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Article

Differences in Vaginal Microbiota Composition Between Infertile and Fertile Patients: A Prospective Study

by
Pei-Chen Chen
1,2,
Shih-Fen Chen
3,
Wei-Tung Hung
3,
Yu-Ying Lin
3,
Ling-Chun Lin
4,5,
Jen-Hung Wang
6 and
Pao-Chu Chen
1,3,*
1
Department of Obstetrics and Gynecology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 97004, Taiwan
2
Master and Ph.D. Programs in Pharmacology and Toxicology, School of Medicine, Tzu Chi University, Hualien 97004, Taiwan
3
Reproductive Health and IVF Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 97004, Taiwan
4
Institute of Medical Sciences, Tzu Chi University, Hualien 97004, Taiwan
5
Master Program in Biomedical Sciences, School of Medicine, Tzu Chi University, Hualien 97004, Taiwan
6
Center for Clinical Epidemiology and Biostatistics, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 97004, Taiwan
*
Author to whom correspondence should be addressed.
Diagnostics 2025, 15(19), 2544; https://doi.org/10.3390/diagnostics15192544
Submission received: 1 July 2025 / Revised: 9 September 2025 / Accepted: 30 September 2025 / Published: 9 October 2025
(This article belongs to the Special Issue New Insights into the Diagnosis of Gynecological Diseases)
Browse Figures
Versions Notes

Abstract

Background/Objectives: Dysbiosis of the vaginal microbiota, particularly the loss of Lactobacillus spp. dominance, is linked to female infertility. While community state types (CSTs) I–III and V have been studied extensively, CST IV remains underexplored. The aim of this prospective study was to compare vaginal microbiota composition—specifically CST IVA and IVB—between fertile and infertile women. Methods: Vaginal samples were collected from 22 women (15 infertile, 7 fertile) using cervical brushes and analyzed via 16S rRNA gene sequencing. DNA was extracted, and V3–V4 regions were sequenced using the Illumina MiSeq platform. Taxonomic classification was performed with QIIME 2 and the Greengenes database. Differences in microbial composition were assessed using the Wilcoxon rank-sum test (p < 0.05) in SPSS v21.0. Results: Infertile women showed lower relative abundances of Lactobacillus spp. (31.54% vs. 42.32%) and Oscillospira spp. relative to fertile women. CST IV was more frequent in the infertile group (29.75% vs. 21.61%). Within CST IV, CST IVA accounted for a higher proportion in infertile women (7.0% vs. 0.94%), with Prevotella spp. representing 95.18% of CST IVA in infertile subjects, as opposed to the figure of 69.77% in fertile counterparts. No clear differences in CST IVB were observed between groups. Conclusions: Increased prevalence of Prevotella spp. in CST IVA may contribute to an unfavorable vaginal environment in infertile women, potentially affecting sperm viability. The presence of Oscillospira spp. in fertile women suggests it is associated with a healthy vaginal microbiota profile.

1. Introduction

Infertility has emerged as a significant public health concern in developing nations, placing a substantial burden on healthcare and economies [1,2]. Globally, infertility affects 15–25% of couples during their reproductive years [3,4], with female infertility accounting for 37% of cases [5]. Female infertility has mainly been attributed to several genetic and pathophysiological mechanisms [3,6,7,8,9]. However, the factors responsible for 15–40% [6] of the cases of infertility in females remain unclear; these cases are often termed “idiopathic infertility” or “unexplained infertility” [7,10,11]. As such, clinicians perpetually endeavor to uncover the causes of these afflictions and, hopefully, increase fertility.
In recent decades, several studies have suggested that dysbiosis of the vaginal microbiota, characterized by cessation of Lactobacillus spp. dominance [12], may have several consequences, including infertility [10,13,14,15,16,17]. The vaginal microbiota can be classified into five community state types (CSTs I–V) [12]. In this field, the roles of CST I, CST II, CST III, and CST V [12,18,19,20,21] have received considerable attention, with some studies reporting greater Lactobacillus spp. abundance in fertile women. However, to date, there are no definitive conclusions regarding the specific microbe or optimal composition of vaginal microbiota in relation to female infertility. This inconsistency highlights the need to investigate less-studied CSTs, such as CST IV. Studies focusing on CST IV are limited, and existing evidence suggests that CST IV may contribute to the development of female infertility [12,21,22,23]. CST IV can be sub-grouped into CST IVA and CST IVB. CST IVA is characterized by the absence of Lactobacillus spp. but richness in Prevotella spp. and Peptoniphilus spp. CST IVB is characterized by low levels of Lactobacillus spp., with low proportions of various anaerobic bacterial microorganisms and an abundance of Atopobium spp., Corynebacterium spp., Finegoldia spp., and Gardnerella spp. [23,24,25]. The association between specific microorganisms of CST IVA or CST IVB in vaginal microbiota and infertility has not been investigated [26].
Thus, in this study, we aimed to analyze the differences in the composition of the vaginal microbiota, with a particular focus on CST IVA and CST IVB, between women with unexplained infertility and spontaneous conception followed by delivery.

2. Materials and Methods

2.1. Ethics Approval

This study was approved by the Research Ethics Committee of the Hualien Tzu Chi Hospital (IRB110-179-A). It was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines. All participants were provided with comprehensive details prior to taking part in this study. Each participant provided written consent to ensure their voluntary agreement to participate in the study.

