Vaginal microbial community state types fail to predict IVF outcomes, whereas <i>Ureaplasma parvum</i> and <i>Lactobacillus iners</i> are negative predictors of implantation, clinical pregnancy and live birth

Abstract STUDY QUESTION Are previously proposed vaginal microbial community state types (CSTs) valid predictors of IVF success, or do alternative microbial signatures provide stronger associations? SUMMARY ANSWER Previously proposed CSTs as predictors of implantation, clinical pregnancy and live birth were not confirmed, while an interaction between Ureaplasma parvum and Lactobacillus iners emerged as a strong negative predictor. WHAT IS KNOWN ALREADY Infertility affects 17% of the global population. Only one-third of treatment cycles of assisted reproductive technologies result in embryo implantation, and even fewer lead to clinical pregnancy or live births. While early findings have spurred the development of microbiome-based tests for success prediction, evidence on supporting their reliability remains inconclusive. STUDY DESIGN, SIZE, DURATION This prospective, single-centre study aimed to validate existing, and identify better, microbial predictors of infertility treatment outcomes. A cohort of 266 infertile female patients (age 18–45 years) undergoing a frozen-thawed embryo transfer cycle in an anovulatory regimen (i.e., a cycle with transfer of an embryo following a previous oocyte retrieval, fertilisation and freezing of embryos) were recruited for the study with a timeframe from 05/2017 to 03/2019. PARTICIPANTS/MATERIALS, SETTING, METHODS The female, infertile patients, aged 18 to 45 years, were undergoing routine care. Vaginal swabs were taken prior to embryo transplantation and subjected to DNA isolation for 16S-based microbiota analysis. Extended demographic and treatment data were recorded. Clinical outcomes were defined as: (i) implantation, confirmed by a positive hCG test, (ii) clinical pregnancy, and (iii) live birth (defined as the birth of a viable infant). Sequencing data were processed in mothur following established pipelines, and microbial composition (taxonomy) as well as microbial diversity (dissimilarity analyses) were determined using the open-source software R. A prediction model for implantation success was built using binary logistic regression based on abundance of putatively predictive microbial taxa. MAIN RESULTS AND THE ROLE OF CHANCE This study suggests that vaginal microbial community state types, alpha-diversity, and the ratio of dominant Lactobacillus species do not correlate in statistical terms or in a clinically meaningful manner with implantation and clinical pregnancy (as a surrogate for endometrial receptivity) or with live birth (as a surrogate for ongoing pregnancy viability). However, Ureaplasma parvum and Lactobacillus iners abundances were identified as negative predictors of embryo implantation, clinical pregnancy and live birth. A subset of women colonized by these taxa experienced drastically reduced embryo implantation and completely failed to achieve clinical pregnancy or give birth to live offspring, suggesting a potential role of these organisms in implantation failure and reproductive outcome, independent of other influencing factors such as age, oestradiol levels, endometrial thickness etc. LARGE SCALE DATA The raw sequencing data used for this manuscript are publicly available at the European Nucleotide Archive under accession number PRJEB107113. LIMITATIONS, REASONS FOR CAUTION This study is a single centre study warranting further validation cohorts. Given the variable nature of the vaginal microbiota, sample sizes need to be enlarged for better refinement of the analyses. Further, the underlying mechanistical basis of our findings is yet elusive and clinical translation has yet to be established. WIDER IMPLICATIONS OF THE FINDINGS While this novel association warrants confirmation, the results caution against reliance on previously suggested CSTs as predictors, and highlight the need for refined, reproducible microbiome-based diagnostics in reproductive medicine. STUDY FUNDING/COMPETING INTEREST(S) No external funding, but financial support was received from the University of Lübeck and the German Center of Infection Research. ML, AM, IP, MD, and JR declare no conflicts of interest. SG discloses personal fees from Organon outside the submitted work. TKE discloses honoraria from Ferring; travel support from Ferring, Merck, Theramex, and Gedeon-Richter; and receipt of equipment/materials/laboratory analyses (to institution) from Arthrex, Besins, Merck and Abbott outside the submitted work. NH discloses personal fees from Gedeon Richter, Ferring and Merck. GG reports that his institution received grants or contracts from Besins, Merck, Abbott, Ferring, and Theramex. He has received personal consulting fees, support for travel fees and meeting attendance, and honoraria for lectures or educational events from Organon, Ferring, Merck, Gedeon-Richter, Theramex, Abbott, ReproNovo, Igyxos, OxoLife, Philipps, ReprodWissen, PregLem, Guerbet, Roche, IBSA, and Besins. He also received support for travel and meeting attendance from Merck, Organon, Ferring, Theramex, Gedeon-Richter, and Abbott. Additionally, he holds unpaid leadership positions as a member of the ESHRE Working Group on RIF, the ESHRE Working Group on clinical KPI, and the ESHRE guideline development group on ovarian stimulation. ASM reports consulting fees and speaker′s fees from Merck, Theramex and Gedeon-Richter as well as travel support from Merck, Theramex, Gedeon-Richter, IBSA, Ferring, and MSD.