The efficacy of vaginal treatment for non‐Lactobacillus dominant endometrial microbiota—A case–control study

Reduced Lactobacillus occupancy in the uterine microflora has been associated with implantation failure. This study aimed to evaluate a treatment for improving the uterine microflora.


INTRODUCTION
Repeated implantation failure (RIF), where pregnancy does not occur even after multiple transfers of good-quality embryos, is often encountered with in vitro fertilization (IVF).One of the main causes of RIF is chromosomal abnormalities in the embryos.However, the pregnancy success rate remains around 60%-70% even when embryos with normal chromosomes are transferred, suggesting the involvement of other causes, such as uterine structure, coagulation capacity, and immunological factors. 1 Recent research has also highlighted the potential association between intrauterine bacterial flora and endometritis, which are environmental factors, as one of the major causes influencing the uterus. 2,3he uterus was initially thought to be sterile 4 until reports from the 1980s revealed the presence of bacteria. 5he Human Microbiome Project further revealed that 9% of the total human microbiome was present in the female reproductive tract. 6,7With advancements in nextgeneration sequencing (NGS), detailed analysis of the bacterial flora in the uterus became possible, revealing its impact on pregnancy rates and IVF outcomes for patients. 8,9 prior study showed that the pregnancy and implantation rates of patients who underwent IVF were significantly higher when the Lactobacillus-dominant (LD) bacterial flora in the uterus was ≥90%. 8Based on this finding, the researchers hypothesized that increasing the Lactobacillus occupancy rate in the uterus might improve IVF outcomes, as the treatment of patients with non-Lactobacillus dominant (NLD) bacteria flora, defined as a Lactobacillus occupancy of <90%, has been reported to be effective with oral or intravaginal administration of antibiotics and probiotics. 10,11n 2020, Kadogami et al. reported NLD among patients with RIF.They demonstrated that a treatment regimen involving both oral and intravaginal administration of antibiotics and probiotics could improve the uterine microflora in a high percentage of affected patients. 10uilding on this approach, a treatment method utilizing vaginal antibiotics and probiotics was developed.This study aimed to compare the efficacy of oral antibiotics and probiotic preparations with that of vaginal antibiotics and probiotics.

Ethics statement
This study was approved by the Ethics Committee of our institution (approval number: H2023-027).Informed consent was not obtained from the patients or their families because the study was based on the analysis of institutional forms, and patient records and information were anonymized prior to the analysis.All methods were performed in accordance with the Declaration of Helsinki and other relevant guidelines.

Study design
This was a single-center case-control study designed to compare the efficacy of intrauterine microflora treatment modalities.

Study population
Patients eligible for this study were those with RIF who underwent assisted reproductive treatment and intrauterine microflora examination at our hospital from November 2018 to January 2023.RIF was defined as the failure to achieve pregnancy after two or more transfers of viable embryos.Patients were classified as NLD if their uterine Lactobacillus occupancy was <90% and LD if it was >90%.Patients with RIF classified as NLD who had undergone treatment to improve their intrauterine microflora and underwent re-examination were included in the study.Those classified as LD and those classified as NLD who underwent only the pre-treatment intrauterine microflora testing were excluded from the study.

Treatment protocol for improvement of intrauterine flora
Patients who underwent intrauterine microflora examination and were diagnosed with NLD received one of the following treatment regimens (Figure 1).
Treatment A consisted of oral antibiotics (metronidazole or amoxicillin-clavulanic acid) for 1 week, selected based on the bacteria detected, followed by a butyric acid bacteria combination drug (3 g/day) as a probiotic for 30-60 days.The intrauterine microflora test was repeated during the luteal phase after commencing oral probiotic administration.The butyric acid bacteria combination drug contained the following components in a 3-g dose: lactomin (Enterococcus faecium T-110 [30 mg]), butyric acid bacteria (Clostridium butyricum TO-A [150 mg]), and saccharomyces (Bacillus subtilis TO-A [150 mg]).
Treatment B consisted of oral (750 mg/day) and vaginal (250 mg/day) administration of metronidazole (Flagyl, Shionogi) as an antibiotic for 1 week, followed by intravaginal administration of probiotic capsules (1 capsule/day) for 1 week and continued administration of oral probiotics (1 capsule/day) after that.Probiotic administration was continued for 3-30 days, and an intrauterine microflora test was repeated during the luteal phase after commencing probiotic administration.The probiotic capsules used as supplements contained two types of lactic acid bacteria in each capsule: Lactobacillus rhamnosus HN001™ (13.4 mg) and Lactobacillus acidophilus La-14 ® (45 mg).

Intrauterine microflora test
Specimens were collected during the luteal phase before treatment.After treatment, specimens were collected during the luteal phase while the patient was taking probiotics.Specimen collection was done by inserting a pipette into the uterus after the vagina was washed with saline solution.The specimens were then submitted to a laboratory for analysis.The intrauterine flora test was performed using either the endometrial microbiome metagenomic analysis test (Igenomix, Spain) or the intrauterine flora test (Varinos Inc., Tokyo).Both of these tests utilize NGS to analyze the 16S rRNA genes of bacteria in intrauterine tissue.

