Trichomonas vaginalis infection and risk of cervical neoplasia: A systematic review and meta-analysis

Objectives The evidence in the literature regarding the relationship between Trichomonas vaginalis (TV) infection and cervical neoplasia is conflicting. The main aim of this study was to evaluate the magnitude of the risk of cervical neoplasia associated with TV infection. Methods A meta-analysis of observational studies, which provided raw data on the association of TV infection with cervical neoplasia, was performed. For this aim, we searched scientific databases (PubMed/Medline, Scopus, the Web of Sciences, and Embase) from inception to March 15, 2023. A random-effects model was applied by Stata 17.0 to calculate the pooled and adjusted odds ratios (ORs) with 95% confidence intervals (CI), including subgroup, sensitivity, and cumulative analyses to explore sources of heterogeneity. Results Of the 2584 records initially identified, 35 eligible studies contributed data for 67,856 women with cervical neoplasia, and 933,697 healthy controls from 14 countries were included. The pooled (2.15; 1.61–2.87; I2 = 87.7%) and adjusted (2.17; 1.82–2.60; I2 = 31.27%) ORs indicated a significant positive association between TV infection and the development of cervical neoplasia. There was no significant change in pooled and adjusted ORs by applying sensitivity and cumulative analyses, indicating the robustness of our findings. The pooled OR was significant in most sub-group analyses. There was no publication bias in the included studies. Conclusion Our findings indicated that women with a TV infection are at significantly greater risk of cervical neoplasia. Future research, particularly longitudinal and experimental studies, should be done to better understand the various aspects of this association.

Introduction the women are asymptomatic, which affects women's health outcomes [18]. Vaginal, cervical, urethral, and pelvic inflammation and pregnancy complications such as ectopic pregnancy, preterm delivery, and infertility are the complications that can be caused by TV infection [19,20]. Although the relationship between TV infection and the development of cervical cancer is still not entirely understood, it is thought that the inflammatory response is the key factor [21,22]. Inflammation is thought to make it easier for HPV to enter the epithelium's basal layer. Thus, viral DNA is incorporated into the host genome. Moreover, TV infection could overexpress viral tumorigenesis, which helps activate cancer-causing pathways [23].
There are several clinical and observational studies evaluating the association between TV infection and cervical neoplasia [24][25][26][27][28][29]. A significant association has been shown in a combined analysis of 24 studies, which most of them were case reports or case series [30]; moreover, a meta-analysis of 17 observational studies in 2018 demonstrated a positive association between TV and the development of cervical cancer [31]. In this study, we determined whether the results of the new studies added had any impact on the results of the previous meta-analysis. As a result, in order to acquire a more in-depth understanding of the potential connection between TV infection and cervical neoplasia, we updated the previous meta-analysis.

Materials and methods
The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) (S1 Checklist) and the Meta-analysis of Observational Studies in Epidemiology (MOOSE) reporting guidelines were followed for this systematic review and meta-analysis [32,33]. In this study, cervical neoplasia was defined as cervical intraepithelial neoplasia ranging from atypical squamous cells of undetermined significance (ASC-US) to invasive cancer confirmed by histopathological methods.

Search strategy and study selection
Two researchers (A.F.N. and K.B.) did a comprehensive systematic search in international scientific databases, including PubMed/Medline, Scopus, Web of Science, and Embase, for all peer-reviewed, published studies assessing the relationship between TV infection and cervical neoplasia from database inception to March 15, 2023. We did not use any language, geographic, or date restrictions. For database searches, the following Medical Subject Headings (MeSH) terms and keywords were combined using Boolean operators (i.e., "OR" and/or "AND"): "Trichomonas vaginalis", "Trichomonas vaginitis", "Trichomonas infections", "Trichomoniasis", "Trichomonas", "sexually transmitted infection", "cervical cancer", "cervical carcinoma", "cervical neoplasm", "cervical abnormality", "cervical dysplasia", "cancer of the cervix", "carcinoma of the cervix", and "cervical intraepithelial neoplasia" (S1 Fig). Additionally, the first 20 pages of Google Scholar and the reference list of relevant studies, including meta-analyses, were reviewed to ensure that no publications, such as gray literature, were missed. 'Google Translate' was used to translate articles from other languages into English. Duplicate citations were removed after exporting by EndNote Reference Manager X8.

Inclusion and exclusion criteria
After deleting duplicates, three trained researchers (K.B., K.B.M., and M.J.T.) independently screened the titles and abstracts and also evaluated the full-text of articles for eligibility. Ambiguous articles were examined by the lead investigator (AR). All the inconsistencies were resolved through discussion. Studies were eligible for inclusion if they investigated the correlation between TV infection and cervical neoplasia using a cross-sectional, cohort, or case-control design; gave a clear explanation of the methods for diagnosis of TV infection and cervical neoplasia; and had sufficient data to calculate the odds ratio (OR) or relative risk (RR) with 95% a confidence interval (95% CI). Studies that evaluated the association following the treatment, conference abstracts, reviews, systematic reviews, editorials, case reports, case series, and papers with not available full-text were excluded. In this meta-analysis, if multiple studies reported on the same population or datasets, the most recent or comprehensive study was chosen for inclusion.

