Endometrial microbiota composition is associated with reproductive outcome in infertile patients

Abstract

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. Video Abstract.

Fig. 1

Flowchart of the study population. Of all patients assessed for eligibility (n = 452), 44 were excluded from the analysis and 66 were lost to follow-up. Thus, 342 patients were ultimately included in our assessment of the impact of the endometrial microbiome on pregnancy outcomes. Abbreviations: BMI, body mass index; ERA, endometrial receptivity analysis; ET, embryo transfer; HRT, hormonal replacement therapy

Fig. 2

Distribution of sequencing data. PCA showing the clustering of the (A) endometrial fluid samples (n = 336) and (B) endometrial biopsy samples (n = 296) and their corresponding blank controls, based on quality parameters such as percentage of empty reads, dispersion index for each sample and the ratio between the filtered and mapped reads. Samples are coloured using a filtered/mapped reads threshold of 0.65 for EF and 0.7 for EB. Abbreviations: BP, biochemical pregnancy; EP, ectopic pregnancy; LB, live birth; CM, clinical miscarriage; NP, no pregnancy

Fig. 3

Co-occurrence bacterial networks in endometrial fluid (A) and endometrial biopsy (B) samples. Each network was created by computing the co-occurring bacteria with significant Pearson correlation coefficients. Samples from all reproductive outcomes are represented. Node properties: (i) circle size, proportional to the normalised and standardised bacterial relative abundances; (ii) colour, communities as retrieved by the Louvain algorithm. Edge properties: (i) thickness, proportional to p value of Pearson correlation coefficient, from the most significant (thicker) to the less significant (thinner); (ii) colour, red for negative and grey for positive Pearson correlation coefficients

Fig. 4

Co-occurrence bacterial networks associated with reproductive outcomes. Co-occurrence bacterial networks in A endometrial fluid samples and B endometrial biopsy samples for each ART outcome. Each network was created by computing the co-occurring bacterial communities with significant Pearson correlation coefficients. Node properties: (i) circle size, proportional to the normalised and standardised bacterial relative abundances; (ii) colour, communities as retrieved by the Louvain algorithm. Edge properties: (i) thickness, proportional to p value of the Pearson correlation coefficient, from the most significant (thicker) to the less significant (thinner); (ii) colour, red for negative and grey for positive Pearson correlation coefficients. For association graphs, the same criteria were applied, with the thickness of the circle and colour intensity being proportional to the corresponding Pearson correlation coefficients. Pairs of bacteria without a circle have no significant Pearson correlation coefficient. BP, biochemical pregnancy; CM, clinical miscarriage; LB, live birth; NP, no pregnancy

Fig. 5

Lactobacillus is more abundant than other taxa in reproductive success vs failure. A Difference between Lactobacillus and other reproductive tract taxa using z-score-normalised values in endometrial fluid (left panel) and endometrial biopsy (right panel) samples. B Predictive model showing the probability of each reproductive outcome based on the EM profile. Posterior predictive distribution density plot of z-score differences between Lactobacillus and other reproductive tract taxa by reproductive outcome. BP, biochemical pregnancy; CM, clinical miscarriage; LB, live birth; NP, no pregnancy

Fig. 6

Pathogenic bacterial profiles significantly associated with reproductive outcome. Box plots showing taxa with significant differential abundance in no pregnancy (NP), biochemical pregnancy (BP) and clinical miscarriage (CM) compared to live birth (LB). Differential abundance was calculated using the distance of each value to the upper (U) or lower (L) bounds for the 95% CI in LB (Supplementary Fig. 3) in A endometrial fluid and B endometrial biopsy samples. Only taxa with significant differential abundance, calculated with a two-sided Mann-Whitney U test, are represented in the graphs. BP, biochemical pregnancy; LB, live birth; CM, clinical miscarriage; NP, no pregnancy

Fig. 7

Chronic endometritis profile associated with reproductive outcomes. Box plots showing differential abundance in chronic endometritis-causing bacteria in no pregnancy (NP), biochemical pregnancy (BP) and clinical miscarriage (CM) compared to live birth (LB). Differential abundance was calculated using the distance of each value to the upper (U) or lower (L) bounds for the 95% CI in LB (Supplementary Fig. 3) in A endometrial fluid and B endometrial biopsy samples. Only taxa with significant differential abundance, calculated with a two-sided Mann-Whitney U test, are represented in the graphs. BP, biochemical pregnancy; LB, live birth; CM, clinical miscarriage; NP, no pregnancy