2.2. Study Design and Subjects

We recruited 30 female participants from the Department of Obstetrics and Gynecology, Hualien Tzu Chi Hospital, from January 2022 to December 2022. This population was subdivided into two cohorts: a fertile cohort (n = 7), whose members had achieved previous spontaneous conception with full-term delivery, and an infertile cohort (n = 15), whose members had been diagnosed with infertility. Samples of fertile cases were collected from routine pap smear examinations. Samples of infertile cases were collected from the infertility clinic. The exclusion criteria included being under 20 years old, menopausal, or afflicted by symptomatic vaginitis, pelvic inflammatory disease, or structural anomalies of the uterus. Baseline characteristic data, including age and body mass index (BMI), were also collected.

2.3. Sample Collection, Bacterial DNA Extraction, and RNA Sequencing

Vaginal discharge was obtained using a cervical brush as part of a speculum examination. The specimens were preserved in DNA preservation liquid. The bacterial DNA from the vaginal discharge samples was extracted using the ZymoBIOMICS DNA Microprep kit (Zymo Research, Irvine, CA, USA) in accordance with the manufacturer’s instructions [27]. Polymerase chain reaction (PCR) amplification and library preparation of the variable regions (V3–V4) of the 16S rRNA gene were performed using the Quick-16STM next-generation sequencing (NGS) library prep kit (Zymo Research, USA). This library was constructed by incorporating unique identification indices for each sample through the use of the Nextera XT sequencing kit (Illumina, San Diego, CA, USA) to obtain and purify the V3–V4 amplicon. Then, the samples were diluted to a concentration of 4 nM for sequencing. The prepared library was quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) prior to sequencing, and the final library concentration was 8 pM. Library sequencing was performed using MiSeq Reagent Kit v3 (Illumina) reagents and MiSeq (Illumina) as the sequencing platform for the metagenomics workflow. Illumina local run manager software (version 3.0) was used for sequencing-data analysis. The index reads were demultiplexed, and FASTQ files were generated. The reads were classified against the Greengenes 16S rRNA gene database (version gg_13_5) [28], which achieved up to species-level sensitivity [29]. The results were generated using the default parameters in the original QIIME 2 software product (version 1.9.1).

2.4. Statistical Data Analysis

Continuous and categorical variables are presented as means ± standard deviations (SD) and frequencies or proportions, respectively. For comparisons of vaginal microbiota composition between the two groups, the relative abundance (percentage) of different bacterial taxa (at the genus or species level, when available) was calculated for each patient. Consequently, the Wilcoxon rank-sum test was adopted to compare the mean differences between the two groups for these taxa. Statistically significant differences were defined as p < 0.05. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) 21.0 (IBM Corp., Armonk, NY, USA).

3. Results

3.1. Flow Chart Pertaining to Study Design and Demographics

Between January 2022 and December 2022, 37 infertile females and 25 fertile females were evaluated to determine their eligibility. Due to unsuccessful DNA extraction, 22 infertile and 18 fertile cases were excluded from further analysis. We ultimately included 22 participants, corresponding to 15 infertile cases and 7 fertile cases, in our investigation (Figure 1).

3.2. Vaginal Microbiota Distribution in the Cohort

The stacked bar graph in Figure 2 shows the distribution of vaginal microbiota by bacterial taxa in infertile females (Sample_1–34), fertile females (Sample_C1–14), the negative control (Sample_N), and the positive control (Sample_P). The numbering system (Sample_1–34 and Sample_C1–14) included both successful and failed DNA extractions; only samples with sufficient sequencing quality were retained for analysis, resulting in 15 infertile samples and 7 fertile samples being included in the final dataset. The unclassified microbiota (depicted in green in Figure 2) was not considered in the analysis of microbiota composition.

3.3. Composition of Vaginal Microbiota in Fertile and Infertile Women

Figure 3 and Figure 4 show the compositions of vaginal microbiota in infertile and fertile females. The top microbiota in infertile females included L. iners, Gardnerella vaginalis, Prevotella amnii, L. crispatus, and Atopobium. The top microbiota in fertile females included L. iners, Gardnerella vaginalis, L. jensenii, L. crispatus, and L. gasseri. The collective proportions of Lactobacillus spp. were 31.54% and 42.32% in the infertile and fertile females, respectively, reflecting a lower abundance of Lactobacillus spp. in infertile females.

3.4. CST IV Composition Between the Infertile and Fertile Groups

Analyzing the differences in the abundance of CST IV bacteria in the vaginal microbiota between the two study groups revealed that infertile females (29.75%) had a higher value than fertile females (21.61%) (Figure 5a). Descriptive analysis revealed that CST IV was more frequent in infertile women (29.75%) than in fertile women (21.61%) (Figure 5a). CST IVA accounted for a higher proportion of the vaginal microbiota in infertile women (7.0%) in comparison with fertile women (0.94%) (Figure 5b), whereas CST IVB distributions appeared similar between the two groups (Figure 5c). At the genus level, Prevotella constituted a higher percentage of CST IVA in infertile women (95.18%) than in fertile women (69.77%) (Figure 6). These findings are based on percentage distributions, and no formal statistical testing was applied.

