European Journal of Cardiovascular Medicine

Unexplained infertility, defined as the absence of an identifiable cause despite comprehensive evaluation of ovulatory function, tubal patency, and semen analysis, affects approximately 10–30% of couples seeking fertility treatment (1). Despite advancements in assisted reproductive technologies (ART), particularly in vitro fertilization (IVF), success rates among women with unexplained infertility remain variable and often suboptimal (2). Recently, the composition of the vaginal microbiota has emerged as a potential factor influencing reproductive outcomes, including IVF success (3).

The vaginal ecosystem is predominantly colonized by Lactobacillus species, which contribute to a low pH environment and suppress the growth of pathogenic bacteria through lactic acid production and other antimicrobial mechanisms (4). A shift from Lactobacillus-dominance to a more diverse microbial composition—commonly referred to as dysbiosis—has been linked to adverse gynecological conditions such as bacterial vaginosis, preterm labor, and implantation failure (5,6). Studies suggest that such microbial imbalances may also interfere with endometrial receptivity, embryo implantation, and pregnancy maintenance (7).

Emerging evidence utilizing 16S rRNA gene sequencing indicates that women with a non-Lactobacillus-dominant microbiota may experience lower IVF success rates, possibly due to inflammatory responses or altered immune tolerance within the reproductive tract (8,9). However, the specific impact of vaginal microbial profiles on IVF outcomes in women with unexplained infertility remains underexplored. Understanding this relationship may offer novel, non-invasive diagnostic and therapeutic approaches to optimize ART outcomes.

This study aims to investigate the association between vaginal microbiota composition and IVF success in women diagnosed with unexplained infertility, with a focus on comparing clinical outcomes between Lactobacillus-dominant and non-Lactobacillus-dominant microbiota profiles.

Study Design and Participants
The study enrolled 100 women aged 25 to 38 years who were clinically diagnosed with unexplained infertility following standard assessments, including hormonal profiling, transvaginal ultrasound, hysterosalpingography, and partner semen analysis. Inclusion criteria comprised regular menstrual cycles, normal ovarian reserve (AMH >1.2 ng/mL), and no history of pelvic inflammatory disease or antibiotic use in the past three months. Patients with endometriosis, uterine anomalies, or chronic systemic illness were excluded.

Sample Collection and Microbiota Analysis
Vaginal swabs were collected from all participants during the follicular phase (days 5–9 of the cycle), prior to ovarian stimulation. Swabs were inserted into the mid-vaginal canal and rotated gently to collect epithelial cells and secretions. Samples were stored at −80 °C until further analysis.

Microbial DNA was extracted using a commercially available kit (Qiagen, Germany) following the manufacturer's protocol. Amplification of the V3–V4 hypervariable regions of the 16S rRNA gene was performed using universal primers. Sequencing was conducted on the Illumina MiSeq platform. Bioinformatic analysis included sequence quality filtering, operational taxonomic unit (OTU) clustering at 97% similarity, and taxonomic classification using the SILVA database.

Grouping and IVF Protocol
Based on microbial analysis, participants were categorized into two groups: Group A (n=58) with Lactobacillus-dominant microbiota (≥80% relative abundance of Lactobacillus spp.) and Group B (n=42) with non-Lactobacillus-dominant profiles. All patients underwent a standard antagonist protocol for controlled ovarian stimulation. Recombinant FSH and hMG were administered with dosage adjustments based on follicular response monitored via ultrasound. Ovulation was triggered with recombinant hCG when leading follicles reached ≥18 mm. Oocyte retrieval was performed 36 hours post-trigger, followed by fertilization via ICSI.

Outcome Measures
Primary outcomes included implantation rate, clinical pregnancy rate, and fertilization rate. Implantation was confirmed by transvaginal ultrasound showing gestational sacs at 5–6 weeks post-transfer. Clinical pregnancy was defined by the presence of fetal cardiac activity. Secondary outcomes included embryo quality and biochemical pregnancy rate.

Statistical Analysis
Data were analyzed using SPSS version 26.0 (IBM Corp., USA). Categorical variables were compared using chi-square or Fisher’s exact test, and continuous variables were analyzed using independent t-tests. Statistical significance was set at p < 0.05. Alpha and beta diversity indices were used to evaluate microbial variation between groups.

