A Correlational Study Between Intraglandular Macrophage-positive And Disorders Of Endometrial Microbiome And Adverse Pregnancy Outcomes

Objective: To investigate whether endometrial intraglandular macrophage(Mφ)-positive phenomenon represented the disorders of endometrial microbiome and were associated with adverse pregnancy outcomes.Methods: Retrospective analysis,patients who underwent endometrial scratching one month before the next natural cycle frozen embryo transplantation(day 7 after ovulation)and with a failed fresh embryo transfer cycle before were included.Endometrial samples were obtained for CD163(Mφ marker)immunohistochemistry staining.According to the number of positive glands and the degree of concentration of Mφ in the glandular lumen,the patients were divided into three groups: Ⅰ(no Mφ in the glandular lumen),Ⅱ(slight infiltration of Mφ in the glandular lumen)and Ⅲ(severe infiltration of Mφ in the glandular lumen).The pregnancy and clinical outcomes of each group were analyzed and compared.On the other hand,metatranscriptomics sequencing was performed on endometrial samples from intraglandular Mφ-positive(n=8)and Mφ-negative(n=3)patients.By comparing microbiome composition between groups and analyzing of host-microbiota functions,searching for the cause of the aggregation of Mφ at intraglandular cavity.And according to the pregnancy outcome,the positive group was divided into pregnant group and non-pregnant group;Microbiome and host transcriptome differences between the two groups were also analyzed to explore the association between the intraglandular Mφ-positive and disorders of endometrial microbiome and adverse pregnancy outcomes.Results: The clinical pregnancy rates of patients with grade Ⅰ,Ⅱand Ⅲ were 75.7%(56/74),81.8%(9/11)and 48.4%(15/31),P=0.018,respectively;and the rate of developing recurrence of implant failure(RIF)was 10.8%(8/74),9.1%(1/11)and 35.5%(11/31),P=0.011,respectively.We identified 83 active microorganisms in the endometrium,including bacteria and viruses,and the dominant group was Escherichia＿coli.The abundance of Bacteroides＿vulgatus and Aeromonas＿veronii decreased significantly in the Mφ-positive group,and abnormal colonization of harmful bacteria such as Staphylococcus,Klebsiella＿pneumoniae,Pseudomonas＿aeruginosa,Shigella＿flexneri,Gardnerella＿vaginalis and Corynebacterium occurred.Expressed genes related to mitochondrial respiratory chain and cholesterol metabolism were significantly down-regulated in the non-pregnant group,while immune inflammatory response and reactive oxygen species(ROS)signaling pathways were activated.Conclusions: The abundant intraglandular Mφ-positive glands or the obvious aggregation of Mφ in glandular lumen was associated with the adverse pregnancy outcome.The intraglandular Mφ-positive phenomenon was associated with the decrease of the abundance of Bacteroides＿vulgatus and the appearance of harmful bacteria in endometrium.The colonization of endometrium by a variety of detrimental pathogens may lead to mitochondrial damage and enhanced immune inflammatory response in endometrium,which may be the cause of the adverse pregnancy outcome.