3.5. Comparison of Vaginal Microbiota Compositions in Infertile and Fertile Females

The compositions of the vaginal microbiota in infertile and fertile females, obtained using the mean value of each vaginal microbiota, are presented in Table 1. The data show that the amounts of Kitasatospora_NA, Oscillospira_eae, Alkaliphilus_crotonatoxidans, L. farciminis, L. brantae, L. japonicus, Staphylococcus_aureus, and Corynebacterium_atypicum were significantly higher in fertile females than in infertile females.

4. Discussion

This study resulted in four major findings. First, Lactobacillus spp. was more abundant in fertile patients than in infertile patients (Figure 3 and Figure 4). Second, CST IV appeared more frequently in infertile women (Figure 5a). Third, within CST IV, Prevotella spp. in CST IVA accounted for a larger proportion in infertile women relative to their fertile counterparts (Figure 6). Fourth, the relative abundance of bacterial taxa was significantly higher in fertile females than in infertile females (Table 1).
Burton et al. first employed the NGS method to characterize the vaginal microbiota in 2002. Since then, an increasing number of studies have focused on the relationship between vaginal microbiota and infertility, especially with respect to idiopathic recurrent pregnancy loss [30,31,32,33]. Most studies have established that the vaginal microbiota was dominated by Lactobacillus spp., classified into CSTs; among them, CST I, II, III, and IV are more abundant in fertile groups [7,12,13,20,28,34,35,36,37].
Although exploring the specific microorganisms of the vaginal microbiota that cause infertility has gained considerable attention, no conclusive findings have been reported [10,26,38]. Wee et al. enrolled 31 participants to compare vaginal microbiota compositions between infertile and fertile females. While no specific cluster showed differences between the two groups, a trend indicated that Ureaplasma and Gardnerella were more abundant in the vagina and cervix, respectively, of infertile females [26]. Sezer et al. also investigated the differences in vaginal microbiota between infertile and fertile females. They found that the amounts of Lactobacillus spp., Staphylococcus spp., Streptococcus spp., and members of the Enterobacteriaceae family were significantly higher in the infertile group under the age of 30. However, the relative abundance of Gardnerella vaginalis, Eubacterium spp., Sneathia, Megasphaera spp., Peptostreptococcus spp., Atopobium vaginae, and Mycoplasma hominis was significantly higher in the infertile group over the age of 30 [38]. Chopra et al. conducted a prospective study involving 80 participants and found that in infertile females, the amounts of Gardnerella spp., Prevotella spp., Atopobium spp., and Enterococcus spp. were significantly higher, whereas the proportions of L. iners were lower [10].
The vaginal microbiota is dynamic, and CST IV bacteria accumulate when Lactobacillus spp. are no longer dominant [35]. CST IVA is characterized by increased relative abundance or rates of Peptoniphilus spp. and Prevotella spp., and CST IVB is dominated by various anaerobic bacterial species related to bacterial vaginosis (BV), such as Atopobium spp., Corynebacterium spp., Finegoldia spp., and Gardnerella spp. [23,39,40,41,42]. Therefore, the presence of CST IVA may indicate that asymptomatic BV and CST IVB may develop into a clinical infection [42]. Previous studies have focused on the association between the presence of CST IV, especially CST IVB, and infertility [12,21,22,23,35]. To the best of our knowledge, no studies have explored the relationship between CST IVA and fertility in infertile and fertile females. In addition, CST IVA may cause asymptomatic BV, which may be a silent cause of female infertility. Furthermore, no studies have compared the differences between specific microorganisms in vaginal microbiota using subgroup analysis of CST IV between infertile and fertile females. We particularly focused on the specific microorganisms in CST IV that may be associated with infertility, given that CST IV plays an important role in infertility. In this study, subgroup analysis of CST IV showed that the most prevalent microorganisms were Gardnerella vaginalis, Prevotella amnii, and Atopobium spp. Among CST IVA compositions, the relative abundance of Prevotella spp. was higher in infertile females (95.18%) than in fertile females (69.77%). This finding suggests a possible association between CST IVA and asymptomatic BV in infertile women. Previous studies reported that Prevotella spp. may negatively affect semen quality, contributing to reduced motility and concentration of sperm [43,44,45,46,47], and correlations between vaginal and seminal microbiota in idiopathic infertile couples have been observed [48,49]. While these reports provide a biological rationale, we did not directly assess semen quality. Therefore, the interpretation that Prevotella spp. may impair sperm function should be regarded as hypothetical. Future studies are needed to directly evaluate semen–vaginal microbiome interactions to clarify this relationship.
We found that several bacterial microorganisms were prominent in fertile females, including Kitasatospora spp., Oscillospira spp., Alkaliphilus crotonatoxidans, L. farciminis, L. brantae, L. japonicus, Staphylococcus aureus, and Corynebacterium atypicum. Among them, Oscillospira is the most clinically important. Wu et al. developed an intrauterine mouse model and found that Oscillospira spp. serve as beneficial vaginal bacteria, increasing the abundance of Lactobacillus and restoring the balance of the vaginal microbiota [50]. Oscillospira spp. could be a promising candidate for next-generation probiotics, offering potential benefits for fertility.
We compared the differences in vaginal microorganisms between infertile and fertile females using CST IV subgroup analysis. Our findings may fill in the existing knowledge gap. Nevertheless, our study still suffers from some limitations. First, the final sample was relatively small (n = 22), reducing statistical power and potentially limiting the generalizability of the findings. Moreover, more than 60% of the original samples did not make it through DNA extraction, and this failure may have introduced selection bias. The potential causes of extraction failure include low microbial biomass, technical variability in specimen handling, or storage-related DNA degradation. Also, there are no standardized methods for DNA extraction for vaginal microbiota [51]. Second, as only participants from a single center in Taiwan were enrolled, the results may not be generalizable to other populations. Third, we only reported relative abundances and percentage differences without providing exact p-values or effect sizes, restricting the statistical transparency of our findings. Another limitation of this study is that information on potential confounding factors such as recent antibiotic use, sexual practices, hormonal treatments, or lifestyle variables was not collected. These factors are known to substantially influence vaginal microbiota composition and could have impacted our findings. Future studies should incorporate detailed records of these variables to better control for their effects. Finally, taxonomic classification relied on the Greengenes database, which is incomplete for vaginal-specific bacteria, leaving some organisms unclassified. These limitations should be addressed in future large-scale, multicenter, and methodologically standardized studies.

