European Journal of Cardiovascular Medicine

Infertility affects 8–12% of couples worldwide and carries substantial psychosocial and economic burden. Male factors are implicated in up to half of infertile partnerships, either as a primary or contributory cause. Beyond classical etiologies—endocrine dysfunction, varicocele, genetic abnormalities, and environmental exposures—evidence increasingly implicates genital tract microbiota and subclinical infections in the disruption of semen quality and function. Bacteria and fungi may adhere to spermatozoa, trigger inflammatory cascades, generate reactive oxygen species (ROS), and alter the biochemical milieu of seminal plasma, thereby impairing motility, vitality, and fertilizing capacity [1–4]. Concurrently, community-onset antimicrobial resistance (AMR) has risen across uropathogens and genital tract organisms, complicating management strategies that rely on empirical therapy.

Seminal microbiota are detectable even in the absence of overt symptoms. Culture-dependent and molecular studies have recovered organisms such as Escherichia coli, Staphylococcus aureus, Ureaplasma species, Mycoplasma genitalium, and Candida spp. from semen. These microorganisms can agglutinate sperm, damage membranes, and increase DNA fragmentation indices—effects linked to reduced natural conception rates and poorer outcomes with assisted reproductive techniques. Importantly, carriage is often asymptomatic; thus, reliance on symptom-based screening misses a substantial fraction of clinically relevant colonization [5–8].

Antibiotic sensitivity landscapes further complicate care. In India and across Asia, fluoroquinolone resistance in Enterobacterales has become pervasive, while extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL) mechanisms threaten cephalosporins and carbapenems, respectively. In Gram-positive organisms, macrolide and lincosamide resistance is common, yet doxycycline and oxazolidinones often retain activity. These trends mandate local surveillance to inform stewardship-aligned, targeted therapy instead of blanket empirical regimens [9–12].

The present prospective analysis from a tertiary care center in India characterizes the burden and pattern of microbial colonization among infertile men, the relationship with semen parameters, and the AMR profile of recovered isolates. Using standardized semen analysis, culture, targeted PCR for sexually transmitted pathogens, and CLSI-concordant susceptibility testing, we sought to (i) delineate the organismal spectrum, (ii) quantify symptomatic versus asymptomatic carriage, and (iii) summarize practical implications for clinical management and partner-based interventions. Our findings underscore the central role of microbiota in male infertility and highlight the urgency of integrated microbiological diagnostics within routine fertility evaluation.

This prospective study enrolled 246 consecutive males undergoing infertility evaluation at a tertiary care center over a two‑year period. Inclusion criteria were age 18–45 years, participation as the male partner in an infertile couple (failure to conceive after ≥12 months of unprotected intercourse), and provision of written informed consent. Exclusion criteria included systemic antibiotic use within the preceding 4 weeks, known genetic or irreversible surgical sterility, symptomatic urinary tract infection, severe systemic illness, current ART cycle at baseline, and BMI >30 kg/m². Ethical approval was obtained from the institutional review board and the study adhered to the Declaration of Helsinki.

Semen collection and analysis: Participants observed 2–7 days of abstinence. Samples were produced by masturbation into sterile containers after hand and genital hygiene. Analyses commenced within one hour of collection following WHO laboratory recommendations. Reported variables included volume (mL), sperm concentration (×10^6/mL), progressive motility (category A), non‑progressive motility (category B), morphology (% normal forms), and vitality (%). Reference interpretation drew on WHO 2010 thresholds, acknowledging subsequent 2021 updates but maintaining internal consistency with the study’s recruitment period.

Microbiology: Semen underwent wet mount and Gram stain for preliminary assessment (leukocytes, yeast, bacterial morphotypes) followed by culture on standard aerobic media (e.g., nutrient agar, MacConkey, blood agar, chocolate agar) and Sabouraud dextrose agar for fungi. Bacteriospermia was defined at ≥10^3 CFU/mL. Targeted STI PCR (when indicated by leukocytospermia or clinical suspicion) addressed Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum, and Chlamydia trachomatis. Growth-positive isolates were identified by conventional biochemical tests. Antibiotic susceptibility testing (AST) employed Kirby–Bauer disc diffusion on Mueller–Hinton agar per CLSI 2021, with agents selected for local relevance. Phenotypic ESBL detection used combined disc synergy, and MBL screening used imipenem–EDTA combined disc testing with standard interpretive cutoffs.

