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

Mowla S; Farahani L; Tharakan T; Davies R; Correia GD; Lee YS; Sudip Kundu; Khanjani S; Sindi E; Raj Rai; Lesley Regan; Khalifa D; Ralf Henkel; Minhas S; Dhillo WS; Ben Nagi J; Phil Bennett; MacIntyre DA; Jayasena CN

doi:10.7554/elife.96090

Male Fertility · Male Factor Causes

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

Mowla S, Farahani L, Tharakan T, Davies R, Correia GD, Lee YS, Kundu S, Khanjani S, Sindi E, Rai R, Regan L, Khalifa D, Henkel R, Minhas S, Dhillo WS, Ben Nagi J, Bennett P, MacIntyre DA, Jayasena CN

Published May 8, 2025 eLife, 13

DOI 10.7554/elife.96090 PMC PMC12061474

Abstract

Several studies have associated seminal microbiota abnormalities with male infertility but have yielded differing results owing to their limited sizes or depths of analyses. The semen microbiota during recurrent pregnancy loss (RPL) has not been investigated. Comprehensively assessing the seminal microbiota in men with reproductive disorders could elucidate its potential role in clinical management. We used semen analysis, terminal-deoxynucleotidyl-transferase-mediated-deoxyuridine-triphosphate-nick-end-labelling, Comet DNA fragmentation, luminol reactive oxidative species (ROS) chemiluminescence, and metataxonomic profiling of semen microbiota by 16S rRNA amplicon sequencing in this prospective, cross-sectional study to investigate composition and bacterial load of seminal bacterial genera and species, semen parameters, ROS, and sperm DNA fragmentation in men with reproductive disorders and proven fathers. 223 men were enrolled, including healthy men with proven paternity (n=63), the male partners in a couple encountering RPL (n=46), men with male factor infertility (n=58), and the male partners of couples with unexplained infertility (n=56). Rates of high sperm DNA fragmentation, elevated ROS, and oligospermia were more prevalent in the study group compared with control. In all groups, semen microbiota clustered into three major genera-dominant groups (1, Streptococcus; 2, Prevotella; 3, Lactobacillus and Gardnerella); no species clusters were identified. Group 2 had the highest microbial richness (p<0.001), alpha-diversity (p<0.001), and bacterial load (p<0.0001). Overall bacterial composition or load has not been found to associate with semen analysis, ROS, or DNA fragmentation. Whilst global perturbation of the seminal microbiota is not associated with male reproductive disorders, men with unidentified seminal Flavobacterium are more likely to have abnormal seminal analysis. Future studies may elucidate if Flavobacterium reduction has therapeutic potential.

Topics

By this author

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology

Björndahl L et al., 2022 Human Reproduction

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Hormone Testing · Serum Panels

Investigating the potential of clinical and biochemical markers to differentiate between functional hypothalamic amenorrhoea and polycystic ovarian syndrome: A retrospective observational study

Abou Sherif S et al., 2021 Clinical endocrinology

Functional hypothalamic amenorrhoea (FHA) is a common cause of amenorrhoea, but diagnosis can be challenging. The aim of this study was to investigate the clinical and biochemical features of FHA, compared to that of polycystic ovarian syndrome (PCOS) and assess the diagnostic performance of the different parameters for differentiating the two conditions. This was a retrospective observational study. We analysed clinical and biochemical parameters of women diagnosed with FHA and PCOS following specialist assessment at the reproductive endocrine gynaecology clinic, St Mary's Hospital. Compared with PCOS, women with FHA had significantly lower body mass index (BMI; 20.1 ± 2.9 vs. 31.1 ± 7.8 kg/m2 ; p< .0001) and a thinner endometrium (3.75 ± 2.23 vs. 6.82 ± 3.32 mm; p< .0001). Women with FHA had significantly lower luteinising hormone (LH; 3.46 ± 7.31 vs. 8.79 ± 4.98 IU/L; p< .0001), and lower LH to follicle-stimulating hormone (FSH) ratio, estradiol, thyroid-stimulating hormone, free thyroxine and prolactin levels; there was no significant difference in FSH levels. BMI had the greatest predictive performance for FHA (area under the curve [AUC]: 0.93; p< .001), followed by estradiol (AUC: 0.89; p< .001), LH (AUC: 0.88; p< .001) and LH:FSH ratio (AUC: 0.86; p< .001). Our data provides quantification for diagnostic accuracy of clinical parameters to differentiate FHA from PCOS, namely low BMI, estradiol, LH and LH:FSH ratio. These data could help clinicians more reliably diagnose FHA in women with secondary amenorrhoea.

