Assessment of testicular microbiota in tobacco-smoking patients with non-obstructive azoospermia
DOI: 10.32364/2587-6821-2026-10-4-6
Assessment of testicular microbiota in tobacco-smoking patients with non-obstructive azoospermia
M.V. Faniev1, Z.A. Kadyrov1, N.G. Kul'chenko1, I.V. Vinogradov2, Ya.V. Prokopiev3
1 RUDN University, Moscow, Russian Federation
2 Scientific Practical Center of Reproductive and Regenerative Medicine, Moscow, Russian Federation
3 Kazan State Medical Academy — Branch of the Russian Medical Academy of Continuous Professional Education, Kazan, Russian Federation
Background: tobacco smoking is recognized as an independent risk factor for deterioration of spermogram parameters including decreased concentration, motility, and morphology of sperm. Actually, impact of tobacco smoking on testicular microbiota is still underinvestigated.
Aim: to evaluate composition of the testicular microbiota in tobacco-smoking patients with non-obstructive azoospermia (NOA).
Materials and Methods: men with NOA (n=72) were enrolled in the study. All patients were divided into 2 groups. In particular, smokers were randomized in the Group 1 (n=33), and non-smokers represented the Group 2 (n=39). According to the indications, all the subjects underwent testicular biopsy by a Micro-TESE (Microsurgical Testicular Sperm Extraction) method in the frame of an assisted reproductive technology program. In the testicular biopsy samples, mcrobiota composition was tested by high-throughput sequencing of a new generation of 16S rRNA gene amplicons.
Results: in patients with NOA, smoking contributes to testicular dysbiosis whose main manifestations are a two-fold increase in abundance of Enterobacteriaceae families, a significant increase in total microbial diversity (α-diversity), and increased number of species of opportunistic bacteria which are not common for normal testicular environment.
Conclusion: although testicular microbiota in tobacco-smoking patients with NOA is becoming more diverse, this resulted from increased proportion of opportunistic pathogens or bacteria unusual for this organ; in turn, this leads to pronounced dysbiosis of the microbiota.
Keywords: microbiome, testicular microbiota, tobacco smoking, microbial diversity, opportunistic microflora, non-obstructive azoospermia, male infertility, infertility, impaired spermatogenesis.
For citation: Faniev M.V., Kadyrov Z.A., Kul'chenko N.G., Vinogradov I.V., Prokopiev Ya.V. Assessment of testicular microbiota in tobacco-smoking patients with non-obstructive azoospermia. Russian Medical Inquiry. 2026;10(4):209–213 (in Russ.). DOI: 10.32364/2587-6821-2026-10-4-6
Background
Infertility affects 1 in 6 married couple, and this resulted from a male factor of infertility in half of the cases [1-3]. Non-obstructive azoospermia (NOA) is the most severe type of male infertility characterized by the absence of spermatozoa in ejaculate due to impaired spermatogenesis in coiled seminiferous tubules [4]. Despite significant progress of assisted reproductive technologies, success of in vitro fertilization and intracytoplasmic sperm injection methods directly depends on the possibility of obtaining viable gametes by Micro-TESE (Microsurgical Testicular Sperm Extraction) [5]. In this regard, identification and management of risk factors of NOA is of primary importance [6]. Among factors leading to NOA, along with endocrine disorders and genetic pathologies [7], researchers are increasingly paying attention to a patient's lifestyle and health effects of his habits (in particular, tobacco smoking) [8, 9]. It is known that tobacco dependence is noted among 37% of married couples seeking medical help at reproductive centers [10].
The issue is urgent because of high prevalence of tobacco smoking among men of reproductive age. Tobacco smoking-induced spermatogenesis impairment results from various factors such as oxidative stress, direct toxic effects on Leydig and Sertoli cells, impaired testicular microcirculation, etc. [11, 12]. In turn, a role of the microbiota, a complex community of microorganisms colonizing various human biotopes, is attracting increasing attention in search for new pathogenetic mechanisms of NOA development [13, 14]. Tobacco smoking has a well-known effect on respiratory and colonic microbiota. However, male reproductive tract microbiota, in particular, microbial composition of testicular tissue and its potential association with NOA, has been poorly studied in tobacco-smoking men.
Aim: to evaluate composition of testicular microbiota in tobacco smokers with NOA.