A total of 100 women with unexplained infertility completed the study and underwent IVF treatment. Based on the vaginal microbiota composition, 58 participants were classified into the Lactobacillus-dominant group (Group A), and 42 into the non-Lactobacillus-dominant group (Group B).

Demographic and Baseline Characteristics

There were no statistically significant differences between the groups in terms of mean age, body mass index (BMI), duration of infertility, or baseline serum AMH levels (p>0.05) (Table 1).

Table 1. Baseline characteristics of the study population

IVF Cycle Outcomes

Clinical outcomes demonstrated significantly better performance in the Lactobacillus-dominant group. The mean implantation rate was higher in Group A (41.3%) compared to Group B (22.5%) (p=0.03). The clinical pregnancy rate in Group A was 56.9%, nearly double that of Group B at 28.6% (p=0.004). Fertilization rates showed no significant difference between groups (p=0.21) (Table 2).

Table 2. IVF outcomes by microbiota composition

*Statistically significant

Microbiota Diversity Analysis

Alpha diversity, measured using the Shannon index, was significantly higher in Group B (3.4 ± 0.5) than in Group A (2.1 ± 0.3), indicating greater microbial heterogeneity in the non-Lactobacillus-dominant group (p<0.001). Beta diversity plots demonstrated distinct clustering of microbiota composition between the two groups (not shown).

These findings suggest a clear association between Lactobacillus-dominant vaginal microbiota and improved IVF outcomes in women with unexplained infertility (Tables 1 and 2).

The present study investigated the influence of vaginal microbiota composition on IVF outcomes in women with unexplained infertility. Our findings demonstrate a significant association between Lactobacillus-dominant microbiota and improved implantation and clinical pregnancy rates. These results support the growing evidence that microbial homeostasis in the vaginal environment plays a pivotal role in reproductive success.

A predominance of Lactobacillus species, particularly L. crispatus and L. jensenii, is considered protective due to their ability to maintain acidic vaginal pH and produce antimicrobial compounds, such as lactic acid and hydrogen peroxide (1,2). In our study, women with Lactobacillus-dominant profiles had nearly twice the clinical pregnancy rate compared to those with dysbiotic microbiota, aligning with earlier reports that associate Lactobacillus dominance with enhanced implantation and embryo survival (3,4).

Conversely, dysbiotic profiles characterized by Gardnerella, Atopobium, and Prevotella were linked to poor IVF outcomes, consistent with prior studies indicating that such microbial communities can provoke subclinical inflammation and disrupt endometrial receptivity (5–7). This is further supported by Romero et al., who found that dysbiosis may increase mucosal permeability and allow bacterial metabolites to interfere with the uterine environment (8).

The observed differences in microbial alpha diversity between groups also mirror findings from recent metagenomic studies, where increased microbial diversity—though generally beneficial in gut ecosystems—has been linked to adverse outcomes in the reproductive tract (9,10). It has been proposed that a stable, low-diversity microbiota dominated by Lactobacillus ensures optimal mucosal immunity and hormonal responsiveness during implantation (11).

Our study found no significant difference in fertilization rates, suggesting that the vaginal microbiota may not directly affect oocyte quality or fertilization potential but primarily influences post-fertilization processes such as implantation and immune modulation (12). Similar conclusions were drawn by Koedooder et al., who emphasized that microbiota-associated implantation failure occurs independently of embryonic development quality (13).

The utility of microbiome profiling as a prognostic tool for IVF success is an emerging concept. Incorporating such evaluations into routine pre-IVF assessments may aid in identifying patients who could benefit from microbiome-modulating interventions, such as probiotics or targeted antibiotics (14). Preliminary trials with Lactobacillus crispatus probiotics have demonstrated improvements in vaginal microbiota and subsequent IVF success, though larger randomized studies are needed to validate these strategies (15).

While our study provides meaningful insights, it has limitations. The sample size, though adequate for initial analysis, may not capture all variations in microbial subtypes. Additionally, the absence of endometrial microbiota assessment limits the interpretation of potential upper tract microbial influence. Longitudinal studies incorporating both vaginal and endometrial sampling are warranted to better understand the interplay between microbiota and reproductive physiology.

In conclusion, our findings reinforce the role of vaginal microbial composition as a critical determinant of IVF success in women with unexplained infertility. Identification and modulation of dysbiosis may offer a non-invasive, adjunctive approach to enhance ART outcomes.

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