5. Conclusions

This study demonstrated that infertile women exhibited a higher prevalence of CST IV, particularly CST IVA, than fertile women. Within CST IVA, the relative abundance of Prevotella spp. was markedly elevated in infertile subjects, suggesting a potential association with an unfavorable vaginal environment. Conversely, Oscillospira spp. were more abundant in fertile women, which may indicate a healthy vaginal microbiota. While previous reports have linked Prevotella spp. to impaired semen parameters, our study did not directly assess semen quality; therefore, such interpretations remain hypothetical. Future large-scale, multicenter studies incorporating integrated analyses of vaginal and seminal microbiota are required in order to validate these findings and explore the potential clinical applications of vaginal microbiota in infertility diagnosis and treatment.

Author Contributions

Conceptualization, P.-C.C. (Pei-Chen Chen) and S.-F.C.; investigation, P.-C.C. (Pao-Chu Chen) and P.-C.C. (Pei-Chen Chen); methodology, S.-F.C., W.-T.H. and Y.-Y.L.; writing, P.-C.C. (Pei-Chen Chen); writing—review and editing, P.-C.C. (Pao-Chu Chen); data curation, L.-C.L.; formal analysis, J.-H.W. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Hualien Tzu Chi Hospital and Buddhist Tzu Chi Medical Foundation (TCRD111-029).

Institutional Review Board Statement

Ethical approval (IRB110-179-A, approved on 1 December 2021) was provided by the Research Ethics Committee of the Hualien Tzu Chi Hospital, Taiwan.

Informed Consent Statement

Informed consent was obtained from all the subjects involved in this study. Written informed consent for publication was obtained from the participants whose data were included.

Data Availability Statement

All data generated or analyzed during this study are available in publicly accessible repositories. The 16S rRNA gene sequencing data used to analyze the vaginal microbiota in this study have been deposited in the DNA Data Bank of Japan (DDBJ) under BioProject accession number PRJDB18901, with sample accession numbers SAMD00824136-SAMD00824157.

Acknowledgments

The authors are grateful to Yu Ru Kou and Dah-Ching Ding for their valuable suggestions regarding the preparation of this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CSTCommunity state type
BMIBody mass index
PCRPolymerase chain reaction
NGSNext-generation sequencing
SDStandard deviation
BVBacterial vaginosis