Data extraction and quality assessment
Three independent investigators (A.F.N., S.R.T., and M.S.) performed data extraction from each relevant study. Consulting with the lead investigator (A. R.) helped resolve any disputes. The following information was extracted (if available): first author's name, publication year, country, geographic region, type of study, diagnostic method for TV infection, number of women with cervical neoplasia as cases, number of women without cervical neoplasia as control group, number of infected women in case and control groups. Where available, adjusted ORs were extracted and used for meta-analysis to reduce confounding, as recommended by the Cochrane Handbook [34]. According to extractable data in included studies, we categorized cervical neoplasia into the following categories: (1) ASC-US; (2) ASC-H; (3) LSIL/CIN1; (4) HSIL/CIN2/CIN3; and (5) invasive cancer. The risk of bias for each study was independently assessed by two investigators using the Joanna Briggs Institute (JBI) Critical Appraisal Tools for cross-sectional, case-control, and cohort studies, and each study was classified as having a low, moderate, or high risk of bias, as recommended for these tools [35-37].

Data synthesis and statistical analysis
Meta-analysis was performed with Stata software version 17 (STATA Corp., College Station, TX, USA). For all tests, two-sided P-values were determined, with P-values less than 0.05 considered statistically significant. Summary pooled and adjusted ORs and related 95% confidence intervals (CIs) were calculated using the DerSimonian-Laird random-effects model (REM) for combining results across studies, which incorporates between-and within-study variance [38]. We used REM because heterogeneity was expected. Cochran's Q test and I 2 statistics were applied to measure between-study heterogeneity. An I 2 value more than 75% was considered significant heterogeneity [39]. We also used subgroup and meta-regression analyses to determine the heterogeneity source. For this purpose, subgroup analyses were performed according to publication year (before 2005, 2006 to 2015, and 2016 to 2023), type of study (cross-sectional, case-control, and cohort studies), diagnostic method of TV infection (polymerase chain reaction (PCR), cytology, microscopy, and other), WHO-defined geographical region (Middle-East and Africa, Europe and North America, South America, Western Pacific, and South-East Asia), country's income level (high, upper middle, and lower middle), and risk of bias for studies (low and moderate). Sensitivity analyses were performed by excluding individual studies to examine the robustness of the results and the effect of each study on the estimated ORs. Cumulative meta-analyses were applied to determine the reliability of the estimated ORs. Publication bias was evaluated using the Egger test and visual inspection of funnel plots [40].

Study selection and characteristics
As shown in the PRISMA flow diagram (Fig 1), our initial search in databases, Google Scholar, and bibliographies of relevant studies identified a total of 2584 records. Following the removal of 865 duplicates, an extra 1626 records were also excluded after reviewing their titles and abstracts. Ninety-three studies were eligible for full-text review. Of these, 58 articles were excluded due to missing crucial data or failing to meet the study's inclusion criteria, thus leaving a total of 35 eligible articles [22][23][24][25][26][27][28][29] Based on methodological quality, 12 and 23 studies were categorized as moderate and low risk of bias studies, respectively ( Table 1). The main characteristics of the included studies are summarized in Table 1. Considering stages of cervical neoplasia, extractable data were available for ASC-US in seven studies, for ASC-H in four studies, for LGSIL/CIN1 in 14 studies, for HGSIL/CIN2/CIN3 in 17 studies, and for invasive cancer in nine studies.

Synthesis of results
As shown in  Fig 2). Employing the Egger's regression test and Funnel plot, we found there is no publication bias in the studies included

