Characterisation and comparison of semen microbiota and bacterial load in men with infertility, recurrent miscarriage, or proven fertility

  1. Shahriar Mowla
  2. Linda Farahani
  3. Tharu Tharakan
  4. Rhianna Davies
  5. Goncalo DS Correia
  6. Yun S Lee
  7. Samit Kundu
  8. Shirin Khanjani
  9. Emad Sindi
  10. Raj Rai
  11. Lesley Regan
  12. Dalia Khalifa
  13. Ralf Henkel
  14. Suks Minhas
  15. Waljit S Dhillo
  16. Jara Ben Nagi
  17. Phillip Bennett
  18. David A MacIntyre  Is a corresponding author
  19. Channa N Jayasena  Is a corresponding author
  1. Institute of Reproductive and Developmental Biology, Imperial College London, United Kingdom
  2. Wolfson Fertility Unit, Department of Gynaecology, St. Mary’s Hospital, Imperial College NHS Trust, United Kingdom
  3. Section of Endocrinology & Investigative Medicine, Imperial College London, United Kingdom
  4. Department of Urology, Charing Cross Hospital, Imperial College NHS Trus, United Kingdom
  5. March of Dimes European Prematurity Research Centre, Imperial College London, United Kingdom
  6. Department of Gynaecology, University College London Hospital, United Kingdom
  7. Tommy's National Centre for Miscarriage Research, Imperial College London, United Kingdom
  8. Department of Andrology, Hammersmith Hospital, Imperial College NHS Trust, United Kingdom
  9. LogixX Pharma, Theale, United Kingdom
  10. Department of Medical Bioscience, University of the Western Cape, South Africa
  11. Centre for Reproductive and Genetic Health (CRGH), United Kingdom
5 figures, 8 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Characterisation of semen microbiota composition at genera level.

(A) Heatmap of Log10 transformed read counts of top 10 most abundant genera identified in semen samples. Samples clustered into three major microbiota groups based mainly on dominance by Streptococcus (Cluster 1), Prevotella (Cluster 2), or Lactobacillus and Gardnerella (Cluster 3) (n=223, Ward’s linkage). (B) Relative abundance of the top 6 most abundant genera within each cluster. (C) Silhouette scores of individual samples within each cluster. (D) Species richness (p<0.0001; Kruskal-Wallis test) and (E) alpha-diversity (p<0.0001; Kruskal-Wallis test) significantly differed across clusters. (F) Assessment of bacterial load using qPCR showed Clusters 2 and 3 have significantly higher bacterial loads compared to Cluster 1. Dunn’s multiple comparison test was used as a post hoc test for between-group comparisons (*p<0.05, ****p<0.0001).

Figure 1—figure supplement 1
Genera-level categorisation of seminal microbiota identified three major clusters using average silhouette scores for number of clusters.
Figure 2 with 1 supplement
Co-occurrence network estimated with SparCC from 16S sequencing counts at species level.

Network representing co-occurrence patterns (edges), between various taxonomic units, assigned at species level (nodes). Edges are coloured by their estimated SparCC correlation coefficient (ρ). Edges with a SparCC bootstrapped p-value<0.05, ρ<0.25, and singleton nodes are not shown. Node colour represents network community membership. Node sizes are proportional to the mean relative abundance of their respective taxon.

Figure 2—figure supplement 1
Characterisation of semen microbiota composition at species level.

(A) Heatmap of Log10 transformed read counts of top 25 most abundant species identified in semen samples. Samples clustered into 15 microbiota groups. (B) Silhouette scores of individual samples in microbial groups. (C) Average silhouette scores for 15 clusters at species level.

Relative abundance and prevalence matrices of Flavobacterium in relation to semen quality and morphology.

(A) Relative abundance of Flavobacterium was significantly higher in samples with abnormal semen (p=0.0002, q=0.02). (B) Detection of Flavobacterium was significantly more prevalent in abnormal semen quality samples (p=0.0003). (C) Flavobacterium relative abundance was significantly higher in samples with <4% morphologically normal forms (p=0.0002, q=0.01). (D) Flavobacterium was also significantly more prevalent in samples with low percentage of morphologically normal sperm (p=0.0009).

Appendix 2—figure 1
Seminal quality and function parameters according to recruited cohorts.

Comparison of microscopic semen parameters (A) concentration (p<0.0001, Kruskal-Wallis rank-sum test), (B) progressive motility (p<0.0001, Pearson’s chi-squared test), and (C) morphology (p<0.0001, Pearson’s chi-squared test) suggested poor semen quality for male factor infertility (MFI) patients. Comparison of clinical semen qualities: (D) reactive oxidative species (ROS), (E) sperm DNA fragmentation index.

Appendix 2—figure 2
Ecological parameters of seminal microbiota for the recruited study cohorts.

