Prevalence of Mycoplasma genitalium and Co-Infections with Chlamydia trachomatis and Neisseria gonorrhoeae Among Japanese Women: A Cross-Sectional Study

Abstract

Background/Objectives: Mycoplasma genitalium is an emerging cause of sexually transmitted infections (STIs) and is increasingly recognized for its association with cervicitis and pelvic inflammatory disease. However, prevalence data in specific Japanese subpopulations, particularly comparing pregnant and non-pregnant women, remains limited. This study aimed to determine the prevalence of M. genitalium and its co-infection rates with Chlamydia trachomatis and Neisseria gonorrhoeae among Japanese women. Methods: A cross-sectional study was conducted using vaginal swab specimens collected between April 2021 and November 2022 from patients visiting two clinics in Gifu, Japan. The study population comprised 2138 non-pregnant women presenting with urogenital symptoms or sexual contact history, and 236 pregnant women undergoing routine antenatal screening. Detection was performed using real-time polymerase chain reaction assays on the cobas^® 8800 system (Roche Diagnostics). Results: Among non-pregnant women, the overall prevalence was 3.8% (82/2138) for M. genitalium, 3.4% (72/2138) for C. trachomatis, and 0.4% (9/2138) for N. gonorrhoeae. Co-infection rates were low; M. genitalium and C. trachomatis co-infection was observed in 0.2% of cases. Among pregnant women, the prevalence was 3.8% (9/236) for both M. genitalium and C. trachomatis, and 0.4% (1/236) for N. gonorrhoeae. No statistically significant differences in prevalence were observed between pregnant and non-pregnant women for any pathogen. Conclusions: The prevalence of M. genitalium in this Japanese cohort was comparable to that of C. trachomatis in both pregnant and non-pregnant women, highlighting its significance as a major STI pathogen. These findings underscore the importance of including M. genitalium in routine STI screening panels for symptomatic women and antenatal care to prevent reproductive health complications. Given the high rates of antimicrobial resistance documented in Japanese M. genitalium strains, specific diagnostic testing is essential to enable targeted, resistance-guided therapy.

1. Introduction

Mycoplasma genitalium has emerged as a significant pathogen responsible for sexually transmitted infections (STIs) globally. Since its first isolation from men with non-gonococcal urethritis (NGU) in the early 1980s, it has been firmly established as a causative agent of NGU in men. It is increasingly associated with cervicitis, pelvic inflammatory disease (PID), preterm birth, and tubal factor infertility in women [1,2]. Despite its clinical importance, M. genitalium is not routinely included in STI screening panels in many countries, including Japan, leading to potential underdiagnosis and untreated infections that may result in serious reproductive health complications.

The global epidemiology of M. genitalium has been extensively documented in recent systematic reviews and meta-analyses. Baumann et al. [3] reported an estimated global prevalence of 1.3% in general populations, with substantially higher rates of 3.9% among attendees of sexual health clinics, 5.2% in men who have sex with men, and 6.9% in female sex workers. Regional variations are notable, with prevalence rates ranging from 1% to 6% reported in the general populations across North America, Europe, and parts of Asia [3,4]. In pregnancy, international data suggest prevalence rates of approximately 1% in high-income settings, though substantially higher rates have been documented in resource-limited settings, including 12.6% in Zambia and 3.6% to 12.5% in Papua New Guinea and South Africa [5,6,7]. These geographic and population-specific variations underscore the importance of local epidemiological data for informing clinical practice and public health policy.

In Japan, the epidemiology of M. genitalium has been predominantly characterized in male populations, with limited data available for women. Multiple studies have documented that M. genitalium accounts for 10% to 20% of non-gonococcal urethritis cases in Japanese men, with detection rates ranging from 13% to 20% in symptomatic male patients [8,9]. A comprehensive review by Deguchi et al. [8] synthesized Japanese data showing consistent detection of M. genitalium in 10% to 20% of men with NGU across multiple centers. Furthermore, Japan has been recognized as an endemic area for antimicrobial-resistant M. genitalium, with recent surveillance in Tokyo documenting alarmingly high rates of macrolide resistance approaching 90% and increasing fluoroquinolone resistance [10,11]. These resistance patterns have significant implications for treatment strategies and highlight the urgent need for diagnostic testing to enable resistance-guided therapy.