Outcomes and analysis: Primary outcomes were (i) frequency distribution of isolates, (ii) symptomatic versus asymptomatic proportions among culture-positive men, and (iii) AST profiles by Gram class. Descriptive statistics summarize means ± SD for continuous variables and frequencies (%) for categorical variables. Results are presented in six tables with accompanying narrative interpretation focused on clinical applicability (screening, empiric therapy, and partner management).

Table 1. Demographic and clinical characteristics of the cohort.

Table 2. Semen parameters (male).

Table 3. Frequency of microbial isolates in male semen samples.

Table 4. Symptomatic vs. asymptomatic distribution among culture-positive males.

Table 5. Antibiotic susceptibility among Gram-positive isolates (counts).

Table 6. Antibiotic susceptibility among Gram-negative isolates (counts).

Across 246 men, culture positivity reached 76.4% with two‑thirds asymptomatic, underscoring the inadequacy of symptom‑only screening. E. coli, S. aureus and Candida predominated. Semen means were within WHO thresholds for volume and concentration but showed borderline progressive motility (32.1%), consistent with subtle functional compromise. Gram‑positive isolates retained susceptibility to doxycycline and linezolid, while Gram‑negatives showed best activity with gentamicin and amoxicillin–clavulanate; resistance to meropenem and ciprofloxacin was notable, aligning with regional AMR trends. These data support routine microbiological work‑up in male infertility and stewardship‑guided therapy with partner co‑management.

This study highlights a substantial burden of genital tract colonization among infertile men in an Indian tertiary setting and demonstrates its practical relevance to semen quality and therapeutic decision‑making. Nearly seven in ten couples in the broader cohort harbored microorganisms, and among men specifically, three in four samples yielded growth with a predominance of asymptomatic carriage. This silent reservoir challenges conventional, symptom‑triggered testing paradigms and argues for routine microbiological assessment in infertility work‑ups.

The organismal spectrum—dominated by Escherichia coli, Staphylococcus aureus and Candida species—maps onto recognized pathophysiologic mechanisms. E. coli and Ureaplasma/Mycoplasma spp. can adhere to spermatozoa, induce reactive oxygen species, and reduce motility; S. aureus produces toxins that impair membrane integrity; and candidal colonization alters seminal biochemistry. In our cohort, progressive motility hovered at the lower reference bound despite adequate counts and morphology, consistent with functional impairment from low‑grade inflammation and oxidative stress. These observations echo reports that bacteriospermia correlates with asthenozoospermia and higher DNA fragmentation indices, adversely affecting both natural conception and ART outcomes.

Antimicrobial susceptibility patterns had clear stewardship implications. Among Gram‑positives, doxycycline and linezolid retained activity, whereas macrolide/lincosamide susceptibility was moderate and gentamicin/ciprofloxacin resistance considerable. For Gram‑negatives, gentamicin and amoxicillin–clavulanate performed comparatively well, while fluoroquinolone and carbapenem resistance—particularly meropenem—was concerning. These trends mirror national surveillance data and suggest that empirical fluoroquinolones or third‑generation cephalosporins are suboptimal for male genital infections in this context. Targeted therapy based on culture plus phenotypic ESBL/MBL detection is preferable, with test‑of‑cure and partner treatment to prevent reinfection cycles.

Strengths of this study include standardized semen analysis, contemporaneous culture with targeted PCR for key sexually transmitted organisms, and systematic AST reporting. Limitations include the lack of quantitative molecular microbiome profiling, the potential under‑detection of fastidious organisms by culture alone, and absence of longitudinal fertility outcomes (pregnancy/live birth). Future work should integrate next‑generation sequencing of the seminal microbiome, oxidative stress biomarkers, and prospectively tracked reproductive outcomes to define causal pathways and therapeutic benefit.

