Culturomics to Investigate the Endometrial Microbiome: Proof-of-Concept

Abstract

The microbiome of the reproductive tract has been associated with (sub)fertility and it has been suggested that dysbiosis reduces success rates and pregnancy outcomes. The endometrial microbiome is of particular interest given the potential impact on the embryo implantation. To date, all endometrial microbiome studies have applied a metagenomics approach. A sequencing-based technique, however, has its limitations, more specifically in adequately exploring low-biomass settings, such as intra-uterine/endometrial samples. In this proof-of-concept study, we demonstrate the applicability of culturomics, a high-throughput culturing approach, to investigate the endometrial microbiome. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy, as part of their routine work-up at Brussels IVF, were included after their informed consent. Biopsies were used to culture microbiota for up to 30 days in multiple aerobic and anaerobic conditions. Subsequent WASPLab^®-assisted culturomics enabled a standardized methodology. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or 16S rRNA sequencing was applied to identify all of bacterial and fungal isolates. Eighty-three bacterial and two fungal species were identified. The detected species were in concordance with previously published metagenomics-based endometrial microbiota analyses as 77 (91%) of them belonged to previously described genera. Nevertheless, highlighting the added value of culturomics to identify most isolates at the species level, 53 (62.4%) of the identified species were described in the endometrial microbiota for the first time. This study shows the applicability and added value of WASPLab^®-assisted culturomics to investigate the low biomass endometrial microbiome at a species level.

Keywords: 16S rRNA; ART; MALDI-TOF; culturomics; embryo implantation; endometrial microbiome.

Figure 1

Venn-diagram of described species in the endometrium microbiome. Left circle: species found with our culturomics protocol. Right circle: species described in the literature [7,16]. Inner circle: species found in both our study and the literature [7,16]. The color describes the prevalence over the 10 samples ranging from present in one sample to present in eight samples. Literature overview includes both infertile and subfertile women.

Figure 2

Venn-diagram of the described genera in the endometrium microbiome. Left circle: genera found with our culturomics protocol. Right circle: genera described in the literature [7,16]. Inner circle: genera found in both our study and the literature [7,16]. Literature overview includes both infertile and subfertile women.

Figure 3

Scattered boxplot for the total number of species found per sample per incubation method (blue: aerobic pre-incubation, orange: anaerobic pre incubation, green: no pre-incubation and red: total).

Figure 4

Scattered boxplot for the number of unique species found within each sample per incubation method (blue: aerobic pre-incubation, orange: anaerobic pre incubation, green: no pre-incubation and red: total).

Figure 5

Scattered boxplot of the number of facultative (blue) and obligate (orange) anaerobic species at different incubation times.

Figure 6

Heatmap of the unweighted UniFrac distances between the samples, along with a dendrogram of the agglomerative hierarchical clustering with a complete linkage method. The numbers from one to 10 on the X and Y axis represent the patient samples. The color in the figure represents phylogenetic relatedness, darker colors equals closer relatedness.

Figure 7

Summary of our culturomics workflow. The endometrial sample is collected and transported in an eSwab^® tube. Following the homogenization of the biopsy with a sterile pestle and mortar, the sample is partly manually inoculated on agar plates. Next, five days later, the colonies on the plates are identified using the MALDI-TOF MS. The other part of the sample is injected in two pre-incubation bottles and incubated for 30 days. Then, after 1, 5, 10 and 30 days, 1 µL of the bottle is inoculated on the agar plates with the WASPLab^®. r Five days later, the colonies on the plates are identified using the MALDI-TOF MS. If the MALDI-TOF MS fails in identifying the strain, a 16S rRNA gene sequencing was performed.

Figure 8

Workflow we used for sterilizing the rumen fluid. No autoclave was used in order to preserve the thermolabile components. Sterility was checked at the end of the process.