In stark contrast to the robust data available for men, epidemiological information on M. genitalium in Japanese women remains remarkably limited. One of the earliest studies, conducted by Uno et al. [12], examined 200 Japanese women and detected M. genitalium in 7.8% of women with cervicitis and 5.7% with adnexitis, but found no infections among 80 asymptomatic pregnant women. Subsequent studies have been scarce, creating a substantial knowledge gap regarding the contemporary prevalence and epidemiological characteristics of M. genitalium infection in Japanese women, particularly in the context of routine gynecological care and antenatal screening. This paucity of data is particularly concerning given the documented associations between M. genitalium and adverse reproductive outcomes in women, including cervicitis, endometritis, PID, and potentially preterm birth and spontaneous abortion [1,2,13,14].

While Chlamydia trachomatis and Neisseria gonorrhoeae have well-established national surveillance systems in Japan, systematic data collection for M. genitalium has not been implemented. Furthermore, diagnostic testing for M. genitalium is not covered by the Japanese national health insurance system, which creates significant barriers to both clinical diagnosis and epidemiological surveillance [8]. This lack of reimbursement has resulted in limited clinical testing, contributing to the sparse epidemiological data available for Japanese women. Understanding local prevalence is critical for guiding empirical treatment strategies, informing screening recommendations, and advocating for policy changes regarding diagnostic test coverage, especially given the rising concern of antimicrobial resistance in M. genitalium strains circulating in Japan.

The clinical significance of M. genitalium extends beyond acute infection. In women, untreated or inadequately treated M. genitalium infections can lead to ascending infection of the upper reproductive tract, resulting in endometritis and PID [1]. These conditions are associated with significant long-term sequelae, including chronic pelvic pain, ectopic pregnancy, and tubal factor infertility [2]. Moreover, emerging evidence suggests that M. genitalium infection during pregnancy may be associated with adverse outcomes such as preterm birth, premature rupture of membranes, and low birth weight, although the strength of these associations varies across studies [5,13,14]. Given these potential complications, understanding the prevalence of M. genitalium in both pregnant and non-pregnant women is essential for developing appropriate screening and management strategies.

This study aims to address the critical knowledge gap regarding M. genitalium epidemiology in Japanese women by investigating the prevalence of M. genitalium, C. trachomatis, and N. gonorrhoeae in a cohort of both pregnant and non-pregnant women attending gynecological clinics in Gifu, Japan. We further analyze the rates of co-infection among these pathogens to provide contemporary epidemiological data that can inform clinical practice, guide treatment decisions, and contribute to the development of evidence-based public health policy for STI screening and management in Japan. By comparing prevalence rates between pregnant and non-pregnant women, we also seek to determine whether M. genitalium screening should be incorporated into routine antenatal care protocols alongside the currently recommended screening for C. trachomatis and N. gonorrhoeae.

2. Materials and Methods

2.1. Study Setting and Population

This retrospective cross-sectional study analyzed data derived from vaginal swabs submitted for STI testing between April 2021 and November 2022. The specimens were obtained from patients visiting the Ai Ladies Clinic and the Takahashi Ladies Clinic, both located in Gifu City, Gifu Prefecture, Japan. These clinics provide general gynecological services, including STI screening, contraceptive counseling, infertility, and antenatal care to the local community. In this retrospective laboratory-based dataset, only limited clinical background information was consistently available. Detailed individual-level demographic variables, such as age distributions, were not uniformly retrievable for all participants.

The study population was categorized into two groups: non-pregnant women (n = 2138) and pregnant women (n = 236). Non-pregnant patients typically presented for consultation due to urogenital symptoms, including urethral symptoms (dysuria or urethral discharge), vaginal symptoms (abnormal vaginal discharge, dysuria, or abnormal bleeding), lower abdominal pain, or as part of contact tracing following notification of a sexual partner’s STI diagnosis. Some asymptomatic women requesting routine STI screening were also included. Pregnant women were screened as part of routine antenatal care procedures, typically during their first prenatal visit, regardless of symptom status. Accordingly, the non-pregnant cohort should be interpreted primarily as a clinically indicated testing population rather than a general screening population. All participants who underwent STI testing during the study period and had valid test results for all three pathogens (M. genitalium, C. trachomatis, and N. gonorrhoeae) were included in the analysis.

