Bacterial communities in semen from men of infertile couples: metagenomic sequencing reveals relationships of seminal microbiota to semen quality

Shun-Long Weng, Chih-Min Chiu, Feng-Mao Lin, Wei-Chih Huang, Chao Liang, Ting Yang, Tzu-Ling Yang, Chia-Yu Liu, Wei-Yun Wu, Yi-An Chang, Tzu-Hao Chang, Hsien-Da Huang, Shun-Long Weng, Chih-Min Chiu, Feng-Mao Lin, Wei-Chih Huang, Chao Liang, Ting Yang, Tzu-Ling Yang, Chia-Yu Liu, Wei-Yun Wu, Yi-An Chang, Tzu-Hao Chang, Hsien-Da Huang

Abstract

Some previous studies have identified bacteria in semen as being a potential factor in male infertility. However, only few types of bacteria were taken into consideration while using PCR-based or culturing methods. Here we present an analysis approach using next-generation sequencing technology and bioinformatics analysis to investigate the associations between bacterial communities and semen quality. Ninety-six semen samples collected were examined for bacterial communities, measuring seven clinical criteria for semen quality (semen volume, sperm concentration, motility, Kruger's strict morphology, antisperm antibody (IgA), Atypical, and leukocytes). Computer-assisted semen analysis (CASA) was also performed. Results showed that the most abundant genera among all samples were Lactobacillus (19.9%), Pseudomonas (9.85%), Prevotella (8.51%) and Gardnerella (4.21%). The proportion of Lactobacillus and Gardnerella was significantly higher in the normal samples, while that of Prevotella was significantly higher in the low quality samples. Unsupervised clustering analysis demonstrated that the seminal bacterial communities were clustered into three main groups: Lactobacillus, Pseudomonas, and Prevotella predominant group. Remarkably, most normal samples (80.6%) were clustered in Lactobacillus predominant group. The analysis results showed seminal bacteria community types were highly associated with semen health. Lactobacillus might not only be a potential probiotic for semen quality maintenance, but also might be helpful in countering the negative influence of Prevotella and Pseudomonas. In this study, we investigated whole seminal bacterial communities and provided the most comprehensive analysis of the association between bacterial community and semen quality. The study significantly contributes to the current understanding of the etiology of male fertility.

Conflict of interest statement

Competing Interests: The authors and the affiliations to Health GeneTech Corporation, along with any other relevant declarations relating to employment, consultancy, patents, products in development or marketed product etc. have declared that no competing interests exist. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Research overview.
Figure 1. Research overview.
Figure 2. Relatively abundant genera in semen…
Figure 2. Relatively abundant genera in semen samples.
Figure 3. Bacterial communities in semen samples.
Figure 3. Bacterial communities in semen samples.
(A) Hierarchical clustering was used to generate a clustering tree depicting the bacterial diversity in 96 men with clinical values. The scale bar represents the sample distance generated by UniFrac. (B) Clinical value status is depicted in the seven horizontal bars. The red and green rectangles respectively represent abnormal and normal clinical values. B1 to B7 respectively represent the clinical value status of semen volume, sperm concentration, motility, Kruger's strict morphology, antisperm antibody (IgA), Atypical, and leucocytes. (C) G1, G2 and G3 represent the three main groups in the clustering results, which were respectively predominated by Pseudomonas, Lactobacillus and Prevotella. (D) The colored bars represent the taxonomic compositions in each sample. Less abundant taxa were grouped in the “Others” category.
Figure 4. Diversity and richness of bacterial…
Figure 4. Diversity and richness of bacterial communities in the G1, G2 and G3 groups.
Figure 5. Genera co-occurring with main contributors…
Figure 5. Genera co-occurring with main contributors and their co-occurring children in the (A) G1, (B) G2 and (C) G3 groups.
Figure 6. Relatively abundant genera in the…
Figure 6. Relatively abundant genera in the (A) normal and (B) case samples.
Figure 7. Relatively abundant bacteria with significantly…
Figure 7. Relatively abundant bacteria with significantly different distributions between the normal and case samples (* p
Figure 8. Diversity and richness of bacterial…
Figure 8. Diversity and richness of bacterial communities in the case and normal samples.
Figure 9. PCoA analysis of case and…
Figure 9. PCoA analysis of case and normal samples.
The blue rectangles and red circles respectively represent the case and normal samples.
Figure 10. Classification rule and potential markers…
Figure 10. Classification rule and potential markers for classifying microbiome communities of semen.

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