vs 0.09 pg/mL (0.0 - 1.05);p = 0.63). The immunoreactivity of HBD1, HBD2 and HBD3 was observed in amnion, chorion and decidua cells from preterm and term pregnancies. Amniochorion membranes are sources of HBD1, HBD2 and HBD3 and their expressions are similar in term and preterm pregnancies.展开更多
Objective: The goal of this study was to determine the prevalence of C. trachomatis in women diagnosed with infertility attending the Outpatient Clinic of Infertility from Botucatu Medical School, UNESP, Brazil. Patie...Objective: The goal of this study was to determine the prevalence of C. trachomatis in women diagnosed with infertility attending the Outpatient Clinic of Infertility from Botucatu Medical School, UNESP, Brazil. Patients and Methods: This molecular study enrolled a total of 112 women. Among these patients, 62 presented primary infertility while 50 presented secondary infertility. The criteria for eligibility included women who were: reproductive-aged;no prior report of seroconversion for HIV;no antibiotic or vaginal cream used in the preceding 30 days;and abstinence from sexual intercourse for 72 hours before the visit. The women were submitted to a gynecological examination and cervical samples were collected with an endocervical cytobrush for molecular analysis of C. trachomatis. Results: The prevalence of chlamydial infection was 8% with similar prevalence between primary (8.1%) and secondary (8.0%) infertility. Conclusion: Considering the asymptomatic nature of chlamydial infection and its association with tubal factor infertility, there is a pressing need to incorporate the screening of C. trachomatis infection as part of the routine investigation for infertility. The early diagnostic by screening can minimize complications and reduce Public Health costs with Assisted Reproductive Technology.展开更多
Expression of Toll-like receptors at the maternal-fetal interface during normal and complicated pregnancies has aroused interest in the last few years. However, despite the importance of TLR-2 and TLR-4, which recogni...Expression of Toll-like receptors at the maternal-fetal interface during normal and complicated pregnancies has aroused interest in the last few years. However, despite the importance of TLR-2 and TLR-4, which recognizes most microorganisms presenting in the amniotic cavity infections associated with prematurity, comparison of the expressions of these receptors is rare in the literature. Thus, the purpose of this study was to compare the gene expression between TLR-2 and TLR-4 in amniochorion membranes of pregnant women with preterm delivery in the presence of histologic chorioamnionitis (HCA). Amniochorion membranes were collected from 40 pregnant women with preterm delivery;20 presented HCA and 20 did not. Fragments of the membranes were submitted to total RNA extraction, followed by cDNA production by reverse transcription. Real time quantitative PCR was used to quantify the gene expression of the TLRs. mRNA concentrations between TLR-2 and TLR-4 were compared using the nonparametric Mann-Whitney test. TLR-2 expression was higher than TLR-4 expression in the presence of HCA. No difference was observed between TLR-2 and TLR-4 expression in membranes in the absence of inflammatory infiltrate. In conclusion, amniochorion membranes express TLR-2 and TLR-4 and higher TLR-2 expression in the presence of histologic chorioamnionitis suggests that microorganisms recognizable by TLR-2 play an important role in the physiopathology of preterm labor.展开更多
基金We acknowledge the volunteers that had donated blood samples for this experi-ment.This work was supported by Sao Paulo Research Foundation(Fapesp)(grants 2016/13616-8,2016/16618-1,2016/01340-8 and 2015/21829-9).
文摘Exosome-like small-extracellular vesicles (sEVs) are extracellular vesicles that act in intercellular communication and are involved in several biologic and pathologic processes. While sEVs increase the stability of their cargo molecules, there is still a need for standardization of sampling and isolation of these microvesicles. We aimed to determine the best sampling method for isolation of sEVs from peripheral blood from reproductive-aged women. Material and Methods: We included samples of plasma from our biobank collected in 2014 by venipuncture in heparin tubes and stored at -80°C. We also included blood samples collected in heparin tubes and Ethylenediamine tetraacetic acid (EDTA) tubes and stored at -80°C for one to two weeks prior processing. All blood samples were collected from the same nine reproductive-aged female volunteers. sEVs were isolated from plasma by ultracentrifugation and filtration and indirectly quantified using Pierce BCA Protein Assay kit. Transmission electron microscopy (TEM) and Nano Tracking Analysis (NTA) were performed to confirm the isolation of sEVs. Results and Discussion: TEM and NTA confirmed the isolation of sEVs. Protein concentration of short-time stored heparin samples was not statistically different from long-time stored heparin samples (1847.2 ± 651.4 vs. 2363.2 ± 1025.1, p = 0.14). There was no difference between heparin and EDTA plasma samples recently collected (2363.2 ± 1025.1 vs. 2044.8 ± 653.2, p = 0.44). In conclusion, blood samples may be collected using heparin or EDTA for isolation of sEVs. Long-time stored plasma samples maintain sEVs integrity and may be used, especially in comparative studies.
基金This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq),Grant#551245/2007-7.
