O-261 The relationship of abnormal vaginal microbiome (dysbiosis of vaginal microbiome) and proinflammatory cytokines in recurrent implantation failure patients
Authors: Hortal M , Lledo B , Ortiz J A , Fabregat A , Moliner B , Bernabeu A , Bernabeu R 
Published: June 2023

Abstract

Study question
Is there any correlation between serum cytokine levels and the composition of the vaginal microbiome in recurrent implantation failure (RIF) patients?

Summary answer
The levels of IFN-γ/IL-10 and TNF-α/IL-6 ratios are higher in patients with dysbiotic microbiome which associates with an imbalance towards inflammation between Th1/Th2 lymphocytes.

What is known already
In recent years, the development of sequencing-based technologies has enabled the evaluation of the vaginal microbiome. Studies have shown that a vaginal microbiota with less than 90% of Lactobacillus is predictive of IVF treatment outcome and suggests that dysbiosis in the reproductive tract negatively affects the reproductive outcome compared to women with a Lactobacillus-dominated microbiome.
Furthermore, it is known that pregnancy is characterized by a Th2-dominant cytokine pattern, and, in contrast, an altered Th1/Th2 ratio, and altered NK cell and macrophage numbers are more prevalent in women with RIF.

Study design, size, duration
Twenty patients were included in a retrospective study between December 2018 and May 2022. Thirteen had normal microbiome and seven had dysbiotic microbiome. In all of them, a vaginal microbiome sample was analyzed and the levels of cytokines IL-2, IL-4, IL- 6, IL-8, IL-10, VEGF, IFNγ, TNFα, IL-1α, IL-1β, MCP-1 and EGF in serum were measured. Patients with uterine malformations, uterine surgery or other known factors which may influence RIF were excluded from the analysis.

Participants/materials, setting, methods
To measure the levels of the different cytokines, a sandwich immunoassay with specific antibodies for the cytokines IL-2, IL-4, IL-6, IL-8, IL-10, VEGF, IFNγ, TNFα, IL -1α, IL-1β, MCP-1 and EGF were used. Vaginal microbiome was analyzed by mass sequencing of the V3V4 region of 16S rRNA. The statistical analysis was performed with R Statistical Software, version 4.0.3 and the bioinformatic analysis of vaginal microbiome was performed using QIIME2 and MicrobiomeAnalyst packages.

Main results and the role of chance
For the ratios between cytokines produced by Th1 lymphocytes and Th2 lymphocytes, we observe that levels of IFN-γ/IL-10 (p = 0,043) and TNF-α/IL-6 (p = 0,021) ratios are higher in patients with dysbiotic microbiome, which have an imbalance towards inflammation.
Comparing levels of cytokines with respect to alpha diversity, no differences are observed either individually or in the ratios between Th1/Th2 cytokines. However, the multivariate model that predicts alpha diversity (p = 0.037; AIC=20.734) does consider the IFN-γ/IL-10 and TNF-α/IL-6 aforementioned ratios. Alpha diversity, in turn, is higher in vaginal samples from patients with dysbiotic microbiome (p = 0.003). Regarding beta diversity, it is also higher in patients with altered microbiome (p = 0.007).
Considering the relative abundance of each bacteria, we can predict the levels of the aforementioned ratios: for TNF-α/IL-6 ratio, the multivariate model reaches 91.37% of prediction including the relative abundance of Burkholderia, Chryseobacterium, Enterococcus, Escherichia, Gemella, Herbaspirillum, Negativicoccus and Staphylococcus. For IFN-γ/IL-10 ratio, the multivariate model reaches 87,65% of prediction including Aerococcus, Burkholderia, Escherichia, Herbaspirillum, Megasphera, Pseudomonas, Ralstonia and Staphylococcus. It is worth noting the absence of Lactobacillus in these models, which suggests that the relative abundance of some pathogenic bacteria is what alter the production of cytokines, not the lower relative abundance of Lactobacillus.

Limitations, reasons for caution
The inherent limitations of a retrospective study design and a limited sample size. In addition, cytokine levels were measured in serum and not in vaginal fluid. Additional studies including a metagenomic approach might be able to determine functional groups of bacteria for analysis.

Wider implications of the findings
These results indicate that pathogenic bacteria, such as Burkholderia and Staphilococcus, which modify cytokine levels and cause inflammation, may also induce an imbalance in the Th1/Th2 ratio. When the vaginal microbiome is altered, the administration of antibiotics or probiotics may improve the immunologic environment, thus, avoiding the use of immunosuppressants.

Trial registration number
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