In conclusion, our data support embedding microbiological diagnostics within routine male infertility evaluation and adopting stewardship‑aligned, partner‑inclusive management to mitigate subclinical colonization and AMR‑driven treatment failure.

Male infertility in this cohort was characterized by high rates of asymptomatic microbial colonization and significant antimicrobial resistance. Borderline motility despite adequate counts points to functional impairment likely mediated by infection‑associated inflammation. Routine culture/PCR, resistance‑guided therapy, and partner co‑management should be standard practice in infertility clinics.

1. Agarwal A, Baskaran S, Parekh N, et al. Male infertility. 2021;397(10271):319–333. doi:10.1016/S0140‑6736(20)32667‑2
2. Farhi J, Ben‑Haroush A. Distribution of causes of infertility in patients attending primary fertility clinics. Isr Med Assoc J. 2011;13(1):51–54.
3. Fraczek M, Kurpisz M. Mechanisms of the harmful effects of bacterial semen infection on ejaculated human spermatozoa. Reprod Biol Endocrinol. 2015;13:51.
4. Rusz A, et al. Seminal microbiome and its impact on male fertility. 2020;8(5):1049–1059.
5. Hou D, et al. Microbiota of the seminal fluid from healthy and infertile men. Fertil Steril. 2013;100(5):1261–1269.
6. Cheung S, et al. Asymptomatic bacteriospermia and semen quality. Hum Reprod Update. 2021;27(6):1–15.
7. Donders GGG, et al. Clinical relevance of Ureaplasma and Mycoplasma in genitourinary infections. Curr Opin Infect Dis. 2017;30(1):87–92.
8. Salonia A, et al. European Association of Urology Guidelines on Sexual and Reproductive Health (Male Infertility). 2023.
9. ICMR‑AMRSN. Antimicrobial resistance surveillance in India — Annual Report 2021/2022.
10. Unemo M, et al. Sexually transmitted infections: challenges and opportunities. Nat Rev Urol. 2017;14(10):586–604.
11. de Almeida V, et al. ESBL‑producing Enterobacterales in community settings: a global perspective. Clin Microbiol Rev. 2022;35(4):e000xx.
12. Llor C, Bjerrum L. Antimicrobial resistance: risk associated with antibiotic overuse. Ther Adv Drug Saf. 2014;5(6):229–241.
13. Laboratory manual for the examination and processing of human semen. 5th/6th ed. Geneva: WHO; 2010/2021.
14. Redelinghuys MJ, et al. The seminal microbiome in health and disease. 2020;8(6):1–14.
15. Fode M, et al. Bacterial infection and male infertility. Nat Rev Urol. 2016;13:35–46.
16. Barbonetti A, et al. Inflammation, oxidative stress and sperm function. Reprod Biol Endocrinol. 2020;18:81.
17. Mejia‑Lancheros C, et al. Antibiotic resistance in uropathogens in South Asia: a review. J Glob Antimicrob Resist. 2019;19:234–245.
18. Rehewy MS, et al. Candida and male fertility: systematic insights. 2019;62(10):826–834.
19. Lis R, Rowhani‑Rahbar A, Manhart LE. Mycoplasma genitalium infection and STI. Clin Infect Dis. 2015;61(S8):S761–S767.
20. Skerk V, et al. Bacteriospermia and semen quality: clinical correlations. World J Mens Health. 2019;37(1):1–12.
21. Smith RP, et al. Sperm DNA fragmentation and infection. 2018;116:23–29.
22. Lunenfeld B, et al. The burden of infertility: an international perspective. Int J Gynaecol Obstet. 2015;131(S1):S3–S8.
23. Zhou M, et al. Carbapenem resistance in Enterobacterales across Asia. J Antimicrob Chemother. 2020;75(2):1–10.
24. Moghaddam A, et al. Partner treatment strategies in male genital infections. 2021;53(3):e139xx.
25. Kissinger P. Partner notification and treatment to control STIs. Sex Transm Dis. 2015;42(2):S55–S60.