2.2. Sampling and Molecular Testing

Vaginal swab specimens were collected by trained clinicians using standardized collection techniques. Each specimen was obtained using a sterile swab inserted into the vaginal canal and rotated against the vaginal wall to ensure adequate cellular material collection. The swabs were immediately transferred to the appropriate transport media provided by the manufacturer. All samples were transported to the testing laboratory on the same day as collection at ambient temperature to maintain specimen integrity.

Molecular detection was performed using nucleic acid amplification tests (NAATs), which are considered the gold standard for STI diagnosis due to their high sensitivity and specificity. For the detection of M. genitalium, C. trachomatis, and N. gonorrhoeae, the cobas^® CT/NG and cobas^® TV/MG assays (Roche Diagnostics, Basel, Switzerland) were utilized. These assays are qualitative in vitro diagnostic tests that employ real-time polymerase chain reaction (PCR) technology to detect specific DNA target regions of the respective pathogens. The cobas^® CT/NG assay simultaneously detects C. trachomatis and N. gonorrhoeae, while the cobas^® TV/MG assay simultaneously detects Trichomonas vaginalis and M. genitalium. All assays were performed on the automated cobas^® 4800 system (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions, with appropriate positive and negative controls included in each run to ensure assay validity.

It should be noted that antimicrobial resistance testing for M. genitalium was not performed in this study, as the primary objective was to determine prevalence rates rather than characterize resistance patterns. However, given the high rates of macrolide and fluoroquinolone resistance documented in Japanese M. genitalium strains, this represents an important limitation of our study.

2.3. Statistical Analysis

Statistical analyses were conducted using RStudio (version 2022.07.1; RStudio, PBC, Boston, MA, USA) with R version 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria). Prevalence rates for each pathogen were calculated as the proportion of positive cases divided by the total number of specimens tested, expressed as percentages with exact 95% confidence intervals (CIs). The exact 95% CIs for prevalence estimates were calculated using the Clopper-Pearson method for binomial proportions, which is particularly appropriate for studies with relatively small numbers of positive cases.

Co-infection rates were determined by identifying specimens that were simultaneously positive for multiple pathogens. The proportions of co-infections among all tested specimens and among pathogen-positive cases were calculated with 95% CIs. To compare prevalence rates between pregnant and non-pregnant women, Fisher’s exact test was employed due to the relatively small number of positive cases in some categories. A two-sided p-value of less than 0.05 was considered statistically significant.

Descriptive statistics were used to summarize the demographic and clinical characteristics of the study population. As this was primarily a descriptive epidemiological study focused on prevalence estimation, formal hypothesis testing was limited to comparisons between the two population groups (pregnant versus non-pregnant women). All statistical analyses were performed in accordance with standard epidemiological methods for cross-sectional studies.

2.4. Ethical Considerations

This retrospective study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Aichi Medical University (ethic code [2021-038], approved on 7 June 2021). Despite the retrospective nature of this study using de-identified laboratory data collected during routine clinical practice, ethical approval was obtained from the institutional review board, and written informed consent was obtained from all participants. All data were anonymized prior to analysis, and patient confidentiality was strictly maintained throughout the study.

3. Results

3.1. Study Population Characteristics

A total of 2374 women were included in this cross-sectional study, comprising 2138 non-pregnant women and 236 pregnant women who underwent STI testing between April 2021 and November 2022. All participants had complete test results for M. genitalium, C. trachomatis, and N. gonorrhoeae. The non-pregnant cohort primarily consisted of women presenting with urogenital symptoms or identified through sexual partner contact tracing, while the pregnant cohort represented women undergoing routine antenatal screening. Because this was a retrospective laboratory-based analysis, quantitative individual-level summaries of age and symptom categories were not consistently available for all participants.

