Kinetics of cytokine production in Mycoplasma hominis and Ureaplasma urealyticum coinfection in chorioamniotic membranes: in vitro study

• Grant number: 18/17890-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2019
• End date: December 31, 2019
• Field of knowledge: Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
• Principal Investigator: Márcia Guimarães da Silva
• Grantee: Geovanna Cristofani Cursino
• Host Institution: Faculdade de Medicina (FMB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Infection of the amniotic cavity and inflammation are associated with spontaneous preterm delivery. In general, these infections are polybacterial and are mainly caused by the bacterial rise from the lower genital tract. Thus, microbial invasion of the amniotic cavity and the risk of premature labor are often related to cervicovaginal colonization by microorganisms such as genital mycoplasmas, which are the main bacteria isolated in the amniotic fluid of preterm labor with intact membranes and preterm premature rupture of membranes. However, the adverse outcome and its association with genital mycoplasmas are inconsistent. For some authors, the moderate innate immune response from the Mollicutes can be trigger by intense regulation after 24 hours of the challenge of the membranes or even by immunogenicity of mycoplasma only. In order to better understand the innate immune response, the objective of this study is to evaluate the kinetic of inflammatory cytokines production in the co-infection by genital mycoplasmas in chorioamniotic membranes. Material and Methods:A total of 10 chorioamnionitis membranes obtained from term pregnancies (e 37 weeks of gestation), in the absence of labor and/or premature rupture of membranes will be collected for in vitro culture stimulation. Chorioamniotic membrane cultures will be stimulated with 10-6 CFU of heat-inactivated bacterial suspension diluted in tissue culture medium with Ureaplasma urealyticum (UU), Mycoplasma hominis (MH) alone or in combination. Samples of chorioamniotic membranes without bacterial stimulation will be used as a negative control, which will be incubated only with the culture medium without bacterial presence, and as a positive control will be used the lipopolysaccharide (LPS).For the evaluation of the production of cytokines in response to mycoplasma infection, the supernatants of each treatment will be collected and stored at -80ºC at the following times: 6 hours, 12 hours, 18 hours, 24 hours and 30 hours. The supernatants obtained from the chorioamniotic membrane cultures will be evaluated by ELISA for the following cytokines: IL-1beta, IL-6, IL-8 e TNF-alpha. Statistical analysis will be performed according to the requirements for the results obtained, and the level of significance will be 5%.