References

  1. Inhorn, M.C.; Patrizio, P. Infertility around the Globe: New Thinking on Gender, Reproductive Technologies and Global Movements in the 21st Century. Hum. Reprod. Update 2015, 21, 411–426. [Google Scholar] [CrossRef]
  2. Bourrion, B.; Panjo, H.; Bithorel, P.-L.; de La Rochebrochard, E.; François, M.; Pelletier-Fleury, N. The Economic Burden of Infertility Treatment and Distribution of Expenditures Overtime in France: A Self-Controlled Pre-Post Study. BMC Health Serv. Res. 2022, 22, 512. [Google Scholar] [CrossRef]
  3. Vander Borght, M.; Wyns, C. Fertility and Infertility: Definition and Epidemiology. Clin. Biochem. 2018, 62, 2–10. [Google Scholar] [CrossRef] [PubMed]
  4. Infertility Workup for the Women’s Health Specialist: ACOG Committee Opinion Summary, Number 781. Obstet. Gynecol. 2019, 133, 1294–1295. [CrossRef] [PubMed]
  5. Walker, M.H.; Tobler, K.J. Female Infertility; StatPearls Publishing: Petersburg, FL, USA, 2021. [Google Scholar]
  6. Carson, S.A.; Kallen, A.N. Diagnosis and Management of Infertility. JAMA 2021, 326, 65. [Google Scholar] [CrossRef]
  7. Hong, X.; Ma, J.; Yin, J.; Fang, S.; Geng, J.; Zhao, H.; Zhu, M.; Ye, M.; Zhu, X.; Xuan, Y.; et al. The Association between Vaginal Microbiota and Female Infertility: A Systematic Review and Meta-Analysis. Arch. Gynecol. Obstet. 2020, 302, 569–578. [Google Scholar] [CrossRef]
  8. Eijkemans, M.J.C.; van Poppel, F.; Habbema, D.F.; Smith, K.R.; Leridon, H.; te Velde, E.R. Too Old to Have Children? Lessons from Natural Fertility Populations. Hum. Reprod. 2014, 29, 1304–1312. [Google Scholar] [CrossRef] [PubMed]
  9. Ravel, J.; Moreno, I.; Simón, C. Bacterial Vaginosis and Its Association with Infertility, Endometritis, and Pelvic Inflammatory Disease. Am. J. Obstet. Gynecol. 2021, 224, 251–257. [Google Scholar] [CrossRef]
  10. Chopra, C.; Kumar, V.; Kumar, M.; Bhushan, I. Role of Vaginal Microbiota in Idiopathic Infertility: A Prospective Study. Microbes Infect. 2024, 26, 105308. [Google Scholar] [CrossRef]
  11. Patel, N.; Patel, N.; Pal, S.; Nathani, N.; Pandit, R.; Patel, M.; Patel, N.; Joshi, C.; Parekh, B. Distinct Gut and Vaginal Microbiota Profile in Women with Recurrent Implantation Failure and Unexplained Infertility. BMC Women’s Health 2022, 22, 113. [Google Scholar] [CrossRef]
  12. Ravel, J.; Gajer, P.; Abdo, Z.; Schneider, G.M.; Koenig, S.S.K.; McCulle, S.L.; Karlebach, S.; Gorle, R.; Russell, J.; Tacket, C.O.; et al. Vaginal Microbiome of Reproductive-Age Women. Proc. Natl. Acad. Sci. USA 2011, 108 (Suppl. S1), 4680–4687. [Google Scholar] [CrossRef]
  13. Xu, J.; Bian, G.; Zheng, M.; Lu, G.; Chan, W.-Y.; Li, W.; Yang, K.; Chen, Z.-J.; Du, Y. Fertility Factors Affect the Vaginal Microbiome in Women of Reproductive Age. Am. J. Reprod. Immunol. 2020, 83, e13220. [Google Scholar] [CrossRef]
  14. Tao, X.; Ge, S.-Q.; Chen, L.; Cai, L.-S.; Hwang, M.-F.; Wang, C.-L. Relationships between Female Infertility and Female Genital Infections and Pelvic Inflammatory Disease: A Population-Based Nested Controlled Study. Clinics 2018, 73, e364. [Google Scholar] [CrossRef]
  15. Takimoto, K.; Yamada, H.; Shimada, S.; Fukushi, Y.; Wada, S. Chronic Endometritis and Uterine Endometrium Microbiota in Recurrent Implantation Failure and Recurrent Pregnancy Loss. Biomedicines 2023, 11, 2391. [Google Scholar] [CrossRef]
  16. de Oliveira, M.S. Infertility and Vaginal Microbiome: Review Study. Braz. J. Sex. Transm. Dis. 2019, 31, 24–29. [Google Scholar]
  17. Gao, X.; Louwers, Y.V.; Laven, J.S.E.; Schoenmakers, S. Clinical Relevance of Vaginal and Endometrial Microbiome Investigation in Women with Repeated Implantation Failure and Recurrent Pregnancy Loss. Int. J. Mol. Sci. 2024, 25, 622. [Google Scholar] [CrossRef]
  18. Green, K.A.; Zarek, S.M.; Catherino, W.H. Gynecologic Health and Disease in Relation to the Microbiome of the Female Reproductive Tract. Fertil. Steril. 2015, 104, 1351–1357. [Google Scholar] [CrossRef] [PubMed]
  19. Venneri, M.A.; Franceschini, E.; Sciarra, F.; Rosato, E.; D’Ettorre, G.; Lenzi, A. Human Genital Tracts Microbiota: Dysbiosis Crucial for Infertility. J. Endocrinol. Investig. 2022, 45, 1151–1160. [Google Scholar] [CrossRef]
  20. Vitale, S.G.; Ferrari, F.; Ciebiera, M.; Zgliczyńska, M.; Rapisarda, A.M.C.; Vecchio, G.M.; Pino, A.; Angelico, G.; Knafel, A.; Riemma, G.; et al. The Role of Genital Tract Microbiome in Fertility: A Systematic Review. Int. J. Mol. Sci. 2021, 23, 180. [Google Scholar] [CrossRef] [PubMed]
  21. Kroon, S.J.; Ravel, J.; Huston, W.M. Cervicovaginal Microbiota, Women’s Health, and Reproductive Outcomes. Fertil. Steril. 2018, 110, 327–336. [Google Scholar] [CrossRef] [PubMed]
  22. Zhang, Y.; Chen, S.; Chen, X.; Zhang, H.; Huang, X.; Xue, X.; Guo, Y.; Ruan, X.; Liu, X.; Deng, G.; et al. Association between Vaginal Gardnerella and Tubal Pregnancy in Women with Symptomatic Early Pregnancies in China: A Nested Case-Control Study. Front. Cell. Infect. Microbiol. 2021, 11, 761153. [Google Scholar] [CrossRef] [PubMed]
  23. Romero, R.; Hassan, S.S.; Gajer, P.; Tarca, A.L.; Fadrosh, D.W.; Nikita, L.; Galuppi, M.; Lamont, R.F.; Chaemsaithong, P.; Miranda, J.; et al. Correction: The Composition and Stability of the Vaginal Microbiota of Normal Pregnant Women Is Different from That of Non-Pregnant Women. Microbiome 2014, 2, 10. [Google Scholar]
  24. Brotman, R.M.; Shardell, M.D.; Gajer, P.; Fadrosh, D.; Chang, K.; Silver, M.I.; Viscidi, R.P.; Burke, A.E.; Ravel, J.; Gravitt, P.E. Association between the Vaginal Microbiota, Menopause Status, and Signs of Vulvovaginal Atrophy. Menopause 2014, 21, 450–458. [Google Scholar] [CrossRef]