3.2. Prevalence of STI Pathogens in Non-Pregnant Women

Among the 2138 non-pregnant women analyzed, the overall positivity rate for M. genitalium was 3.8% (82/2138; 95% CI: 3.1–4.7%), which was slightly higher than that of C. trachomatis at 3.4% (72/2138; 95% CI: 2.7–4.2%). N. gonorrhoeae was the least common pathogen, detected in only 0.4% (9/2138; 95% CI: 0.2–0.8%) of the women. The comparable prevalence rates of M. genitalium and C. trachomatis highlight the clinical significance of M. genitalium as a major STI pathogen in this population.

Regarding mono-infections, M. genitalium was detected as a single pathogen in 3.5% (76/2138; 95% CI: 2.8–4.4%) of cases, while C. trachomatis mono-infection was found in 3.1% (66/2138; 95% CI: 2.4–3.9%). N. gonorrhoeae mono-infection was detected in only 0.2% (4/2138; 95% CI: 0.05–0.5%) of cases.

Co-infections were relatively rare in this cohort. Simultaneous infection with C. trachomatis and M. genitalium occurred in 0.2% (6/2138; 95% CI: 0.09–0.5%) of patients, representing 7.3% (6/82) of M. genitalium-positive cases and 8.3% (6/72) of C. trachomatis-positive cases. Co-infection with C. trachomatis and N. gonorrhoeae was observed in 0.2% (5/2138; 95% CI: 0.08–0.5%) of the cohort. Notably, there were no cases of co-infection involving M. genitalium and N. gonorrhoeae without C. trachomatis, and no triple infections (all three pathogens simultaneously) were identified. Detailed distributions of infection patterns are presented in

Table 1. Prevalence of urogenital pathogens among non-pregnant women (n = 2138).

Infection Status	Count (n)	Percentage (%)	95% CI
Mono-infections
M. genitalium only	76	3.5	2.8–4.4
C. trachomatis only	66	3.1	2.4–3.9
N. gonorrhoeae only	4	0.2	0.05–0.5
Co-infections
C. trachomatis + M. genitalium	6	0.2	0.09–0.5
C. trachomatis + N. gonorrhoeae	5	0.2	0.08–0.5
M. genitalium + N. gonorrhoeae	0	0.0	0.0–0.2
Triple infection (CT + NG + MG)	0	0.0	0.0–0.2
Total Positivity (Overall)
M. genitalium Total	82	3.8	3.1–4.7
C. trachomatis Total	72	3.4	2.7–4.2
N. gonorrhoeae Total	9	0.4	0.2–0.8

CI, confidence interval; CT, Chlamydia trachomatis; MG, Mycoplasma genitalium; NG, Neisseria gonorrhoeae.

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3.3. Prevalence of STI Pathogens in Pregnant Women

Among the 236 pregnant women screened during routine antenatal care, the prevalence patterns were remarkably similar to those observed in the non-pregnant group. M. genitalium and C. trachomatis both showed an overall positivity rate of 3.8% (9/236; 95% CI: 1.8–7.1%), while N. gonorrhoeae was detected in only one case (0.4%; 95% CI: 0.01–2.3%).

Single infections of M. genitalium and C. trachomatis were both identified in 3.4% (8/236; 95% CI: 1.5–6.5%) of the pregnant cohort. N. gonorrhoeae mono-infection was detected in one case (0.4%; 95% CI: 0.01–2.3%). Only one case of co-infection (C. trachomatis and M. genitalium) was identified, representing 0.4% (1/236; 95% CI: 0.01–2.3%) of the pregnant population. This co-infection accounted for 11.1% (1/9) of M. genitalium-positive cases and 11.1% (1/9) of C. trachomatis-positive cases among pregnant women. No other co-infection combinations were observed, and no triple infections were detected. Detailed distributions of infection patterns in pregnant women are presented in

Table 2. Prevalence of urogenital pathogens among pregnant women (n = 236).

