P-327 A multicenter retrospective study of the investigation of detailed immunological inflammatory grading for chronic endometritis and their correlation with the endometrial microbiome
Authors: Mariya T , Ogawa M , Minase G , Mizuuchi M , Sanai H , Henmi H , Kanaya M , Ikeda U , Tanaka E S , Kinjo S , Shimada M , Endo T , Minase T , Torigoe T , Saito T 
Published: July 2024

Abstract

Study question
There exists refractory chronic endometritis which does not respond to antibiotic treatment. This study aims to elucidate its pathogenesis and develop detection methods.

Summary answer
Through treatment intervention for chronic endometritis (CE), the inflammatory grade decreased overall and the bacteria most associated with endometritis were Lactobacillus iners.

What is known already
CE and endometrial dysbiosis have been reported to be associated with recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF), but there is insufficient data confirming the interrelation between them. Moreover, the diagnosis of CE typically involves single immunohistochemical staining with CD138, but the diagnostic criteria vary across reports (e.g., McQueen’s method, Liu’s method, etc.) and moreover, use of this classical inflammatory assessment methods raises concerns about their sufficiency in evaluating the pathogenesis of endometritis in cases of RPL and RIF.

Study design, size, duration
A retrospective analysis was conducted on 206 samples of endometrial specimens collected from infertile patients at seven participating assisted reproductive healthcare facilities from 2021 to 2023. Among these, 73 samples (from 48 cases) had undergone both histological examination and simultaneous endometrial microbiome testing, and data on pregnancy outcomes (including implantation success and ongoing pregnancy rate) were collected until December 2023 for these cases.

Participants/materials, setting, methods
The endometrial specimens from cases with RIF or RPL were obtained through intrauterine aspiration in luteal phase. Immunohistochemical staining using CD138, CD56, and CD8 antibodies was performed. A composite grading for endometritis, based on histological findings and immunohistochemistry intensity, was established (INF grading; grade 0 to 5). Furthermore, a portion of the specimens underwent microbiome analysis using next-generation sequencing targeting the 16SrRNA, exploring correlations between bacterial taxa, abundance, histological findings, and pregnancy outcomes.

Main results and the role of chance
Among analyzed 48 cases, 11 cases underwent repeated endometrial microbiome analysis post-treatment again, while 7 cases had additional testing without improvement in chronic endometritis (CE) or endometrial dysbiosis. The McQueen’s method diagnosed 27 cases with CE, while the Liu method identified 4 cases. According to our INF grading, 11 cases were classified as endometritis grade 3 or above. Successful embryo implantation was achieved in 18 cases (37.5%), with ongoing pregnancy in 13 cases (27.1%). CE treatment involved broad-spectrum oral antibiotics, mainly doxycycline and metronidazole, resulting in improvement to grade 2 or below in 9 out of 11 cases with INF grade 3 or above. However, clear associations between pregnancy outcomes and INF grades were not apparent, with the most significant correlation found in CE diagnosis using Liu’s method. Analyzing biomarkers for CE through LEfSe (Linear discriminant analysis effect size), both McQueen’s method and INF grading 2 or above were associated with the intrauterine presence of Lactobacillus iners. Estimating endometrial inflammation based on intrauterine Lactobacillus iners presence yielded an AUC of 0.733 for McQueen’s CE and 0.723 for INF grade 2 or above.

Limitations, reasons for caution
This study is a retrospective analysis, and the treatment protocols and intervention criteria for chronic endometritis vary among different facilities. The number of cases is also limited, and it is desirable to conduct a unified prospective multicenter cohort study with consistent protocols.

Wider implications of the findings
The combination of the endometrial microbiome testing and detailed inflammatory grading suggests the potential for establishing appropriate treatment intervention strategies and predicting the success rate of infertile treatment. Since Lactobacillus iners tends to colonize extensively in the vaginal environment of East Asian women, it is necessary to explore region-specific measures.

Trial registration number
not appicable