  25. Chopra, C.; Bhushan, I.; Mehta, M.; Koushal, T.; Gupta, A.; Sharma, S.; Kumar, M.; Khodor, S.A.; Sharma, S. Vaginal Microbiome: Considerations for Reproductive Health. Future Microbiol. 2022, 17, 1501–1513. [Google Scholar] [CrossRef] [PubMed]
  26. Wee, B.A.; Thomas, M.; Sweeney, E.L.; Frentiu, F.D.; Samios, M.; Ravel, J.; Gajer, P.; Myers, G.; Timms, P.; Allan, J.A.; et al. A Retrospective Pilot Study to Determine Whether the Reproductive Tract Microbiota Differs between Women with a History of Infertility and Fertile Women. Aust. N. Z. J. Obstet. Gynaecol. 2018, 58, 341–348. [Google Scholar] [CrossRef]
  27. ZymoBIOMICSTM DNA Miniprep Kit Instruction Manual Ver 1.4.1. 27 August 2020. 2020. Available online: https://zymoresearch.eu/products/zymobiomics-dna-miniprep-kit (accessed on 7 October 2025).
  28. DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie, E.L.; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L. Greengenes, a Chimera-Checked 16S RRNA Gene Database and Workbench Compatible with ARB. Appl. Environ. Microbiol. 2006, 72, 5069–5072. [Google Scholar] [CrossRef]
  29. Chang, Y.-S.; Hsu, M.-H.; Tu, S.-J.; Yen, J.-C.; Lee, Y.-T.; Fang, H.-Y.; Chang, J.-G. Metatranscriptomic Analysis of Human Lung Metagenomes from Patients with Lung Cancer. Genes 2021, 12, 1458. [Google Scholar] [CrossRef]
  30. Burton, J.P.; Reid, G. Evaluation of the Bacterial Vaginal Flora of 20 Postmenopausal Women by Direct (Nugent Score) and Molecular (Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis) Techniques. J. Infect. Dis. 2002, 186, 1770–1780. [Google Scholar] [CrossRef]
  31. Zhao, C.; Wei, Z.; Yang, J.; Zhang, J.; Yu, C.; Yang, A.; Zhang, M.; Zhang, L.; Wang, Y.; Mu, X.; et al. Characterization of the Vaginal Microbiome in Women with Infertility and Its Potential Correlation with Hormone Stimulation during in Vitro Fertilization Surgery. mSystems 2020, 5, e00450-20. [Google Scholar] [CrossRef] [PubMed]
  32. Haahr, T.; Zacho, J.; Bräuner, M.; Shathmigha, K.; Skov Jensen, J.; Humaidan, P. Reproductive Outcome of Patients Undergoing in Vitro Fertilisation Treatment and Diagnosed with Bacterial Vaginosis or Abnormal Vaginal Microbiota: A Systematic PRISMA Review and Meta-Analysis. BJOG 2019, 126, 200–207. [Google Scholar] [CrossRef]
  33. Campisciano, G.; Florian, F.; D’Eustacchio, A.; Stanković, D.; Ricci, G.; De Seta, F.; Comar, M. Subclinical Alteration of the Cervical-Vaginal Microbiome in Women with Idiopathic Infertility. J. Cell. Physiol. 2017, 232, 1681–1688. [Google Scholar] [CrossRef]
  34. Zhou, X.; Brown, C.J.; Abdo, Z.; Davis, C.C.; Hansmann, M.A.; Joyce, P.; Foster, J.A.; Forney, L.J. Differences in the Composition of Vaginal Microbial Communities Found in Healthy Caucasian and Black Women. ISME J. 2007, 1, 121–133. [Google Scholar] [CrossRef]
  35. Gajer, P.; Brotman, R.M.; Bai, G.; Sakamoto, J.; Schütte, U.M.E.; Zhong, X.; Koenig, S.S.K.; Fu, L.; Ma, Z.S.; Zhou, X.; et al. Temporal Dynamics of the Human Vaginal Microbiota. Sci. Transl. Med. 2012, 4, 132ra52. [Google Scholar] [CrossRef]
  36. El-Sayed, A.; Aleya, L.; Kamel, M. Microbiota’s Role in Health and Diseases. Environ. Sci. Pollut. Res. Int. 2021, 28, 36967–36983. [Google Scholar] [CrossRef] [PubMed]
  37. Koedooder, R.; Singer, M.; Schoenmakers, S.; Savelkoul, P.H.M.; Morré, S.A.; de Jonge, J.D.; Poort, L.; Cuypers, W.J.S.S.; Beckers, N.G.M.; Broekmans, F.J.M.; et al. The Vaginal Microbiome as a Predictor for Outcome of in Vitro Fertilization with or without Intracytoplasmic Sperm Injection: A Prospective Study. Hum. Reprod. 2019, 34, 1042–1054. [Google Scholar] [CrossRef] [PubMed]
  38. Sezer, O.; Soyer Çalışkan, C.; Celik, S.; Kilic, S.S.; Kuruoglu, T.; Unluguzel Ustun, G.; Yurtcu, N. Assessment of Vaginal and Endometrial Microbiota by Real-Time PCR in Women with Unexplained Infertility. J. Obstet. Gynaecol. Res. 2022, 48, 129–139. [Google Scholar] [CrossRef]
  39. Shen, L.; Zhang, W.; Yuan, Y.; Zhu, W.; Shang, A. Vaginal Microecological Characteristics of Women in Different Physiological and Pathological Period. Front. Cell. Infect. Microbiol. 2022, 12, 959793. [Google Scholar] [CrossRef]
  40. Cocomazzi, G.; De Stefani, S.; Del Pup, L.; Palini, S.; Buccheri, M.; Primiterra, M.; Sciannamè, N.; Faioli, R.; Maglione, A.; Baldini, G.M.; et al. The Impact of the Female Genital Microbiota on the Outcome of Assisted Reproduction Treatments. Microorganisms 2023, 11, 1443. [Google Scholar] [CrossRef]
  41. Lledo, B.; Fuentes, A.; Lozano, F.M.; Cascales, A.; Morales, R.; Hortal, M.; Sellers, F.; Palacios-Marques, A.; Bermejo, R.; Quereda, F.; et al. Identification of Vaginal Microbiome Associated with IVF Pregnancy. Sci. Rep. 2022, 12, 6807. [Google Scholar] [CrossRef]
  42. Zhang, F.; Dai, J.; Chen, T. Role of Lactobacillus in Female Infertility via Modulating Sperm Agglutination and Immobilization. Front. Cell. Infect. Microbiol. 2020, 10, 620529. [Google Scholar] [CrossRef] [PubMed]
  43. Baud, D.; Zuber, A.; Peric, A.; Pluchino, N.; Vulliemoz, N.; Stojanov, M. Impact of Semen Microbiota on the Composition of Seminal Plasma. Microbiol. Spectr. 2024, 12, e0291123. [Google Scholar] [CrossRef]
  44. Weng, S.-L.; Chiu, C.-M.; Lin, F.-M.; Huang, W.-C.; Liang, C.; Yang, T.; Yang, T.-L.; Liu, C.-Y.; Wu, W.-Y.; Chang, Y.-A.; et al. Bacterial Communities in Semen from Men of Infertile Couples: Metagenomic Sequencing Reveals Relationships of Seminal Microbiota to Semen Quality. PLoS ONE 2014, 9, e110152. [Google Scholar] [CrossRef]
