Inflammatory cytokines in the lower genital tract: analyses by absolute cytokine levels and normalized cytokine/total protein ratios result in different outcomes

Abstract

OBJECTIVE: Cytokines are increasingly used to study the impact of microbicides on immune response in the lower female genital tract. The objective was to test if 2 methods of analyzing cytokine levels in the reproductive tract gave different results.DESIGN: Randomized, assessor-blinded crossover trial.MATERIALS AND METHODS: In a clinical trial assessing the impact of inert and pro-inflammatory topical gels on the vagina and endometrium, 14 subjects underwent baseline evaluation without gel exposure and at least one (and up to two) more set of vaginal lavage (VL) and endometrial lavage (EL) after exposure to two possible conditions: 3 days of nonoxynol-9 (N-9) or placebo gel. 8 pro-inflammatory cytokines (Il-1rα, Il-6, Il-8, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α 3 anti-inflammatory cytokines (Il-1rα, Il-10, Slp-1) and total protein levels were assayed in all VL and EL specimens (RayBio Human Cytokines Array). Cytokines were analyzed as absolute values and cytokine/total protein ratios. These measures were compared by gel exposure using signrank tests.RESULTS: Compared to baseline and placebo, N-9 exposure changed both absolute cytokine levels and cytokine/total protein ratios in the vagina and the endometrium. However, both the magnitude and direction of change in cytokines differed between analyses using absolute values versus normalized ratios. For example, compared to placebo, N-9 was associated with significantly decreased Il-6 (p=0.01), MCP-1 (p=0.02), MIP-1α (p=0.02), TNF-α (p=0.05) Il-10 (p=0.02) and Slp-1 (p=0.03) cytokine/total protein ratios in the vagina. In contrast, N-9 was only associated with decreased Il-6 (p=0.05) and increased Il-8 (p=0.001)when analyzed by absolute levels.CONCLUSIONS: When analyzing the inflammatory impact of topical vaginal agents, results are highly impacted by the method of cytokine analyses. If the evaluation of microbicides candidates is to incorporate surrogates of markers of inflammation, a standardization of methodology is paramount. OBJECTIVE: Cytokines are increasingly used to study the impact of microbicides on immune response in the lower female genital tract. The objective was to test if 2 methods of analyzing cytokine levels in the reproductive tract gave different results. DESIGN: Randomized, assessor-blinded crossover trial. MATERIALS AND METHODS: In a clinical trial assessing the impact of inert and pro-inflammatory topical gels on the vagina and endometrium, 14 subjects underwent baseline evaluation without gel exposure and at least one (and up to two) more set of vaginal lavage (VL) and endometrial lavage (EL) after exposure to two possible conditions: 3 days of nonoxynol-9 (N-9) or placebo gel. 8 pro-inflammatory cytokines (Il-1rα, Il-6, Il-8, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α 3 anti-inflammatory cytokines (Il-1rα, Il-10, Slp-1) and total protein levels were assayed in all VL and EL specimens (RayBio Human Cytokines Array). Cytokines were analyzed as absolute values and cytokine/total protein ratios. These measures were compared by gel exposure using signrank tests. RESULTS: Compared to baseline and placebo, N-9 exposure changed both absolute cytokine levels and cytokine/total protein ratios in the vagina and the endometrium. However, both the magnitude and direction of change in cytokines differed between analyses using absolute values versus normalized ratios. For example, compared to placebo, N-9 was associated with significantly decreased Il-6 (p=0.01), MCP-1 (p=0.02), MIP-1α (p=0.02), TNF-α (p=0.05) Il-10 (p=0.02) and Slp-1 (p=0.03) cytokine/total protein ratios in the vagina. In contrast, N-9 was only associated with decreased Il-6 (p=0.05) and increased Il-8 (p=0.001)when analyzed by absolute levels. CONCLUSIONS: When analyzing the inflammatory impact of topical vaginal agents, results are highly impacted by the method of cytokine analyses. If the evaluation of microbicides candidates is to incorporate surrogates of markers of inflammation, a standardization of methodology is paramount.