Materials and Methods
A prospective cohort longitudinal parallel-group study (an extract from Protocol No. 24 of the Meeting of the Ethics Committee of the RUDN University dt January 18, 2024) included males aged from 23 to 56 years (n=72) with NOA diagnosed according to spermogram data and clinical test results who signed a voluntary informed consent. Non-inclusion criteria: obstructive azoospermia, acute lower urinary tract infections, cancer, varicocele, antimicrobial treatment for inflammatory diseases of the reproductive tract received less than 3 months ago, abnormal results of a general analysis of prostate secretions and a bacterial culture test of ejaculate.
All the patients were divided into 2 groups depending on a social habit (i.e., tobacco smoking, ≤1 pack/day according to a history): Group 1 - tobacco smokers (n=33), Group 2 - non-smokers (n=39)
According to the indications, a Micro-TESE testicular biopsy was performed in all the subjects as a part of an ART program. The testicular specimens were examined for composition of the microbiota. For this purpose, high-performance Next-Generation Sequencing (NGS) of 16S rRNA gene amplicons was applied by a patented technique [15]. The data were processed by QIIME (Quantitative Insights into Microbial Ecology) software. During primary processing, version 1.9.1 was used to filter and cluster the sequences into operational taxonomic units. To improve accuracy of the taxonomic classification, the transition to QIIME 2 was performed with the GreenGenes database. The software combination provided accurate and reproducible study results. Statistical data analysis was performed by specialized Statistica software (version 10.0, StatSoft Inc., USA). The primary analysis of quantitative indicators was carried out with calculation of the median (Me), as well as the 1st and 3rd quartiles [Q1; Q3] to describe a main trend and determine data variation. To assess significance of the differences between independent groups, nonparametric methods were used as the most appropriate for data that did not correspond to normal distribution and/or for small samples. For a pairwise comparison of the two groups (including obtaining a significant result for a Kraskel — Wallis test), the Mann — Whitney U-test was used. The results were considered significant at p<0.05.
Results
In the Group 1, NOA patients were significantly older than in the Group 2 (42 years vs 35 years, respectively) (p<0.05). Perhaps, this is due to the fact that tobacco smoking prevalence is initially higher among the elderly because of the lack of widespread promotion of a healthy lifestyle two decades ago.
In the testicular microbiota of NOA patients, there is a relative abundance mainly of Proteobacteria, Actinobacteria, Bacteroidetes, Bacillota types (formerly Firmicutes). The Figure 1 shows the bacterial families whose relative abundance, on average, exceeds 1.8%.
In non-smokers, testicular microbiota showed pronounced dominance of 3 families (Enterobacteriaceae, Burkholderiaceae and Lactobacillaceae), and microbiota composition changed radically in tobacco smokers. In particular, relative abundance of Enterobacteriaceae demonstrated clear predominance, and other bacterial families lost their dominance. As for tobacco-smoking patients, the microbiota composition was more diverse and uniform while abundance of many other bacterial families (such as Peptoniphilaceae, Mycobacteriaceae, and Staphylococcaceae) increased markedly (see Fig. 1). Thus, both study groups demonstrated significant high pure abundance of Enterobacteriaceae (Escherichia) in testicular tissue. At the same time, infertile non-smokers showed relative Escherichia coli abundance of 11% while it was 21% in smokers (according to a Kraskel — Wallis test: p<0.05). As for other bacterial families, mean relative abundance was <0.3% of the total microbial community in both observation groups.
To quantify α-diversity, the Shannon index calculated by QIIME2 software was used. Thus, α-diversity index of the testicular microbiota in infertile non-smokers was lower than that in tobacco smokers (p>0.05) (Fig. 2). In non-smokers, an unexpressed diversity of the microbiota was reported. This may be caused by dominance of 1-2 commensal bacterial species or by very low bacterial colonization level in testicular tissue. Higher α-diversity of testicular microbiota in infertile tobacco smokers indicates a change in local immune environment that may lead to chronic inflammation and impaired spermatogenesis.
Discussion
Every day, millions of males expose themselves to several thousand toxic compounds contained in cigarette smoke voluntarily without a second thought about their direct impact on reproductive function. It is known that tobacco smoke consists of more than 7 thousand chemical compounds including nicotine, combustion products, heavy metals (cadmium, lead) and free radicals [16] impairing spermatogenesis.