(A) Species richness (p=0.30) and (B) Simpson’s diversity index (p=0.49) were not significantly different based on recruited study cohorts. Kruskal-Wallis tests with Dunn’s multiple comparison p-values demonstrated on the plots. (C) Bacterial load of seminal microbiota in recruited study cohorts. There were no significant differences in bacterial load based on recruited study cohorts using the number of 16S rRNA genes per 1 ml of semen (p=0.22, Kruskal-Wallis test).

Tables

Table 1
Patient demographics and notable parameters of seminal quality and function for controls and study subjects.

Fisher’s exact tests for all except age. Chi-squared test for age (n=223).

FactorCategoriesControlsStudy casesp-Value
DNA fragmentation indexLow45/114 (40%)69/114 (60%)0.0002***
High12/82 (15%)70/82 (85%)
ROS<3.77 RLU/s53/143 (37%)90/143 (63%)0.02*
>3.77 RLU/s5/33 (15%)28/33 (85%)
Semen volumeOptimal55/208 (26%)153/208 (84%)0.03*
Suboptimal8/15 (53%)7/15 (47%)
Age<3411/49 (22%)38/49 (88%)0.04*
34–4131/124 (25%)93/124 (85%)
>4121/50 (42%)29/50 (58%)
EthnicityCaucasian39/156 (25%)117/156 (75%)0.10
Non-Caucasian24/67 (36%)43/67 (64%)
Concentration>15 M/ml58/182 (32%)124/182 (68%)0.01*
<15 M/ml5/41 (21%)36/41 (79%)
Progressive motility>32%60/207 (29%)147/207 (71%)0.56
<32%3/16 (19%)13/16 (81%)
Sperm morphology>4%22/74 (30%)52/74 (70%)0.87
<4%41/144 (28%)103/144 (72%)
Semen qualityOptimal24/78 (31%)54/78 (69%)0.53
Suboptimal39/145 (27%)106/145 (73%)
Table 2
Differential abundance analysis for bacterial genera with seminal quality and functional parameters.

Positive t-values indicate a positive relationship, and a negative t-value describes a negative relationship between relative abundance of taxa and seminal quality and function parameters. Significant relationships are indicated using p-values. q-Values represent Benjamini-Hochberg false discovery rate corrected p-values for multiple comparisons.

Sperm quality and function parametersGeneraWelch’s t-statisticp-Valueq-Value
Sperm DNA fragmentationFinegoldia–2.360.01*0.27
Cutibacterium–2.200.02*0.27
Porphyromonas2.160.03*0.27
Varibaculum2.110.03*0.27
ROSLactobacillus2.180.02*0.66
Corynebacterium–2.040.04*0.66
Semen qualityFlavobacterium3.390.0008***0.02*
Prevotella2.260.02*0.38
Sperm concentrationPorphyromonas–2.080.03*0.61
Sperm morphologyFlavobacterium3.640.0003***0.01*
Prevotella2.030.04*0.67
Semen volumeCorynebacterium2.270.02*0.32
Actinotigum–2.200.02*0.32
Varibaculum–2.160.03*0.32
Table 3
Differential abundance analysis for bacterial species with seminal quality and functional parameters.

Positive t-values indicate a positive relationship, and a negative t-value describes a negative relationship between relative abundance of taxa and seminal quality and function parameters. Significant relationships are indicated using p-values. q-Values represent Benjamini-Hochberg false discovery rate corrected p-values for multiple comparisons.

Clinical factorSpeciesWelch’s t-statisticp-Valueq-Value
Sperm DNA fragmentationPeptostreptococcaceae bacterium2.180.03*0.91
ROSLactobacillus iners2.240.02*0.94
Unidentified Anaerococcus–2.030.04*0.94
Semen qualityUnidentified Flavobacterium3.760.0002***0.01*
Corynebacterium tuberculostearicum–2.060.04*0.82
Semen volumeCorynebacterium tuberculostearicum2.640.0080.24
Unidentified Varibaculum–2.480.010.24
Staphylococcus epidermidis2.350.010.24
Unidentified Peptoniphilus–2.320.020.24
Dialister propionicifaciens–2.240.020.24
Prevotella colorans–2.140.030.26
CohortsStaphylococcus haemolyticus0.040.020.97
Table 4
Differential abundance analysis for specific taxa at genera level for controls and cases with male factor infertility.

Positive t-values indicate a relationship, and a negative t-value describes a negative relationship between relative abundance of taxa and seminal quality and function parameters. Significant relationships are indicated using p-values. q-Values represent Benjamini-Hochberg false discovery rate corrected p-values for multiple comparisons.

Clinical factorGeneraWelch’s t-statisticp-Valueq-Value
Sperm DNA fragmentationCutibacterium–2.560.01*0.31
Porphyromonas2.340.02*0.31
Varibaculum1.960.0510.53
ROSFinegoldia–1.990.04*0.77
Sperm concentrationFinegoldia2.040.04*0.71
Sperm morphologyFlavobacterium3.640.0003***0.01*
Prevotella2.030.04*0.67
Semen volumeFacklamia2.990.003**0.10
Actinotignum–2.200.02*0.36
Dialister–1.990.04*0.36
Table 5
Differential abundance analysis for specific taxa at species for controls and male factor infertility.