Data collection and evaluation
Patient background information, including age, body mass index, history of pregnancy and childbirth, smoking history, and history of infertility treatment, was collected.Additionally, information on the constitutive microflora before and after treatment was collected from patients with abnormal uterine microflora.
The efficacy of Treatment A and B in improving the intrauterine flora was evaluated and compared."Cure" was defined as a transition from NLD in the pre-treatment intrauterine microflora test to LD in the post-treatment intrauterine microflora test, and "improvement" was defined as an increase in Lactobacillus occupancy by >20%.

Comparison of pre-treatment flora in treatment responders and non-responders
Within the Treatment A and Treatment B groups, patients determined to be cured were classified as the "response group" (responders), and those without satisfactory results were classified as the "non-response group" (non-responders)-the presence or absence of specific bacteria before treatment was compared between response and non-response groups.

Statistical analysis
All analyses were performed using JMP Pro version 15 (SAS Institute, Tokyo, Japan), with p < 0.05 considered statistically significant.The data were analyzed using the chi-square, Mann-Whitney U, and Wilcoxon signed-rank sum tests.

RESULTS
Intrauterine microflora examination was performed for 113 patients with implantation failure.Among these patients, 17 and 10 with NLD received Treatments A and B, respectively (Figure 2).

Patient background
Notably, no significant differences were observed between the two treatment groups in terms of body mass index, smoking, primiparity, history of miscarriage, Lactobacillus occupancy before treatment, and number of embryo transplants before treatment (Table 1).However, the mean age was significantly higher in the Treatment A group (39.8 ± 0.9 years) compared to the Treatment B group (35.5 ± 1.2 years).Additionally, the Lactobacillus occupancy before treatment was slightly lower in the Treatment A group compared to the Treatment B group, but the difference was not statistically significant.

Comparison of treatment effectiveness in improving intrauterine microflora
The number of patients in the Treatment A and Treatment B groups determined to be cured or improved based on the assessment of uterine microflora was 7 (41.2%)and 9 (90.0%),respectively (p = 0.0127) (Table 2).Additionally, the Lactobacillus occupancy after treatment was significantly higher in the Treatment B group (62.9% ± 12.7%) compared to the Treatment A group (5.7% ± 9.8%) (p = 0.0242).Based on these findings, Treatment B demonstrated higher effectiveness than Treatment A in achieving both cure or improvement and higher Lactobacillus occupancy.

Lactobacillus occupancy and classification after treatment B
Nine out of the 10 patients who received treatment B showed increased Lactobacillus occupancy following treatment, whereas the remaining patient experienced a decline (Table 3).Of the nine patients with improvement, seven showed an increase in one Lactobacillus species, and two showed an increase in two Lactobacillus species.Lactobacillus occupancy after treatment (%) 5.7 ± 9.8 62.9 ± 12.7 0.0016

Note:
The data was analyzed using the chi-square test.
Lactobacillus acidophilus was the most common Lactobacillus species observed in this study, followed by Lactobacillus jensenii.Lactobacillus species from the probiotics were detected in the patients who were re-examined immediately after treatment.In comparison, diverse Lactobacilli with an inclination toward an increase in the original vaginal Lactobacillus species were observed in patients re-examined after 3-4 weeks of oral probiotic administration.
Comparison of pre-treatment bacterial flora in treatment responders and non-responders A comparison of the pre-treatment bacterial flora between treatment responders and non-responders showed no significant differences in the presence of any bacterial classifications (Figure 3).