Infection Status	Count (n)	Percentage (%)	95% CI
Mono-infections
M. genitalium only	8	3.4	1.5–6.5
C. trachomatis only	8	3.4	1.5–6.5
N. gonorrhoeae only	1	0.4	0.01–2.3
Co-infections
C. trachomatis + M. genitalium	1	0.4	0.01–2.3
C. trachomatis + N. gonorrhoeae	0	0.0	0.0–1.5
M. genitalium + N. gonorrhoeae	0	0.0	0.0–1.5
Triple infection (CT + NG + MG)	0	0.0	0.0–1.5
Total Positivity (Overall)
M. genitalium Total	9	3.8	1.8–7.1
C. trachomatis Total	9	3.8	1.8–7.1
N. gonorrhoeae Total	1	0.4	0.01–2.3

CI, confidence interval; CT, Chlamydia trachomatis; MG, Mycoplasma genitalium; NG, Neisseria gonorrhoeae.

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3.4. Comparison Between Pregnant and Non-Pregnant Women

Comparative analysis between pregnant and non-pregnant women revealed no statistically significant differences in the prevalence of any of the three pathogens examined. For M. genitalium, the prevalence was identical in both groups (3.8%; p = 1.000, Fisher’s exact test). Similarly, no significant difference was observed for C. trachomatis (3.4% in non-pregnant versus 3.8% in pregnant women; p = 0.744) or N. gonorrhoeae (0.4% in both groups; p = 1.000). These findings suggest that pregnancy status does not significantly influence the prevalence of these STI pathogens in women attending gynecological clinics in this region. However, the relatively small sample size of pregnant women limits the statistical power to detect modest differences.

The co-infection rate between M. genitalium and C. trachomatis was also similar between the two groups (0.2% in non-pregnant versus 0.4% in pregnant women; p = 0.527), indicating that co-infection patterns do not differ substantially by pregnancy status.

4. Discussion

This study provides contemporary data on the prevalence of M. genitalium in Japanese women, revealing a positivity rate of 3.8% in both pregnant and non-pregnant women attending gynecological clinics in Gifu, Japan. These findings are particularly significant as they demonstrate that M. genitalium prevalence is comparable to that of C. trachomatis (3.4% in non-pregnant and 3.8% in pregnant women) in this population, highlighting M. genitalium as a major STI pathogen that warrants greater clinical attention. However, given the single-region clinic-based design of this study, our findings should be interpreted as supportive evidence for further evaluation of targeted screening strategies rather than as definitive evidence for universal routine screening.

Our results align with recent international studies indicating that M. genitalium prevalence often rivals that of C. trachomatis in certain symptomatic populations and clinical settings. A systematic review and meta-analysis by Baumann et al. [3] reported global M. genitalium prevalence estimates of 1.3% in general populations, with higher rates of 3.9% among attendees of sexual health clinics and substantially elevated rates in high-risk groups. The prevalence of 3.8% in our non-pregnant cohort—primarily comprising women with urogenital symptoms or contact history—is consistent with recent literature from Asia and other regions. A 2024 study among Vietnamese participants in an HIV pre-exposure prophylaxis program reported high M. genitalium prevalence rates [4], while studies from other Asian regions have documented prevalences ranging from 3% to 13% depending on population characteristics and testing methodologies [15]. The consistency of our findings with international data supports the validity of our results and suggests that M. genitalium is a common STI pathogen in Japanese women, comparable to other well-established pathogens.

The similarity in infection rates between pregnant and non-pregnant women in our study (3.8% in both groups) is particularly noteworthy and has important implications for antenatal care. While pregnant women are routinely screened for C. trachomatis and N. gonorrhoeae as part of standard antenatal care in Japan, M. genitalium is not included in standard screening protocols. Recent evidence has strengthened the association between M. genitalium and adverse pregnancy outcomes. A 2022 systematic review and meta-analysis by Frenzer et al. [13] found that M. genitalium infection may be associated with an increased risk of preterm birth, although the authors noted heterogeneity across studies. Additionally, a 2022 study by Jonduo et al. [14] demonstrated associations between genital mycoplasmas and various adverse pregnancy outcomes, though the authors acknowledged limitations in drawing definitive conclusions due to confounding factors. More recently, a 2025 study from Zambia by Schröder et al. [5] reported a 12.6% prevalence of M. genitalium among pregnant women, emphasizing the potential for adverse pregnancy outcomes and the importance of appropriate screening and treatment. A 2024 study by Scoullar et al. [16] examining M. genitalium in pregnancy specifically addressed co-infections with other STIs, noting that 18–35% of M. genitalium-positive individuals had concurrent STIs, with C. trachomatis being the most common. These collective findings support the consideration of broader screening protocols in antenatal care, particularly for populations with symptoms, risk factors, or in regions with high prevalence rates.

