Iranian Journal of Medical Microbiology

Table 1. Utilized primers in the present study.

Each Real-Time PCR reaction was performed in a final volume of 20 µl, containing 0.6 μM of each primer, 10 μL of 2X Q-PCR Master Mix (SYBR, ROX) (SMOBIO, Taiwan), and 5.8 μL of sterilized ultra-pure water. The input DNA was 3 ng/reaction. The cycling conditions were as follows: initial denaturation at 95°C for 5 min; followed by 40 cycles at 95°C for 10 s, and annealing/extension at 59–61°C for 60 s. Reactions were run on the Rotor-Gene 6000 real-time PCR cycler (Qiagen Corbett, Germany). For negative controls, all ingredients of the reaction mixture were used except for template DNA.
To verify primer specificities, melting curves were generated at the end of each PCR reaction. Fluorescent data were acquired during the extension phase. After 40 cycles, a melting curve for each gene was generated by increasing the temperature from 60 to 95°C (1°C per step), while the fluorescence was measured. Samples were run in duplicates.
For the determination of the number of  L. crispatus, A. vaginae, P. bivia, and G. vaginalis present in each sample, standard curves were constructed corresponding to 10^¹ to 10^¹⁰ copies/ml (23). These curves were created based on the normalized copy number of the 16S rRNA gene for each species (Biosystems, 2013) and Applied Biosystems tutorials. The bacterial concentrations from each sample were calculated from the threshold cycle values (CT) obtained from the standard curves. According to previous studies (22, 23), bacterial standard strains were selected from the American Type Culture Collection (ATCC).

2.6. Statistical Analysis

Statistical analyses were conducted using GraphPad Prism 8.4.3 (GraphPad Software, San Diego, CA, USA). The Shapiro–Wilk test was used to assess the normality of the data. The Mann-Whitney U test or t-test was applied to compare two groups of continuous numerical data. Additionally, Fisher's exact test was utilized to assess the association between two categorical datasets. A p-value of less than 0.05 was considered statistically significant for the analyses.