Positive t-values indicate a positive relationship, and a negative t-value describes a negative relationship between relative abundance of taxa and seminal quality and function parameters. Significant relationships are indicated using p-values. q-Values represent Benjamini-Hochberg false discovery rate corrected p-values for multiple comparisons.

Clinical factorSpeciesWelch’s t-statisticp-Valueq-Value
Sperm DNA fragmentationStaphylococcus hominis–2.320.02*0.68
ROSUnidentified Flavobacterium2.420.010.54
Unidentified Anaerococcus–2.120.030.54
Schaalia radingae–2.120.03*0.54
Haemophilus parainfluenza2.020.04*0.54
Semen qualityUnidentified Flavobacterium2.360.01*0.91
Semen volumeDialister micraerophilus–2.660.008**0.41
Corynebacterium tuberculostearicum2.270.02*0.44
Staphylococcus epidermidis2.220.02*0.44
Actinotignum schaalii–2.000.04*0.45
CohortsStaphylococcus haemolyticus0.040.01*0.68
Appendix 1—table 1
Comparison of mean age and prevalence of ethnicities in study recruitment cohorts.

Ethnicity representation amongst recruited cohorts was not significantly different (p=0.38, chi-squared test). RPL: recurrent pregnancy loss, MFI: male factor infertility, UI: unexplained infertility.

Study cohortAge (mean ±SD)Ethnicity
Control (n=63)40.1±839/63 (62%) Caucasian
24/63 (38%) Non-Caucasian
RPL (n=46)38.2±535/46 (76%) Caucasian
11/46 (24%) Non-Caucasian
MFI (n=58)36.3±4.541/58 (70%) Caucasian
17/58 (30%) Non-Caucasian
UI (n=56)37±4.741/56 (73%) Caucasian
15/56 (27%) Non-Caucasian
Appendix 1—table 2
Distribution of clinical factors, microscopic seminal parameters, confounding factors, and recruitment cohorts according to genera clusters.

Chi-squared tests.

FactorsThresholdsCluster 1Cluster 2Cluster 3p-Value
DNA frag indexLow60 (53%)39 (34%)15 (13%)0.47
High37 (45%)35 (43%)10 (12%)
ROS<3.77 RLU/s74 (52%)56 (39%)13 (9%)0.81
>3.77 RLU/s19 (58%)11 (33%)3 (9%)
Semen volumeOptimal105 (50%)80 (38%)23 (12%)0.12
Suboptimal8 (53%)3 (20%)4 (27%)
CohortsControl36 (57%)22 (35%)5 (8%)0.76
MFI26 (45%)25 (45%)7 (10%)
RPL23 (50%)17 (37%)6 (13%)
UI28 (50%)19 (34%)9 (16%)
Age<3430 (61%)14 (29%)5 (10%)0.58
34–4159 (48%)49 (40%)16 (12%)
>4124 (48%)20 (40%)6 (12%)
EthnicityCaucasian82 (53%)57 (37%)17 (10%)0.58
Non-Caucasian31 (46%)26 (39%)10 (15%)
Concentration>15 M/ml93 (51%)67 (37%)22 (12%)0.96
<15 M/ml20 (49%)16 (39%)5 (12%)
Progressive motility>32%105 (51%)78 (38%)24 (11%)0.67
<32%8 (50%)5 (31%)3 (19%)
Morphology>4%37 (50%)24 (32%)13 (18%)0.19
<4%72 (50%)58 (40%)14 (10%)
Semen qualityOptimal41 (53%)24 (31%)13 (16%)0.17
Suboptimal72 (50%)59 (17%)14 (33%)
Appendix 1—table 3
Richness and diversity of seminal bacterial based on seminal quality and function parameters.

Categorical classifications of seminal parameters were based on the clinically defined thresholds. Mann-Whitney tests for all except age. Kruskal-Wallis test was used for age.

FactorsRichness p-valueDiversity p-value
DNA frag index0.680.89
ROS0.250.23
Semen volume0.540.85
Age0.140.12
Ethnicity0.310.24
Concentration0.790.66
Progressive motility0.380.54
Morphology0.820.97
Semen quality0.740.90

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  1. Shahriar Mowla
  2. Linda Farahani
  3. Tharu Tharakan
  4. Rhianna Davies
  5. Goncalo DS Correia
  6. Yun S Lee
  7. Samit Kundu
  8. Shirin Khanjani
  9. Emad Sindi
  10. Raj Rai
  11. Lesley Regan
  12. Dalia Khalifa
  13. Ralf Henkel
  14. Suks Minhas
  15. Waljit S Dhillo
  16. Jara Ben Nagi
  17. Phillip Bennett
  18. David A MacIntyre
  19. Channa N Jayasena
(2025)
Characterisation and comparison of semen microbiota and bacterial load in men with infertility, recurrent miscarriage, or proven fertility
eLife 13:RP96090.
https://doi.org/10.7554/eLife.96090.4