  45. Zeyad, A.; Hamad, M.; Amor, H.; Hammadeh, M.E. Relationships between Bacteriospermia, DNA Integrity, Nuclear Protamine Alteration, Sperm Quality and ICSI Outcome. Reprod. Biol. 2018, 18, 115–121. [Google Scholar] [CrossRef]
  46. Pagliuca, C.; Cariati, F.; Bagnulo, F.; Scaglione, E.; Carotenuto, C.; Farina, F.; D’Argenio, V.; Carraturo, F.; D’Aprile, P.; Vitiello, M.; et al. Microbiological Evaluation and Sperm DNA Fragmentation in Semen Samples of Patients Undergoing Fertility Investigation. Genes 2021, 12, 654. [Google Scholar] [CrossRef] [PubMed]
  47. Campisciano, G.; Iebba, V.; Zito, G.; Luppi, S.; Martinelli, M.; Fischer, L.; De Seta, F.; Basile, G.; Ricci, G.; Comar, M. Lactobacillus Iners and Gasseri, Prevotella Bivia and HPV Belong to the Microbiological Signature Negatively Affecting Human Reproduction. Microorganisms 2020, 9, 39. [Google Scholar] [CrossRef]
  48. Mändar, R.; Punab, M.; Borovkova, N.; Lapp, E.; Kiiker, R.; Korrovits, P.; Metspalu, A.; Krjutškov, K.; Nõlvak, H.; Preem, J.-K.; et al. Complementary Seminovaginal Microbiome in Couples. Res. Microbiol. 2015, 166, 440–447. [Google Scholar] [CrossRef]
  49. Amato, V.; Papaleo, E.; Pasciuta, R.; Viganò, P.; Ferrarese, R.; Clementi, N.; Sanchez, A.M.; Quaranta, L.; Burioni, R.; Ambrosi, A.; et al. Differential Composition of Vaginal Microbiome, but Not of Seminal Microbiome, Is Associated with Successful Intrauterine Insemination in Couples with Idiopathic Infertility: A Prospective Observational Study. Open Forum Infect. Dis. 2020, 7, ofz525. [Google Scholar] [CrossRef] [PubMed]
  50. Wu, F.; Kong, Y.; Chen, W.; Liang, D.; Xiao, Q.; Hu, L.; Tan, X.; Wei, J.; Liu, Y.; Deng, X.; et al. Improvement of Vaginal Probiotics Lactobacillus Crispatus on Intrauterine Adhesion in Mice Model and in Clinical Practice. BMC Microbiol. 2023, 23, 78. [Google Scholar] [CrossRef] [PubMed]
  51. Singer, M.; Koedooder, R.; Bos, M.P.; Poort, L.; Schoenmakers, S.; Savelkoul, P.H.M.; Laven, J.S.E.; de Jonge, J.D.; Morré, S.A.; Budding, A.E. The Profiling of Microbiota in Vaginal Swab Samples Using 16S RRNA Gene Sequencing and IS-pro Analysis. BMC Microbiol. 2021, 21, 100. [Google Scholar] [CrossRef]
Diagnostics 15 02544 g001
Figure 1. Flow chart of the study design.
Figure 1. Flow chart of the study design.
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Figure 2. Stacked bar graph of the distribution of microbiota by bacterial taxa in infertile (Sample_1–34) and fertile females (Sample_C1–14), the negative control (Sample_N), and the positive control (Sample_P).
Figure 2. Stacked bar graph of the distribution of microbiota by bacterial taxa in infertile (Sample_1–34) and fertile females (Sample_C1–14), the negative control (Sample_N), and the positive control (Sample_P).
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Figure 3. The composition of the vaginal microbiota in the infertile group.
Figure 3. The composition of the vaginal microbiota in the infertile group.
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Figure 4. The composition of the vaginal microbiota in the fertile group.
Figure 4. The composition of the vaginal microbiota in the fertile group.
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Figure 5. Percentage distribution of vaginal microbiota community state types (CSTs) between infertile and fertile women: (a) overall distribution of CST IV; (b) relative proportion of CST IVA; and (c) relative proportion of CST IVB. Data are presented descriptively without formal statistical testing. CST: community state type.
Figure 5. Percentage distribution of vaginal microbiota community state types (CSTs) between infertile and fertile women: (a) overall distribution of CST IV; (b) relative proportion of CST IVA; and (c) relative proportion of CST IVB. Data are presented descriptively without formal statistical testing. CST: community state type.
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Figure 6. Percentage distribution of dominant bacterial genera in CST IVA (Prevotella and Peptoniphilus) and CST IVB (Atopobium, Corynebacterium, Finegoldia, and Gardnerella) among infertile and fertile women. Data are presented descriptively without formal statistical testing. CST: community state type.
Figure 6. Percentage distribution of dominant bacterial genera in CST IVA (Prevotella and Peptoniphilus) and CST IVB (Atopobium, Corynebacterium, Finegoldia, and Gardnerella) among infertile and fertile women. Data are presented descriptively without formal statistical testing. CST: community state type.
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Table 1. The compositions of the vaginal microbiota in infertile and fertile females, presenting the mean values for each vaginal microbiota.
Table 1. The compositions of the vaginal microbiota in infertile and fertile females, presenting the mean values for each vaginal microbiota.
Bacterial TaxaInfertility (n = 15)Fertility (n = 7)Totalp-Value
MeanSDMeanSDMeanSD
Kitasatospora_NA0.0000.0000.0780.0810.0250.0570.001
Oscillospira_eae0.0040.0170.0410.0550.0160.0370.025
Alkaliphilus_crotonatoxidans0.0000.0000.0180.0310.0050.0190.030
Lactobacillus_farciminis0.0000.0000.0340.0590.0110.0350.030
Lactobacillus_brantae0.0080.0330.1250.2010.0110.0350.037
Lactobacillus_Japonicus0.0000.0000.0250.0460.0080.0270.039
Staphylococcus_aureus0.0100.0270.0640.0910.0270.0590.044
Corynebacterium_atypicum0.0000.0000.0850.1580.0270.0940.045
Data are presented as rates per ten thousand (‱).
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MDPI and ACS Style