DISCUSSION
This study compared the efficacy of two treatment methods for improving intrauterine microflora in patients with IVF repeat failures and abnormal intrauterine microflora.The findings showed that vaginal antibiotic and probiotic formulations significantly increased Lactobacillus occupancy in the uterine microflora compared to oral formulations.Additionally, it was confirmed that the Lactobacillus species that increased in the uterus following treatment were not exclusively the species administered.Furthermore, an examination of the presence of bacterial species that were less likely to respond to treatment showed no clear differences in treatment efficacy regardless of the treatment method employed.To the best of our knowledge, this is the first report that examines not only Lactobacillus occupancy but also specific Lactobacillus species after uterine flora treatment.
Research has shown that abnormalities in the endometrial flora negatively affect IVF outcomes; hence, many attempts have been made to treat abnormal intrauterine flora.Although few reports exist on endometrial flora treatment, the most common means of treating intrauterine inflammation in these studies is antibiotic administration. 12However, antibiotics such as Lactobacilli affect both pathogenic and protective bacteria, and their sole administration is not recommended.Therefore, to replenish the Lactobacilli reduced by antibiotics, a probiotic formulation of Lactobacilli should be administered in combination, as previously employed in the treatment of the intrauterine microflora. 10his study demonstrated the crucial role of intravaginal administration of antibiotics and probiotics in improving treatment efficacy.Although the uterine cavity was previously thought to be sterile, 4 research has shown that the uterus has a unique microflora, mostly composed of Lactobacilli. 13Moreover, recent studies have reported that cervical mucus plugs do not completely prevent infection 14 and that the concordance rate between human uterine and vaginal flora is 80%, 8 indicating a close connection between the two. 15Furthermore, animal studies have also reported that both bacterial and vaginal inflammation increase the number of inflammatory cells in the uterine cavity. 16These findings collectively demonstrate the influence of the vaginal microflora on the uterine microflora.
Unlike oral formulations, probiotic vaginal formulations bypass the gastrointestinal tract and directly affect the uterus and vagina.Therefore, the increase in the number of Lactobacilli in the vagina may have directly led to an increase in the number of Lactobacilli in the uterine lumen, or the anti-inflammatory effect on the vagina may have improved the intrauterine environment and promoted the growth of Lactobacilli.Additionally, Lactobacilli in the vagina produce lactic acid and inhibit the growth of pathogenic bacteria by lowering the intravaginal pH and enhancing self-cleaning through adhesion to the vaginal epithelium and modulation of immune responses. 17These actions are considered to possibly alter the vaginal and uterine environments to favor Lactobacilli growth.
The significance of the intravaginal administration of antibiotics in this study was the treatment of abnormal vaginal flora.The eradication of abnormal vaginal bacteria supported the growth of Lactobacillus species via the administered probiotics.Furthermore, the use of metronidazole as the antibiotic of choice may have contributed to the improved treatment efficacy observed.Although metronidazole has a sufficient spectrum as a therapeutic antibiotic for bacterial vaginosis, its low bactericidal activity against Lactobacillus may have created an environment conducive to the growth of Lactobacillus species, and Treatment B, which was administered vaginally, may have augmented this effect.
In this study, the occupancy of the administered Lactobacillus species and other Lactobacillus species was evaluated using an intrauterine flora test.It was found that the increase in the number of Lactobacillus species observed in the uterine lumen was not solely based on the species administered.The Lactobacillus species detected were more diverse, and the occupancy rate of species other than the administered Lactobacillus species tended to increase in patients re-examined after about 3-4 weeks of continued oral treatment compared to those detected in patients re-examined immediately after the start of oral treatment.This suggests that the administered Lactobacillus species may have created an intrauterine environment where Lactobacillus can easily multiply and that the Lactobacillus species originally present in the uterine cavity may later increase.
Although it has been reported that increasing the Lactobacillus occupancy rate in the uterus leads to improved IVF outcomes, no published studies have directly investigated this association.Moreover, the effectiveness of the Lactobacillus species (L.acidophilus La-14 and L. rhamnosus HN001) contained in the probiotics used in this study have not been reported, and further investigation, including changes over time in Lactobacillus species following treatment, is needed.
The bacterial species associated with poor IVF outcomes were Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Haemophilus, Klebsiella, Chryseobacterium, Neisseria, Staphylococcus, and Streptococcus spp. 18These strains were analyzed with the expectation that they might be associated with treatment resistance, especially those related to IVF prognoses.The presence of these abovementioned abnormal flora species was not consistently observed in patients who did not respond to treatment in this study.However, the number of cases in this study may have been insufficient; hence, further studies with a larger sample size are warranted.
The main limitation of this study is that it was retrospective, with a small sample size and a non-randomized design.Additionally, assessing the number of bacteria using the intrauterine microflora test was difficult.It has been pointed out that the composition and total number of bacteria are both important factors when evaluating the effects of bacterial flora. 19However, quantifying bacteria using any of the currently available intrauterine flora tests is difficult, and this is considered to be an issue for future research.Furthermore, there were issues with the transvaginal collection of samples.Although contact with the vaginal wall was minimized at the time of collection, and the cervix was thoroughly washed before collection, the possibility that this might have affected the endometrial microbiota results cannot be ruled out.Regardless, transvaginal collection is the only clinically feasible method for obtaining endometrial samples, and it is unlikely that this method significantly impacted the present results. 18,20Additionally, this study evaluated the effect of treatment on the endometrial flora but did not examine the persistence of the therapeutic effect.Likewise, the pregnancy rate and outcomes of the treated patients were not analyzed.Therefore, future studies should examine the effects of this treatment on IVF outcomes, as it is necessary to examine whether intrauterine Lactobacillus species affect the time to pregnancy and IVF outcomes.It is also important to identify Lactobacillus species that are favorable for treatment and consider treatments that increase their occupancy.
In conclusion, this study demonstrated the effectiveness of vaginal antibiotic and probiotic formulations in treating abnormal uterine microflora.However, its impact on IVF outcomes is still unknown and needs to be further studied in the future.

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I G U R E 1 Treatment regimen for each treatment group.

F I G U R E 2
Study flowchart of the patients.T A B L E 1 Patient background.Treatment A group (n = 17) Treatment B group (n = 10) Data were analyzed using the chi-square and Mann-Whitney U tests. a Including bifidobacterium.T A B L E 2 Intrauterine flora improvement after treatment.Treatment A group (n = 17) Treatment B group (n = 10)

T A B L E 3
Change in Lactobacillus occupancy and classification in Treatment B group.

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I G U R E 3 Comparison pre-treatment types of bacteria in treatment responders and non-responders.