P-384 Efficacy of endometrial microbiome metagenomic analysis with recurrent implantation failure and recurrent pregnancy loss: multicenter study in Japan
Authors: Iwami N , Komiya S , Asada Y , Tatsumi K , Habara T , Kuramoto T , Seki M , Yoshida H , Takeuchi K , Shiotani M , Mukaida T , Odawara Y , Mio Y , Kamiya H 
Published: June 2022

Abstract

Study question
Does the result of endometrial microbiome metagenomic analysis (EMMA), a next generation sequencing (NGS)-based test of the intrauterine microbiome, have an impact on pregnancy rate after the test?

Summary answer
After recommend treatment with antimicrobial and probiotic therapy, the group diagnosed as dysbiosis by EMMA achieved pregnancy significantly earlier than the group with Normal result.

What is known already
Using NGS technology, EMMA testing can determine the composition of the endometrial microbiome by analysing bacterial 16S ribosomal RNA with a focus on the lactobacillus population. Endometrial flora in patients undergoing in vitro fertilization (IVF) is often composed of pathogenic microorganisms which decrease implantation rates, such as the Enterobacteriaceae family, Staphylococcus spp., Escherichia coli and Gram-negative bacteria. Other studies also indicate that Lactobacillus spp. is a major microorganism in the endometrium. Especially, lactobacillus-dominated microbiota (LDM, defined as &gt; 90% Lactobacillus spp.) in the endometrium has been reported to lead to better pregnancy outcomes than non-LDM (&lt;90% Lactobacillus spp.).

Study design, size, duration
This study was a prospective, multicenter cohort study of 527 patients (under 42 years old) with recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) at 14 IVF treatment facilities in Japan from June 2019 to August 2021. Defining RIF as three or more failed implantation attempts and RPL as two or more miscarriages, we examined the prognosis of two additional embryo transfers (ETs) after the EMMA test in patients who underwent the test.

Participants/materials, setting, methods
Endometrial tissue was obtained by aspiration from patients in day 15-25 of their menstrual cycles, and sample tissues were analyzed by NGS for EMMA. Participant centers treated patients according to the therapies specified in the reports, including antibiotic treatments, probiotic treatments, re-analysis, and embryo transfer. Multivariate analysis was performed using a generalized linear model with the endpoint of ongoing pregnancy. For the time-to-event analyses, we used Kaplan-Meier survival analysis to compare time to ongoing pregnancy.

Main results and the role of chance
The results of the first EMMA were as follows: 229 patients (43.4%) were normal with Lactobacillus spp. &gt;90% (Normal group), 110 patients (20.9%) were abnormal with less than 90% Lactobacillus spp. and predominantly pathogenic bacteria (Abnormal group), and 188 patients (35.7%) were mild with low absolute amounts of bacteria and ultralow biomass indicating almost sterile (Mild + Ultralow group). There were no significant differences in background factors such as age, duration of infertility, number of previous ETs, or history of deliveries among the three groups. Gardnerella was the most pathogenic bacteria detected in patients with Abnormal EMMA results. All patients in the Abnormal group were treated with antimicrobials and probiotics, and those in Mild + Ultralow group were treated with probiotics. Odds ratio for ongoing pregnancy rate was 1.10(95%CI 0.67-1.82, p = 0.699) in Abnormal group and 1.23(95%CI 0.80-1.89, p = 0.342) in Mild + Ultralow group, respectively. After the intervention, ongoing pregnancies were comparable to those in Normal group.Analysis of time to pregnancy using Kaplan-Meier survival curves showed that Abnormal group had a significantly higher rate of ongoing pregnancies during the observation period than the other groups (p = 0.031).

Limitations, reasons for caution
Since this study was not necessarily limited to euploid embryos transferred after testing, an aging bias cannot be excluded. Since this study was conducted with all patients receiving EMMA, the effectiveness of the test needs to be further validated by comparison to patients without EMMA testing.

Wider implications of the findings
This study is the first multicenter study to demonstrate that the intervention based on EMMA reports improve pregnancy outcome in the patients with RIF and RPL. We suggest that the EMMA procedure, which aims at establishing an appropriate uterine microbiome, may be important for implantation and pregnancy continuation.

Trial registration number
UMIN000036917