Early Pregnancy · Progesterone Support

Micronized vaginal progesterone to prevent miscarriage: a critical evaluation of randomized evidence

Coomarasamy A et al., 2020 Am J Obstet Gynecol Free full text on PubMed Central

Progesterone is essential for the maintenance of pregnancy. Several small trials have suggested that progesterone supplementation may reduce the risk of miscarriage in women with recurrent or threatened miscarriage. Cochrane Reviews summarized the evidence and found that the trials were small with substantial methodologic weaknesses. Since then, the effects of first-trimester use of vaginal micronized progesterone have been evaluated in 2 large, high-quality, multicenter placebo-controlled trials, one targeting women with unexplained recurrent miscarriages (the PROMISE [PROgesterone in recurrent MIScarriagE] trial) and the other targeting women with early pregnancy bleeding (the PRISM [PRogesterone In Spontaneous Miscarriage] trial). The PROMISE trial studied 836 women from 45 hospitals in the United Kingdom and the Netherlands and found a 3% greater live birth rate with progesterone but with substantial statistical uncertainty. The PRISM trial studied 4153 women from 48 hospitals in the United Kingdom and found a 3% greater live birth rate with progesterone, but with a P value of .08. A key finding, first observed in the PROMISE trial, and then replicated in the PRISM trial, was that treatment with vaginal micronized progesterone 400 mg twice daily was associated with increasing live birth rates according to the number of previous miscarriages. Prespecified PRISM trial subgroup analysis in women with the dual risk factors of previous miscarriage(s) and current pregnancy bleeding fulfilled all 11 conditions for credible subgroup analysis. For the subgroup of women with a history of 1 or more miscarriage(s) and current pregnancy bleeding, the live birth rate was 75% (689/914) with progesterone vs 70% (619/886) with placebo (rate difference 5%; risk ratio, 1.09, 95% confidence interval, 1.03-1.15; P=.003). The benefit was greater for the subgroup of women with 3 or more previous miscarriages and current pregnancy bleeding; live birth rate was 72% (98/137) with progesterone vs 57% (85/148) with placebo (rate difference 15%; risk ratio, 1.28, 95% confidence interval, 1.08-1.51; P=.004). No short-term safety concerns were identified from the PROMISE and PRISM trials. Therefore, women with a history of miscarriage who present with bleeding in early pregnancy may benefit from the use of vaginal micronized progesterone 400 mg twice daily. Women and their care providers should use the findings for shared decision-making.

Fertility and Outcomes · Conception After Excision

Reproductive capacity and recurrence of disease after surgery for moderate and severe endometriosis - a retrospective single center analysis

Schippert C et al., 2020 BMC Women's Health Free full text on PubMed Central

Background Endometriosis can be associated with considerable pain and sterility. After surgical excision of moderate or severe endometriosis lesions, the rate of recurrence reaches up to 67%. The objective of this retrospective study was to establish the recurrence and pregnancy rates following surgical resection of stage III/IV endometriosis lesions. Indications for operation were endometriosis symptoms, sonographic findings and/or infertility. Methods A total of 456 patients who underwent stage III/IV endometriosis surgery between 2004 and 2014 were sent a questionnaire relating to their postoperative medical treatment, pregnancies, relief of symptoms and recurrence. Responses of 206 patients (45.2%) and their clinical data were analysed for this study. Results A total of 66.5% ( N = 137) of patients had stage III disease, and 33.5% ( N = 69) had stage IV disease. The average age was 37 years (17–59). A total of 63.1% ( N = 130) of surgeries were performed by laparoscopy, 21.8% ( N = 45) were performed by laparotomy and 15% ( N = 31) were performed by conversion. Complete resection of endometriosis lesions was achieved in 90.8% of patients ( N = 187). After surgery, 48.5% ( N = 100) of the women did not receive hormonal treatment; the main reason was the desire for children in 53%. Complete or partial relief in complaints was achieved in 93.2% ( N = 192). The rate of recurrence was 21.8% ( N = 45). The statistically significant factors that was associated with a higher risk to develop recurrence was an age < 35 ( p < 0.005). After surgery, 65.8% (79/120) of patients who wished to have children became pregnant. There was a statistically significant association among a higher postoperative pregnancy rate and age < 35 ( p < 0.003) in multivariate logistic regression analysis and laparoscopic surgical access in univariate logistic regression analysis ( p < 0.01). Conclusion We assessed the high percentage of complete or partial relief of symptoms of 93.2%, the high postoperative pregnancy rate of 65.8% and the low rate of recurrence of 21.8% compared to international literature to be very encouraging for women suffering from moderate and severe endometriosis. Though laparoscopy is considered the ‘gold standard’of endometriosis surgery, laparotomy still may be indicated in patients with extensive endometriosis especially to preserve reproductive function.

