Background Previous evidence indicates associations between the female reproductive tract microbiome composition and reproductive outcome in infertile patients undergoing assisted reproduction. We aimed to determine whether the endometrial microbiota composition is associated with reproductive outcomes of live birth, biochemical pregnancy, clinical miscarriage or no pregnancy. Methods Here, we present a multicentre prospective observational study using 16S rRNA gene sequencing to analyse endometrial fluid and biopsy samples before embryo transfer in a cohort of 342 infertile patients asymptomatic for infection undergoing assisted reproductive treatments. Results A dysbiotic endometrial microbiota profile composed of Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Haemophilus, Klebsiella, Neisseria, Staphylococcus and Streptococcus was associated with unsuccessful outcomes. In contrast, Lactobacillus was consistently enriched in patients with live birth outcomes. Conclusions Our findings indicate that endometrial microbiota composition before embryo transfer is a useful biomarker to predict reproductive outcome, offering an opportunity to further improve diagnosis and treatment strategies.

Investigation of the microbial community in the female reproductive tract has revealed that the replacement of a community dominated by Lactobacillus with pathogenic bacteria may be associated with implantation failure or early spontaneous abortion in patients undergoing assisted reproductive technology (ART) treatment. Herein we describe taxonomically and functionally the endometrial microbiome of an infertile patient with repeated reproductive failures (involving an ectopic pregnancy and two clinical miscarriages). The microbiological follow-up is presented over 18-month in which the microbiota was evaluated in six endometrial fluid samples. The microbial profile of 16S rRNA gene sequencing showed a persistent infection with Gardnerella and other bacterial taxa such as Atopobium and Bifidobacterium. In addition, taxonomic and functional analysis by whole metagenome sequencing in the endometrial fluid sample collected before one clinical miscarriage suggested the presence of multiple Gardnerella vaginalis clades with a greater abundance of clade 4, usually associated with metronidazole resistance. These results revealed a persistent G. vaginalis endometrial colonization presenting genetic features consistent with antimicrobial resistance, biofilm formation, and other virulence factors, which could be related to the reproductive failure observed.

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Background: Previous evidence indicates associations between the female reproductive tract microbiome composition and reproductive outcome in infertile patients undergoing assisted reproduction. We aimed to determine whether the endometrial microbiota composition is associated with reproductive outcomes of live birth, biochemical pregnancy, clinical miscarriage, or no pregnancy. Methods: Here we present a multicentre prospective observational study using 16S rRNA gene sequencing to analyse endometrial fluid and biopsy samples before embryo transfer in a cohort of 342 infertile patients asymptomatic for infection undergoing assisted reproductive treatments. Results: A dysbiotic endometrial microbiota profile composed of Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Haemophilus, Klebsiella, Neisseria, Staphylococcus and Streptococcus was associated with unsuccessful outcomes. In contrast, Lactobacillus was consistently enriched in patients with live birth outcomes. Conclusions: Our findings indicate that endometrial microbiota composition before embryo transfer is a useful biomarker to predict reproductive outcome, offering an opportunity to further improve diagnosis and treatment strategies.

Study question Is there an association between the composition of the endometrial microbiota and the reproductive outcomes in infertile patients undergoing in vitro fertilization (IVF)? Summary answer The composition of the endometrial microbiota (EM) prior to embryo transfer is associated with the different reproductive outcomes: live birth, no pregnancy or clinical miscarriage. What is known already The investigation of bacterial communities in the female reproductive tract using molecular methods has revealed the existence of a continuum microbiota that extends from the vagina to the upper genital tract. Previous evidence suggests the existence of an association between the vaginal and endometrial microbiome composition with reproductive and obstetrical outcomes. Specifically, the presence of specific pathogens together with low abundance of Lactobacilli has been associated with poor IVF outcomes. Study design, size, duration Multicentre prospective observational clinical study analysing the EM of infertile patients undergoing IVF (with maternal age ≤40) or ovum donation (≤50 years). A total of 452 infertile patients undergoing IVF/ovum donation were assessed for eligibility in 13 reproductive clinics in Europe, America, and Asia. The duration of the study was 30 months and the recruitment period extended between August 2017 and February 2019 (ct.gov 03330444). Participants/materials, setting, methods Endometrial fluid and endometrial biopsy were collected during a hormonal replacement therapy cycle after 5 days of progesterone (P) administration prior to a frozen embryo transfer cycle. Endometrial microbiota (EM) composition was analyzed using 16S rRNA gene sequencing using compositional data to transform scale-invariant values in both sample types. The EM in fluid and biopsy was associated with live birth, biochemical pregnancy, clinical miscarriage, or no pregnancy. Main results and the role of chance Of the 452 patients assessed, 44 did not meet the selection criteria and were excluded for the study and 66 patients were lost to follow-up. Of the 342 remaining patients, 198 (57.9%) became pregnant [141 (41.2%) had a live birth, 27 (7.9%) had a biochemical pregnancy, 2 (0.6%) had an ectopic pregnancy, and 28 (8.2%) a clinical miscarriage], while 144 (42.1%) did not become pregnant. The baseline characteristics, clinical and embryological variables were homogeneous and no bias toward the clinical outcome categories was observed. Our association study showed that the composition of the EM was associated with the reproductive outcome in both endometrial fluid and biopsy. A dysbiotic endometrial microbiota profile composed of Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Haemophilus, Klebsiella, Neisseria, Staphylococcus and Streptococcus was significantly associated with unsuccessful outcomes, especially no pregnancy and clinical miscarriage. In contrast, Lactobacillus was consistently enriched in patients with live birth outcomes. The EM in endometrial fluid did not fully reflect that in endometrial biopsy, although their association with clinical outcome was consistent. Limitations, reasons for caution The main limitation was the small number of biochemical pregnancy and clinical miscarriage analysed. During transcervical collection of endometrial samples caution was taken to avoid contamination with the cervix although cervical contamination cannot be fully discarded. Wider implications of the findings Our data indicate that EM dysbiosis is associated with poor clinical outcome in ART. Thus, the EM composition before embryo transfer could be a useful biomarker to consider offering an opportunity to further improve diagnosis and treatment strategies. Trial registration number Clinical trials.gov 03330444

RESULTS: 1301 RNA species were common to both the mouse and human DEGs. 318 of these genes were directly bound by FOXO1. Pathway analysis identified Wnt/B-catenin signaling (-log(p-value) 4.14, Z score -0.626), estrogen mediated proliferation (-log(p-value) 10, Z score -3.05), and IL-6 signaling (-log(p-value) 4.73, Z score 0.943) as significantly altered by both human cycle phase and murine FOXO1 deletion, strongly suggesting a role of FOXO1 in these critical pathways for normal endometrial function. ChIP-Seq demonstrated direct FOXO1 binding to multiple regulated genes involved in estrogen-mediated proliferation, IL-6 signaling, and Wnt/b-catenin signaling, supporting a direct action of FOXO1 on these essential signaling pathways. FOXO1 was also found to directly bind several upstream regulators critical to endometrial receptivity, including CEBPB and CCND1.CONCLUSIONS: Epithelial FOXO1 directly regulates key pathways necessary for human uterine receptivity.References: 1. Vasquez YM, Wang X, Wetendorf M, et al. FOXO1 regulates uterine epithelial integrity and progesterone receptor expression critical for embryo implantation.