文摘Background: Adolescents are disproportionally affected by sexually transmitted infections (STI). Chlamydia trachomatis (CT) and Trichomonas vaginalis (TV) are the most frequent curable STI in adolescents, causing serious consequences for their reproductive health. Therefore, we aimed to determine the prevalence and incidence of CT and TV, as well as their risk factors in pregnant adolescents from Belém, northern Brazilian Amazon. Methods: This prospective study enrolled 199 adolescents up to 20 weeks of pregnancy. They were scheduled for follow-up visit between 28 and 29 weeks of pregnancy. Sociodemographic and behavioral data were obtained by interview. Cervicovaginal samples were taken to test for TV, CT, Neisseria gonorrhoeae and bacterial vaginosis. Univariate and multivariate analyses were performed to test the association of prevalent/incident CT and TV with the variables. Results: Prevalence of cervical CT infection was 33.7% (n = 67/ 199), and for trichomoniasis it was 4.0% (n = 8/199). Cervical ectopy increased the risk for prevalent CT (OR, 1.93;95% CI, 1.01 - 3.70), while having treated vaginal discharge in the past (OR, 0.51;95% CI, 0.26 - 0.98) and being married (OR, 0.10;95% CI, 0.01 - 0.83) were protective against current CT and TV, respectively. Among the 95 (47.7%) adolescents who completed follow-up, 15 cases of incident CT were identified. Incident CT was associated with having a formal or informal job (OR, 28.4;95% CI, 2.1 - 391.6) and bacterial vaginosis treatment at the baseline (OR, 0.08;95% CI, 0.01 - 0.69). Conclusion: Prevalence and incidence rates of TV and CT are high in this population devoid of STI routine screening. Treatment of bacterial vaginosis may benefit this population by reducing risk for CT acquisition.
文摘Amniochorion membranes were collected from 25 pregnant women at preterm labor, in the presence or not of Preterm Premature Rupture of Membranes (PPROM) and 27 pregnant women at term in the presence at labor, in order to quantify the expression and to evaluate the immunoreactivity of human beta defensins (HBD)1, HBD2, HBD3 and HBD4 in amniochorion membranes from pregnancies complicated by spontaneous prematurity. The HBDs were evaluated by immunohistochemistry, real time quantitative PCR and ELISA. Statistical analyses were performed using Chi-squared and Mann Whitney tests. There was no significant difference in HBDs expression between study and control groups: HBD1 (Md = 0.62 (0.0 - 105.0) vs Md = 0.80 (0.02 - 25.0);p = 0.85), HBD2 (Md = 0.17 (0.0 - 5.2) vs Md = 0.0 (0.0 - 43.2);p = 0.16), HBD3 (Md = 0.11 (0.0 - 140.5) vs Md = 0.06 (0.0 - 972.1);p = 0.91). Also, HBD1, HBD2 and HBD3 protein expression was not significant different between the groups: HBD1 (1.32 pg/mL (0.0 - 1.85) vs 1.08 pg/mL (0.04 - 2.22);p = 0.67), HBD2 (0.00 pg/mL (0.0 - 1.74) vs 0.02 pg/mL (0.0 - 1.24);p = 0.69), HBD3 (0.04 pg/mL (0.0 - 1.05) vs 0.09 pg/mL (0.0 - 1.05);p = 0.63). The immunoreactivity of HBD1, HBD2 and HBD3 was observed in amnion, chorion and decidua cells from preterm and term pregnancies. Amniochorion membranes are sources of HBD1, HBD2 and HBD3 and their expressions are similar in term and preterm pregnancies.
文摘Objective: The goal of this study was to determine the prevalence of C. trachomatis in women diagnosed with infertility attending the Outpatient Clinic of Infertility from Botucatu Medical School, UNESP, Brazil. Patients and Methods: This molecular study enrolled a total of 112 women. Among these patients, 62 presented primary infertility while 50 presented secondary infertility. The criteria for eligibility included women who were: reproductive-aged;no prior report of seroconversion for HIV;no antibiotic or vaginal cream used in the preceding 30 days;and abstinence from sexual intercourse for 72 hours before the visit. The women were submitted to a gynecological examination and cervical samples were collected with an endocervical cytobrush for molecular analysis of C. trachomatis. Results: The prevalence of chlamydial infection was 8% with similar prevalence between primary (8.1%) and secondary (8.0%) infertility. Conclusion: Considering the asymptomatic nature of chlamydial infection and its association with tubal factor infertility, there is a pressing need to incorporate the screening of C. trachomatis infection as part of the routine investigation for infertility. The early diagnostic by screening can minimize complications and reduce Public Health costs with Assisted Reproductive Technology.
基金São Paulo Research Foundation(Fapesp)grants numbers 2008/55418-1 and 2008/53382-0.
文摘Expression of Toll-like receptors at the maternal-fetal interface during normal and complicated pregnancies has aroused interest in the last few years. However, despite the importance of TLR-2 and TLR-4, which recognizes most microorganisms presenting in the amniotic cavity infections associated with prematurity, comparison of the expressions of these receptors is rare in the literature. Thus, the purpose of this study was to compare the gene expression between TLR-2 and TLR-4 in amniochorion membranes of pregnant women with preterm delivery in the presence of histologic chorioamnionitis (HCA). Amniochorion membranes were collected from 40 pregnant women with preterm delivery;20 presented HCA and 20 did not. Fragments of the membranes were submitted to total RNA extraction, followed by cDNA production by reverse transcription. Real time quantitative PCR was used to quantify the gene expression of the TLRs. mRNA concentrations between TLR-2 and TLR-4 were compared using the nonparametric Mann-Whitney test. TLR-2 expression was higher than TLR-4 expression in the presence of HCA. No difference was observed between TLR-2 and TLR-4 expression in membranes in the absence of inflammatory infiltrate. In conclusion, amniochorion membranes express TLR-2 and TLR-4 and higher TLR-2 expression in the presence of histologic chorioamnionitis suggests that microorganisms recognizable by TLR-2 play an important role in the physiopathology of preterm labor.