Related research

Male Factor Causes · Lifestyle and Environment

Effect of environmental factors on seminal microbiome and impact on sperm quality

Neto FTL et al., 2024 Frontiers in endocrinology Free full text on PubMed Central

This review provides a comprehensive overview of the existing research on the seminal microbiome and its association with male infertility, while also highlighting areas that warrant further investigation. A narrative review was conducted, encompassing all relevant studies published between 1980-2023 on the male reproductive tract microbiome in humans. This review considered studies utilizing culture-based, polymerase chain reaction (PCR)-based, and next-generation sequencing (NGS)-based methodologies to analyze the microbiome. Data extraction encompassed sample types (semen or testicular tissue), study designs, participant characteristics, employed techniques, and critical findings. We included 37 studies comprising 9,310 participants. Among these, 16 studies used culture-based methods, 16 utilized NGS, and five employed a combination of methods for microorganism identification. Notably, none of the studies assessed fungi or viruses. All NGS-based studies identified the presence of bacteria in all semen samples. Two notable characteristics of the seminal microbiome were observed: substantial variability in species composition among individuals and the formation of microbial communities with a dominant species. Studies examining the testicular microbiome revealed that the testicular compartment is not sterile. Interestingly, sexually active couples shared 56% of predominant genera, and among couples with positive cultures in both partners, 61% of them shared at least one genital pathogen. In couples with infertility of known causes, there was an overlap in bacterial composition between the seminal and vaginal microbiomes, featuring an increased prevalence of Staphylococcus and Streptococcus genera. Furthermore, the seminal microbiome had discernible effects on reproductive outcomes. However, bacteria in IVF culture media did not seem to impact pregnancy rates. Existing literature underscores that various genera of bacteria colonize the male reproductive tract. These organisms do not exist independently; instead, they play a pivotal role in regulating functions and maintaining hemostasis. Future research should prioritize longitudinal and prospective studies and investigations into the influence of infertility causes and commonly prescribed medication to enhance our understanding of the seminal microbiota's role in reproductive health.

Male Factor Causes · Lifestyle and Environment

Male Tract Microbiota and Male Infertility

Grande G et al., 2024 Cells Free full text on PubMed Central

In recent years, several studies have analyzed the composition of the male genital tract microbiota and its changes in infertility or in different situations associated with infertility. The aim of this narrative review is to obtain more insight on this topic; in particular, to describe actual evidence about changes in the semen microbiota in patients with infertility, male tract infections, or HPV infections. In semen, an increase in semen Prevotella spp. is associated with oligozoospermia and with obesity-associated asthenozoospermia; an increase in Pseudomonas is more frequently associated with asthenozoospermia and oligozoospermia; a reduction in Lactobacilli spp. (namely in Lactobacillus crispatus) may represent a marker of low semen quality. However, an increase in Lactobacillus iners is considered a risk factor for a reduced sperm concentration. In patients with prostatitis, there is a reduction in Lactobacillus spp. and an increase in Streptococcus spp., opening important perspectives about the role of probiotic treatments in these patients. Finally, an increase in Fusobacteria spp. was observed in patients with an HPV infection. In the conclusion, we underline the interactions between the seminal and vaginal microbiota, so that further studies should focus on the "couple genital microbiota".