Chen, P.-C.; Chen, S.-F.; Hung, W.-T.; Lin, Y.-Y.; Lin, L.-C.; Wang, J.-H.; Chen, P.-C. Differences in Vaginal Microbiota Composition Between Infertile and Fertile Patients: A Prospective Study. Diagnostics 2025, 15, 2544. https://doi.org/10.3390/diagnostics15192544

AMA Style

Chen P-C, Chen S-F, Hung W-T, Lin Y-Y, Lin L-C, Wang J-H, Chen P-C. Differences in Vaginal Microbiota Composition Between Infertile and Fertile Patients: A Prospective Study. Diagnostics. 2025; 15(19):2544. https://doi.org/10.3390/diagnostics15192544

Chicago/Turabian Style

Chen, Pei-Chen, Shih-Fen Chen, Wei-Tung Hung, Yu-Ying Lin, Ling-Chun Lin, Jen-Hung Wang, and Pao-Chu Chen. 2025. "Differences in Vaginal Microbiota Composition Between Infertile and Fertile Patients: A Prospective Study" Diagnostics 15, no. 19: 2544. https://doi.org/10.3390/diagnostics15192544

APA Style

Chen, P.-C., Chen, S.-F., Hung, W.-T., Lin, Y.-Y., Lin, L.-C., Wang, J.-H., & Chen, P.-C. (2025). Differences in Vaginal Microbiota Composition Between Infertile and Fertile Patients: A Prospective Study. Diagnostics, 15(19), 2544. https://doi.org/10.3390/diagnostics15192544

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