Endometrial microbiome at the time of embryo transfer: next-generation sequencing of the 16S ribosomal subunit

Abstract

Purpose: Characterization of the human microbiome has become more precise with the application of powerful molecular tools utilizing the unique 16S ribosomal subunit's hypervariable regions to greatly increase sensitivity. The microbiome of the lower genital tract can prognosticate obstetrical outcome while the upper reproductive tract remains poorly characterized. Here, the endometrial microbiome at the time of single embryo transfer (SET) is characterized by reproductive outcome.

Methods: Consecutive patients undergoing euploid, SET was included in the analysis. After embryo transfer, performed as per routine, the most distal 5-mm portion of the transfer catheter was sterilely placed in a DNA free PCR tube. Next-generation sequencing of the bacteria specific 16S ribosome gene was performed, allowing genus and species calls for microorganisms.

Results: Taxonomy assignments were made on 35 samples from 33 patients and 2 Escherichia coli controls. Of the 33 patients, 18 had ongoing pregnancies and 15 did not. There were a total of 278 different genus calls present across patient samples. The microbiome at time of transfer for those patients with ongoing pregnancy vs. those without ongoing pregnancy was characterized by top genera by sum fraction. Lactobacillus was the top species call for both outcomes.

Conclusions: The data presented here show the microbiome at the time of embryo transfer can successfully be characterized without altering standard clinical practice. This novel approach, both in specimen collection and analysis, is the first step toward the goal of determining physiologic from pathophysiologic microbiota. Further studies will help delineate if differences in the microbiome at the time of embryo transfer have a reliable impact on pregnancy outcome.

Keywords: 16S ribosomal subunit; Microbiome, embryo transfer; Next-generation sequencing.

Fig. 1

Examples of the overall fraction of reads supporting the genera are shown for Acinetobacter (a), Pseudomonas (b), Lactobacillus (c), and Flavobacterium (d) in the non-ongoing and ongoing categories. Uncorrected p values are shown

Fig. 2

The microbiome at time of transfer for those patients with ongoing pregnancy vs. those without ongoing pregnancy characterized by top genera. Key represents fraction of reads given to a genus among all reads assigned

Fig. 3

To characterize the diversity of the samples, two alpha diversity metrics were utilized: the Shannon diversity index (SDI) and chao1. The indices for the V3 hypervariable region are shown here for the samples obtained from the ongoing vs. non-ongoing pregnancy outcomes

Fig. 4

The p values for all comparison groups are plotted along with their max mean fraction within status group. Given the large number of variables tested for association with patient ongoing status in this initial study, no differences between the two status groups were large enough to survive multiple test corrections