Semen Analysis · Sperm Function Testing

Sperm DNA fragmentation and recurrent pregnancy loss: a systematic review and meta-analysis

McQueen DB et al., 2019 Fertil Steril

To investigate the rate of sperm DNA fragmentation in male partners of women with recurrent pregnancy loss and fertile control women. Systematic review and meta-analysis. Not applicable. PATIENT(S): A total of 579 male partners of women with recurrent pregnancy loss and 434 male partners fertile control women. INTERVENTION(S): Prospective studies were identified through a Pubmed search. Recurrent pregnancy loss was defined as two or more previous pregnancy losses. Fertile control women had a history of a live birth or ongoing pregnancy. MAIN OUTCOME MEASURE(S): The primary outcome was the rate of sperm DNA fragmentation. The summary measures were reported as mean difference with 95% confidence interval (CI). RESULT(S): Fifteen prospective studies were included in a qualitative review. Pooled data from 13 studies with sufficient data for meta-analysis suggest that male partners of women with a history of recurrent pregnancy loss have a significantly higher rate of sperm DNA fragmentation compared to the partners of fertile control women: mean difference 11.91, 95% CI 4.97-18.86. CONCLUSION(S): These findings support an association between sperm DNA fragmentation and recurrent pregnancy loss. However, given the significant heterogeneity between studies and lack of prospective pregnancy outcome data, further large prospective studies are needed.

Male Endocrine and Genetic Factors · Genetic Causes of Male Infertility

Decreased fecundity and sperm DNA methylation patterns

Jenkins TG et al., 2016 Fertil Steril Free full text on PubMed Central

To evaluate the relationship between epigenetic patterns in sperm and fecundity. Prospective study. Academic andrology and in vitro fertilization laboratory. PATIENT(S): Twenty-seven semen samples from couples who conceived within 2 months of attempting a pregnancy and 29 semen samples from couples unable to achieve a pregnancy within 12 months. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genomewide assessment of differential sperm DNA methylation and standard semen analysis. RESULT(S): We analyzed DNA methylation alterations associated with fecundity in 124 semen samples, and identified regions of interest in 27 semen samples from couples who conceived within 2 months of attempting a pregnancy and a total of 29 semen samples from couples who were unable to achieve a pregnancy within 12 months. No differences in sperm count, sperm morphology, or semen volume were observed between the patients achieving a pregnancy within 2 months of study time and those not obtaining a pregnancy within 12 months. However, using data from the human methylation 450k array analysis we did identify two genomic regions with statistically significantly decreased (false discovery rate <0.01) methylation and three genomic regions with statistically significantly increased methylation in the failure-to-conceive group. The only two sites where decreased methylation was associated with reduced fecundity are at closely related genes known to be expressed in sperm, HSPA1L and HSPA1B. CONCLUSION(S): Our data suggest that there are genomic loci where DNA methylation alterations are associated with decreased fecundity. We have thus identified candidate loci for future study to verify these results and investigate the causative or contributory relationship between altered sperm methylation and decreased fecundity.

Male Fertility › Male Factor Causes › Lifestyle and Environment · Infertility › Recurrent Pregnancy Loss › Immune and Thrombophilia Factors

Sudip Kundu, Raj Rai, Lesley Regan, Ralf Henkel, Phil Bennett

S Kundu, R Rai, L Regan, R Henkel, Philip Bennett, Phillip Bennett, P Bennett

DOI 10.7554/elife.96090 10.7554/elife.96090 Mowla et al. 2025, Mowla 2025

Cite this article

Mowla, S., Farahani, L., Tharakan, T., Davies, R., Correia, G. D., Lee, Y. S., Kundu, S., Khanjani, S., Sindi, E., Rai, R., Regan, L., Khalifa, D., Henkel, R., Minhas, S., Dhillo, W. S., Ben Nagi, J., Bennett, P., MacIntyre, D. A., & Jayasena, C. N. (2025). Characterisation and comparison of semen microbiota and bacterial load in men with infertility, recurrent miscarriage, or proven fertility. eLife, 13. https://doi.org/10.7554/elife.96090