Appearance and progression of oxidative stress are primary pathogenetic factors of pathospermia in smokers. Tobacco smoke contains very high levels of pro-oxidants (free radicals). At the same time, smokers demonstrate decreased levels of antioxidants (vitamins C, E, and glutathione) in seminal plasma. The imbalance leads to excessive production of reactive oxygen species whose high levels contribute to destruction of sperm lipid membranes along with mitochondrial damage, and, most dangerously, cause fragmentation of nuclear DNA [17].
Smoke components such as cadmium and nicotine are tropic to the testicles. Accumulating in them, cadmium inhibits testosterone synthesis by Leydig cells directly and damages Sertoli cells [10, 18]. This results in hormonal imbalance and destruction of the best possible environment for sperm development. In addition, nicotine causes vasospasm and impairs blood microcirculation. In the testicles, this induces chronic hypoxia that additionally inhibits cellular metabolism and germ cell division.
So, an experimental mouse study of L. Zhong et al. [19] demonstrated that 12-week exposure to cigarette smoke led to a significant decrease in testicular dimensions and weight (p<0.05), and total sperm motility decreased by 48.08% compared with the control group (p<0.01). In testicles of animals inhaled cigarette smoke, relative abundance of Firmicutes (or Bacillota according to a current taxonomic name) and Actinobacteria increased significantly (p<0.05); the same indicator decreased in Bacteroidetes and Proteobacteria.
The authors' study showed that, in patients with NOA, smoking is associated with deep dysbiosis (imbalance) of testicular microbiota manifested by a two-fold predominance of Enterobacteriaceae and a significant increase in microbial diversity due to growth of opportunistic or foreign bacteria for testicles. Such a change in the testicular microbial community in infertile men may have negative consequences for tissue immunity and cause irreversible spermatogenesis impairment.
Conclusion
In patients with NOA, smoking is associated with a significant change in the testicular microbiota leading to pronounced dysbiosis: it increases a proportion of Enterobacteriaceae by 2 times, enhances overall microbial diversity in the interstitium and promotes gonadal colonization by opportunistic and foreign microflora.
The study results improve understanding of male infertility pathogenesis. Testicular microbiota investigation enables not only conventional consideration of NOA as a hormonal and genetic disorder, but also a view with respect to microbial ecology of reproductive organs (especially, in smokers).
About the authors:
Mikhail V. Faniev — C. Sc. (Med.), Assistant Professor of the Department of Endoscopic Urology, Faculty of Advanced Training for Medical Practitioners, Medical Institute, RUDN University; 6, Miklukho-Maklaya str., Moscow, 117198, Russian Federation; ORCID iD 0000-0002-7323-3126
Zieratsho A. Kadyrov — Dr. Sc. (Med.), Professor, Head of the Department of Endoscopic Urology and Ultrasound Diagnosis, Faculty of Continuing Medical Education, Medical Institute, RUDN University; 6, Miklukho-Maklaya str., Moscow, 117198, Russian Federation; ORCID iD 0000-0002-1108-8138
Nina G. Kul'chenko — C. Sc. (Med.), Urologist, Ultrasound Doctor, Leading Scientific Officer at the Scientific and Educational Resource Center for Innovative Technologies of Immunophenotyping, Digital Spatial Profiling and Ultrastructural Analysis (Molecular Morphology), RUDN University; 6, Miklukho-Maklaya str., Moscow, 117198, Russian Federation; ORCID iD 0000-0002-4468-3670
Igor V. Vinogradov — Dr. Sc. (Med.), Academic Adviser of the Scientific Practical Center of Reproductive and Regenerative Medicine; 7, Begovaya str., Moscow, 125284, Russian Federation; ORCID iD 0000-0001-7469-3952
Yaroslav V. Prokopiev — C. Sc. (Med.), Assistant Professor of the Department of Urology and Nephrology, Kazan State Medical Academy — Branch of the Russian Medical Academy of Continuous Professional Education; 11, Mushtari str., Kazan, 420012, Russian Federation; ORCID iD 0000-0002-4345-127X
Contact information: Yaroslav V. Prokopiev, e-mail: yar80@mail.ru
Financial Disclosure: no authors have a financial or property interest in any material or method mentioned.
There is no conflict of interest.
Received 01.02.2026.
Revised 25.02.2026.
Accepted 23.03.2026.
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