Premature delivery is the primary cause of morbidity and mortality during pregnancy in most countries (24). Bacterial vaginosis has been implicated in developing PTB and subsequent complications (25). G.vaginalis, A.vaginae, and P.bivia have been introduced as the main culprits for developing bacterial vaginosis (26). Since the microbiome has a considerable variety in different geographical areas and races, we studied the impact of those bacteria PTB in Iranian mothers. 
Classical diagnostic methods, such as the Amsel criteria and Nugent scoring systems, are the practical and cost-effective options for diagnosing bacterial vaginosis (27, 28). Although culture is considered the standard diagnostic approach for many bacterial infections, it is not recommended for bacterial vaginosis due to the challenges in isolation and their scarcity in normal vaginal flora (29). As an alternative diagnostic method, polymerase chain reaction (PCR) can identify the type of bacteria (30). Of interest, our results have depicted significant trends among the load of G.vaginalis, A.vaginae, P.bivia, and L. Crispatus with clinical signs and symptoms of bacterial vaginosis in our samples. The present study has shown that the prevalence of bacterial vaginosis based on Amsel's criteria is 36/36% in our 55 included cases. Consistent with this, Ruh Bakhsh et al. reported that the prevalence of bacterial vaginosis was 31% in the Gilan province of Iran in 2019 (31). The estimated prevalence of the present study was higher than the reports from Ethiopia (19.4%) and India (20.5%) and close to the reports from Kenya (37%) and Zimbabwe (32.5%) (32-35). Thus, the prevalence of bacterial vaginosis is remarkable in different geographical regions.
In this study, we demonstrated the high sensitivity and specificity of the qPCR method. Although qPCR requires specialized equipment, it proves to be more efficient and less labor-intensive than Nugent scoring, which relies on the manual assessment of Gram-stained smears. This makes qPCR a suitable option in settings with limited skilled personnel but access to basic molecular biology tools (36). While Amsel's criteria are straightforward and require minimal equipment, they exhibit lower sensitivity and specificity, increasing the risk of misdiagnosis, particularly in complicated cases. qPCR provides rapid results, which are crucial for timely treatment initiation—an essential factor in low-resource settings where diagnostic delays can worsen health outcomes (37). Furthermore, qPCR's ability to simultaneously detect multiple pathogens, including those responsible for BV, trichomoniasis, and vulvovaginal candidiasis, enables a comprehensive approach to diagnosis. This is particularly beneficial for identifying co-infections, which are common and can complicate treatment strategies (38). Overall, qPCR presents a promising tool for improving diagnostic accuracy and patient care in a variety of clinical settings.
The present study has demonstrated that mothers with bacterial vaginosis have higher levels of G. vaginalis, P. bivia, and A. vaginae bacteria, but these mothers have lower levels of L. crispatus bacteria. Our results have not identified any significant relationships between the presence of studied bacteria with PTB; this is in line with the study by Adesiji et al. (39).
The vaginal microbiome composition in our study (prevalence of Lactobacillus species) is consistent with the prevalence and microbial profile findings from the Kenyan and Zimbabwean studies, but slightly higher than from India and Ethiopia (40, 41). This discrepancy warrants further exploration into the potential influence of ethnic variations, behavioral practices (such as hygiene and dietary habits), and even diagnostic methodologies employed across these diverse populations. Roohbakhsh et al.'s 2019 study within Iran provides a valuable internal comparison, and the noted differences with other regions underscore the importance of considering the multifaceted factors that can shape the vaginal microbiome (42). Future research could benefit from standardized protocols and larger, multi-center studies to disentangle these complex interactions and provide a more comprehensive understanding of global variations in the vaginal microbiome (42).
Consistent with this, Livani et al. have reported that there is no considerable difference between the levels of G. vaginalis and A. vaginae bacteria in mothers with PTB compared to those with term delivery (43). However, Lim et al. have reported that the presence of A. vaginae is higher in patients with preterm delivery or premature rupture of membranes (44). Also, Keli et al. have shown that women with preterm delivery have low levels of Lactobacillus species and higher levels of Gardnerella, Atopobium, Megasphaera, and Streptococcus in the lower genital tract (45). Besides, Prodan-Barbulescu et al. have demonstrated that the presence of G. vaginalis is substantially associated with PTB (46). Also, Nguyen et al. have indicated that bacterial vaginosis, unlike fungal infection, increases the risk of PTB and preterm premature rupture of membranes (47).
The null association between BV-associated bacteria and PTB observed in our studies presents a paradox with other research findings. This discrepancy may arise from variability in diagnostic criteria, which can contribute to conflicting results (48). Additionally, the timing of diagnosis is crucial: BV detected before 16 weeks’ gestation shows a strong correlation with PTB, while diagnoses made later demonstrate weaker associations (49).  Moreover, pathogen specificity may play a role; subclinical infections or polymicrobial interactions (50), such as those involving G. vaginalis and Mycoplasma hominis, could increase risk more than BV alone (49). For instance, one study reported a 2.1-fold increased risk of PTB when both pathogens were present (49).  The host immune response is also a key factor, as BV-associated bacteria can trigger inflammatory cytokines (e.g., IL-1β, IL-6), which weaken fetal membranes (51). However, genetic or immunological variability across different populations may modulate the effects of these inflammatory responses, potentially explaining the inconsistencies observed in various studies (51).
Cultural and behavioral factors in Iran may explain these differences. For instance, lower alcohol consumption and smoking rates among Iranian women, compared to their Western counterparts, could reduce confounding behavioral risks for PTB (52).
The present study suffers from several limitations. First, we only studied bacterial vaginosis in the third trimester. Given the dynamic nature of bacterial vaginosis in pregnant women, longitudinal studies are needed to comprehensively investigate the impact of bacterial vaginosis on preterm delivery throughout all trimesters. Second, Given the cohort of only 55 participants and a prevalence of PTB at 36.36%, this study may lack the statistical power necessary to detect subtle associations, highlighting the need for  more substantial sample size in future research.
Third, we utilized real-time PCR to quantify the abundance of specific bacterial groups; however, this method does not provide information about the metabolic activities of each bacterium in bacterial vaginosis. Overall, the current study provides novel insights into the bacterial vaginosis prevalence and the impact of G. vaginalis, L.crispatus, P.bivia, and A.vaginae in preterm labor of Iranian pregnant women.

 

In our included pregnant women, the prevalence of bacterial vaginosis is 36.36% according to the Amsel criteria. Our results have shed light on the significant trends between the load of G.vaginalis, A.vaginae, P.bivia, and L. Crispatus with clinical signs and symptoms of bacterial vaginosis in pregnant women. However, there has been no significant difference in the load of G.vaginalis, A.vaginae, P.bivia, and L. Crispatus in the lower genital tract of patients with PTB compared to those with term delivery.

 

The authors would like to thank the Shahid Akbarabadi Clinical Research Development Unit (ShACRDU), School Of Medicine Iran University of Medical Sciences(IUMS), Tehran, Iran for their specialists who helped collect vaginal swab samples throughout the preiod of study (IR.IUMS.FMD.REC.1401.242). All the authors have been reading and concurring with this manuscript.
 
 

Ethical Considerations

The study received ethical approval from the Ethics Committee of the Iran University of Medical Sciences (IR.IUMS.FMD.REC.1401.242) and was conducted in accordance with the principles of the Helsinki Declaration.

Authors’ Contributions

Parisa Rahimi: Writing – original draft, Project administration, Investigation. Shirin Dashtbin: Supervision. Shiva Mirkalantari: Writing – review & editing. Maryam Kashanian: Writing – review & editing. Nooshin Eshraghi: Writing – review & editing. Faramarz Masjedian Jazi: Writing – review & editing, Resources, Project administration, Conceptualization. All authors read and approved the final manuscript.

Conflicts of Interest

This study was financially supported in Iran University of Medical Sciences (Tehran, Iran).
 
 

The authors were not utilized AI Tools.