Discussion
Cervical cancer incidence is rising quickly, and cervical cancer deaths are anticipated to be almost 0.5 million by 2030 globally [68]. Accordingly, this cancer is a serious health concern worldwide. Traditional treatments, such as chemotherapy, radiotherapy, and surgery, can be given for cervical cancer at an early stage, but these treatments are not efficient for late-stage cancer [69]. The recurrence rate of cervical cancer after surgery in CIN 2+ is 5-10% at 5 years [70][71][72]. Additionally, these treatments are unsafe. An increased risk of preterm delivery, lower birth weight, and preterm premature rupture of the membrane before 37 weeks of pregnancy have been shown to be linked with surgical treatment of the CIN [73]. As a result, despite major therapy advancements, cervical cancer mortality remains high, and the 5-year survival rate for women with advanced-stage cervical cancer is just 17% [74,75]. These findings show the importance of finding gaps in cervical cancer risk factors to prevent this cancer, which has a high mortality rate.
In the present updated systematic review and meta-analysis of the association between TV infection and cervical neoplasia, we found that women with TV infection had a significantly higher risk of developing cervical cancer. This significant correlation was found in most subgroup analyses, including cohort and case-control studies, most geographical regions, all time periods, and studies with both moderate and low risk of bias. Consistent with our findings, a Infection with TV could also be related to the development of other types of urogenital abnormalities, including prostate and bladder cancers [76,77]. A previous meta-analysis investigated the correlation between prostate cancer and TV infection as well and found that patients with TV infection had a 1.17-fold higher risk of prostate cancer, indicating the potential role of TV infection in the development of different types of cancer [77]. Our results are consistent with those of other meta-analyses, which also found that other STIs play a significant role in cervical cancer development. For example, Chlamydia trachomatis infection was significantly correlated with cervical cancer in meta-analyses [78][79][80]. In another study, genital mycoplasma infection was reported to be linked to HPV-related cervical neoplasia. They demonstrated that Mycoplasma genitalium and Ureaplasma urealyticum were correlated with an increased risk of infection with high-risk HPV viruses. A significantly elevated risk of abnormal cervical cytopathology has also been linked to Ureaplasma urealyticum, Ureaplasma parvum, and Mycoplasma hominis [81]. It has also been shown that HIV infection is linked to slower clearance and higher HPV infection rates. As a result of increased HPV persistence in HIV patients, higher rates of LSIL and HSIL and an increased incidence of cervical cancer were observed [82]. Even though epidemiological studies found a positive correlation between HSV-2 infection and cervical cancer risk, subgroup analysis in cohort studies did not find a significant association between HSV-2 infection and cervical cancer [83]. These findings indicate that cervical cancer progression is influenced by STIs other than HPV. The role and mechanism of these infections in cervical cancer need to be studied further.
Although the exact mechanisms by which TV causes cervical cancer are unknown, it seems likely that it is caused by a combination of factors that work together in a complicated way. Several studies have found molecular links between TV infection and cervical cancer progression. Chronic inflammation of the cervix is one possible mechanism by which TV contributes to cervical cancer. This leads to immune cell-attracting cytokines and chemokines, increased production of reactive oxygen species, which damage DNA, and genetic mutations that raise the risk of cancer development [19, [84][85][86]. TV infection is associated with the development of high-grade CIN due to damage and alterations to the cervical epithelium, which create a conducive environment for the organism's proliferation. The immune response generated against the invading organism involves the recruitment of numerous leukocytes, leading to inflammation, which can promote DNA damage and mutations [11,87]. Besides, TV generates cytotoxic chemicals that stimulate epithelial atypia and dysplasia, such as cell-detaching factors and Nnitrosamines. The organism thrives on tissue debris and serous exudate and produces significant tissue damage [88]. The pH level in the vagina also rises during TV infection, which further creates a favorable environment for the organism to proliferate [89]. In addition, TV can interact with HPV, a known risk factor for cervical cancer. The parasite can enhance HPV's ability to infect host cells and promote the persistence of the virus in infected cells, which can increase the risk of cervical cancer [22,23]. Another proposed mechanism is through nitric oxide (NO). The parasite can induce the production of NO by neutrophils in the cervix, which can cause DNA damage and promote the growth of abnormal cells. This can lead to precancerous lesions and eventually cervical cancer. Moreover, HPV infection has also been shown to induce NO release, which may contribute to cervical cancer [90,91].
This study has important strengths. By including 35 studies that examined 67,856 women with cervical neoplasia and 933,697 healthy controls and applying several subgroup, sensitivity, and cumulative analyses, this study provides the most up-to-date and comprehensive evidence considering the relationship between TV infection and the development of cervical neoplasia. We found no significant change for the pooled and adjusted ORs in sensitivity and cumulative analyses, which indicates the robustness of our findings. This study also has some limitations. Despite our comprehensive search, it is likely that some studies-mostly those that were published in non-indexed local journals-were overlooked. The information for potentially relevant confounding factors such as age, alcohol and tobacco use, high-risk behavior in sexual activity, and co-infection with HPV, HIV, and other STIs were not available or consistently reported in most studies; therefore, we were unable to extract and analyze these covariates. Nevertheless, we analyzed adjusted ORs reported in 13 studies, so this limitation has been addressed to a large extent. Most studies were cross-sectional or case-control, and there were only five cohort studies. Therefore, it is difficult to draw conclusions about the natural history of TV infection and cervical neoplasia due to the lack of longitudinal data. We found substantial heterogeneity in most of the meta-analytic estimates, although low non-significant heterogeneity was recorded for cohort studies (I 2 = 16%) and adjusted ORs (I 2 = 31%). Variation in diagnostic methods for the diagnosis of TV infection, definition and stages of cervical neoplasia, geographical region, country's income levels, and publication year are likely to have contributed to the heterogeneity (Table 3).

Conclusion
This updated systematic review and meta-analysis confirmed findings of the previous metaanalysis and indicated that women with TV infection are at a higher risk of cervical neoplasia. Future research, particularly longitudinal and experimental studies, should be done to better understand various aspects of this association. Moreover, more investigation is needed to determine how TV interacts with other infectious agents and environmental covariates that may predispose women to cervical neoplasia, as well as to identify amenable factors and effective interventions that can be addressed by the public health system.