The co-infection rate between M. genitalium and C. trachomatis was relatively low in our study (0.2% in non-pregnant women and 0.4% in pregnant women, representing 7.3% and 11.1% of M. genitalium-positive cases, respectively). This observation suggests that while these infections share similar transmission routes and risk factors, they may be circulating somewhat independently within this community rather than strictly clustering in the same individuals. However, the clinical significance of even low rates of co-infection should not be underestimated, as co-infected individuals may require modified treatment strategies to address both pathogens effectively. The distinction between mono-infection and co-infection is important for clinical management and treatment selection. Notably, 55.6% (5/9) of N. gonorrhoeae-positive cases in the non-pregnant cohort occurred as co-infections with C. trachomatis, a pattern that is broadly consistent with reports from other clinical cohorts worldwide.

The clinical implications of our findings are substantial for Japanese healthcare practice. Currently, M. genitalium testing is not covered by the Japanese national health insurance system, creating significant barriers to both clinical diagnosis and epidemiological surveillance [8]. Given that M. genitalium prevalence in our study was comparable to that of C. trachomatis, which is routinely screened and reimbursed, our results support consideration of incorporating M. genitalium into targeted STI testing strategies, particularly for symptomatic women and selected antenatal care settings where local prevalence, clinical risk, and cost-effectiveness justify such an approach. The potential cost-effectiveness of such screening should be evaluated in future studies, considering the documented associations between M. genitalium infection and adverse reproductive outcomes, including cervicitis, pelvic inflammatory disease, preterm birth, and potential long-term sequelae such as tubal factor infertility [1,2,13]. Economic analyses incorporating direct medical costs, indirect costs related to complications, and quality-adjusted life years would provide valuable data to inform policy decisions regarding insurance coverage for M. genitalium testing.

Furthermore, the therapeutic implications of our findings cannot be overstated in the context of Japan’s alarming antimicrobial resistance rates. Current international guidelines, including those from the Centers for Disease Control and Prevention (CDC) and European guidelines, do not recommend routine screening for asymptomatic M. genitalium infection, but emphasize that testing should be considered in symptomatic patients, particularly when initial treatment for urethritis or cervicitis fails [17,18]. However, the syndromic management approach of treating urogenital symptoms with empirical azithromycin monotherapy—a common strategy for presumed chlamydial infections—may be inadequate for patients with M. genitalium infection. Recent surveillance data from Tokyo have documented macrolide resistance approaching 90% and rising fluoroquinolone resistance in M. genitalium isolates [10,11]. A comprehensive 2025 systematic review by Chua et al. [19] documented evolving patterns of macrolide and fluoroquinolone resistance in M. genitalium worldwide, with Japan identified as a region of particular concern. Most recently, a 2025 study by Kikuchi et al. [10] found a high prevalence of macrolide resistance-associated mutations and emerging fluoroquinolone resistance, with dual-resistant strains spreading in certain populations in Tokyo. This alarming resistance profile underscores the critical need for specific M. genitalium diagnostic testing followed by resistance-guided therapy to prevent treatment failures, reduce complications, and limit further transmission of resistant strains. The use of empirical azithromycin without documented M. genitalium testing may inadvertently contribute to the selection and spread of macrolide-resistant strains.

The issue of antimicrobial resistance in M. genitalium has become increasingly concerning globally and represents one of the most pressing challenges in STI management. A comprehensive 2025 systematic review by Chua et al. [19] documented evolving patterns of macrolide and fluoroquinolone resistance worldwide, with some regions experiencing dual-class resistance exceeding 10%. In Japan specifically, the situation is particularly dire. The work by Jensen and Unemo [20] highlighted that antimicrobial treatment and resistance in sexually transmitted bacterial infections, including M. genitalium, represent a growing threat to public health. The high resistance rates documented in Japan suggest that treatment protocols need urgent revision, with consideration of first-line therapies such as extended-course moxifloxacin (for quinolone-susceptible strains) or newer agents such as pristinamycin or lefamulin, where available. Ideally, resistance testing should be performed prior to treatment initiation, although practical and economic barriers currently limit widespread implementation of this approach in Japan.

