Endometrial Molecular Microbiome Profiling: Empowering Reproductive Outcomes through Enhanced Pathogen and Lactobacillus Diagnostics

Background: Chronic endometritis (CE) is a subtle but clinically relevant inflammatory condition that adversely impacts fertility and reproductive outcomes. Conventional diagnostic methods often fail to detect key pathogens, highlighting the need for molecular approaches. This study evaluated the diagnostic utility of the EMPOWER panel, a real-time polymerase chain reaction (RT-PCR) assay targeting 23 pathogens (including Acinetobacter baumannii, Candida albicans, Chlamydia trachomatis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Gardnella vaginalis, Klebsiella oxytoca, Klebsiella pneumoniae, Mycobacterium tuberculosis (MTB) , Mycoplasma genitalium, Mycoplasma hominis, Neisseria gonorrhoeae , Nontuberculous mycobacteria, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Treponema pallidum, Trichomonas vaginalis, Ureaplasma parvum and Ureaplasma urealyticum ) and five Lactobacillus species ( Lactobacillus crispatus, Lactobacillus jenseni, Lactobacillus brevis, Lactobacillus acidophilus and Lactobacillus plantarum ), in endometrial biopsy (EB) specimens from Indian women with reproductive challenges. Methods: A retrospective analysis of 385 EB samples was conducted. Patients presented with abnormal uterine bleeding (AUB), recurrent implantation failure, recurrent miscarriage, recurrent pregnancy loss, or in vitro fertilization failure. Specimens were subjected to both microbial culture and molecular testing (RT-PCR). Pathogen prevalence, Lactobacillus abundance, and demographic distributions were analyzed. Results: Pathogens were identified in 230 out of 385 cases (59.7%) by RT-PCR, which was substantially higher than the 37% positivity found by conventional culture. The most prevalent organisms included U. parvum (13.5%), E. faecalis (11.7%), and M. genitalium (10.4%). Polymicrobial infections were detected in 5.4% of cases, commonly S. agalactiae with C. albicans . No cases of N. gonorrhoeae or MTB were detected. Higher Lactobacillus abundance (>100,000 colony-forming units per milliliter was associated with pathogen-negative samples, suggesting a protective role in reproductive health. CE prevalence increased with age, reaching 100% in women over 45 years. Conclusions: RT-PCR significantly enhances the detection of CE-associated pathogens compared with conventional culture. Ureaplasma parvum, E. faecalis, and M. genitalium were dominant contributors. The findings highlight the protective role of Lactobacillus species in maintaining endometrial health. Incorporating molecular diagnostics into routine workflows may improve infertility management, particularly in Indian women with unexplained reproductive failure.