This study has several important limitations that should be acknowledged. First, the relatively small sample size of pregnant women (n = 236) compared to the non-pregnant group (n = 2138) limits the precision of prevalence estimates in the obstetric population and reduces statistical power to detect modest differences between groups. The wide confidence intervals for prevalence estimates in pregnant women reflect this limitation. Second, as a laboratory-based retrospective study, detailed clinical correlates such as specific symptom profiles, symptom duration and severity, comprehensive sexual behavior history (including number of sexual partners, condom use, and history of previous STIs), and pregnancy outcomes were not available for individual-level analysis. In particular, quantitative age-stratified summaries and standardized symptom-status data were not consistently retrievable for all participants from the available dataset. Such information would have provided valuable insights into risk factors and clinical presentations associated with M. genitalium infection. Third, and most importantly, antimicrobial resistance testing was not performed in this dataset. Given the documented high rates of macrolide and fluoroquinolone resistance in Japan, future prospective studies should incorporate molecular resistance testing (detection of mutations in the 23S rRNA gene for macrolide resistance and parC gene for quinolone resistance) to guide appropriate treatment strategies and monitor resistance trends over time.

Additionally, our study population was derived exclusively from two gynecological clinics in Gifu city, which may limit the generalizability of our findings to other geographic regions of Japan or to populations accessing care through different healthcare settings such as public health centers, university hospitals, or specialized STI clinics. The prevalence estimates may not be representative of asymptomatic women in the general population, as the non-pregnant cohort primarily consisted of symptomatic patients or those identified through contact tracing. The pregnant cohort, while screened as part of routine antenatal care, may not represent the general pregnant population, as women who access prenatal care at private clinics may differ in socioeconomic status and health-seeking behaviors from those accessing care through public facilities. Finally, we did not collect detailed information on sexual behaviors, number of sexual partners, history of previous STIs, or other important risk factors that could have provided additional epidemiological insights and enabled risk stratification analyses. Future studies should incorporate comprehensive questionnaires to identify specific risk factors for M. genitalium infection among Japanese women and to characterize high-risk populations that may benefit most from targeted screening.

Despite these limitations, our study provides valuable contemporary data on M. genitalium prevalence in a substantial cohort of Japanese women and contributes important epidemiological information to the limited existing literature. The use of highly sensitive and specific nucleic acid amplification tests (Aptima™ TMA assays) enhances the reliability of our prevalence estimates. The inclusion of both pregnant and non-pregnant women allows for comparative analysis and provides data relevant to different clinical settings and screening scenarios.

In conclusion, M. genitalium was identified in 3.8% of both pregnant and non-pregnant women in this Japanese cohort from Gifu, a prevalence comparable to that of C. trachomatis and substantially higher than that of N. gonorrhoeae. The significant presence of M. genitalium warrants increased clinical awareness and incorporation of diagnostic testing into clinical algorithms for women presenting with urogenital symptoms. Our findings also support further investigation of whether selected antenatal care populations may benefit from expanded testing, rather than establishing a recommendation for universal routine screening on the basis of this study alone. Given the high antimicrobial resistance rates documented in Japanese M. genitalium strains, specific diagnostic testing is essential to enable targeted, resistance-guided therapy and prevent treatment failures that could lead to persistent infection, ongoing transmission, and serious reproductive health complications. Future research should focus on characterizing antimicrobial resistance patterns in M. genitalium isolates from Japanese women, evaluating the cost-effectiveness of expanded screening programs, investigating clinical outcomes and pregnancy complications associated with M. genitalium infection, and assessing optimal treatment strategies in the context of high-level antimicrobial resistance. Policy initiatives to include M. genitalium testing in national health insurance coverage should be pursued, given the growing evidence of its clinical significance and public health impact.