Role of Ureaplasma urealyticum and Ureaplasma parvum as Risk Factors for Cervical Dysplasia with Human Papillomavirus

Human papillomavirus (HPV) can be transmitted sexually and causes cervical malignancies in women. Among the risk factors, sexually transmitted infections (STIs) caused by bacteria have raised concerns because they are asymptomatic and persistent and can increase the risk of HPV infection. This study assessed the correlation among Ureaplasma urealyticum , Ureaplasma parvum, Chlamydia trachomatis, and Mycoplasma hominis infections towards abnormal cervical cells positive for HPV. Seventy outpatients at the Colposcopy Outpatient Clinic, Dr. Cipto Mangunkusumo Hospital in Jakarta, Indonesia, were enrolled in this cross-sectional study. Viruses and bacteria were detected using polymerase chain reaction and compared with liquid-based cytology results for cervical cytology. Of the 70 patients, 24 (34.28%) showed abnormal cervical cytology. Among those with abnormal cervical cytology, C.trachomatis was reported in 1 patient (4.2%), M.hominis in 6 patients (25%), U.urealyticum in 13 patients (54.2%), and U.parvum in 10 patients (41.7%). Statistical analysis demonstrated an association between U.urealyticum and U.parvum infections and HPV infection ( U.urealyticum p = 0.012; U.parvum p = 0.022). U.urealyticum and U.parvum were more prevalent than C.trachomatis and M.hominis in HPV-positive women, suggesting their role in HPV infection.


INTRODUCTION
Human papillomavirus (HPV) is an etiological agent of cervical malignancy in women. [1][2][3] HPV has been classified based on specific genomic regions, such as the E7, E6, and L1 protein-coding regions. More than 200 HPV types have been identified to date, 4,5 which have been grouped into low-risk HPV (LR-HPV) and high-risk HPV (HR-HPV) based on the malignant transformation of infected cells. 4,6 HR-HPV has oncogenic features and includes types 16,18,3 1,33,35,39,45,51,52,56,58,59,68,73 and 82. 2,4,7 Most HPV infections are cleared by cell-mediated immunity within 2 years of exposure. 1,4 However, certain types of HPV show persistent infections, such as HPV-16 and HPV-18. 2,4,8 Long-term persistence of HR-HPV causes a decline in viral clearance and indicates the change of cervical cells to pre-cancerous forms or even cancer. 4,8 Besides the persistence of HPV infection, other risk factors also play an essential role in the progression of cervical precancer and cancer, including the early onset of sexual activity, high parity, smoking, hormonal contraception, immunosuppression, and bacterial sexually transmitted infections (STIs). 9,10 Among these, bacterial STIs have raised concern because they are asymptomatic and persistent and can increase the risk of HPV infection. 10-12 Several bacterial STIs have been reported as risk factors, including Ureaplasma urealyticum,Ureaplasma parvum, Chlamydia trachomatis, and Mycoplasma hominis. 3,[12][13][14] A study showed that C.trachomatis infection was reported more frequently in HPV-positive women than that in HPV-negative women. 3,14,15 A study on genital mycoplasma infections and cervical cytology abnormalities showed that M.hominis and U.urealyticum were found in samples with abnormal cervical cytology. [16][17][18] There are no established data from Indonesia showing a correlation among U.urealyticum,U.parvum,C.trachomatis, and M.hominis infections with abnormal HPV-positive cervical cytology. Therefore, a cross-sectional study was conducted to assess the relationship among U.urealyticum, U.parvum, C.trachomatis, and M.hominis infections and abnormal HPVpositive cervical cytology.

Patient and Specimen Collection
This was a cross-sectional study with a minimum sample size of 30 participants. Seventy women were recruited from the Colposcopy Outpatient Clinic of Dr. Cipto Mangunkusumo Hospital, Jakarta, Indonesia, between June 2016 and June 2017. Respondents were recruited through consecutive sampling. All participants were sexually active, 20-67 years old, nonpregnant, and provided written informed consent prior to enrollment in the study. An obstetrician obtained a cervical sample and placed it in a Liqui-PREP liquid-based cytology (LBC) vial (LGM International Inc., Melbourne, FL, USA). The sample was subsequently divided into 5 mL portions for cytological examination and polymerase chain reaction (PCR). The samples were stored at a temperature of 4°C and were processed within 2 days of collection.

Liquid-Based Cytology Pap Smear
Liquid-based cytology Pap smears were performed based on the corporation's protocols under the supervision of a pathologist. 19 The cervical cytology was categorized using the 2014 Bethesda system: high-grade squamous intraepithelial lesions (HSIL); low-grade squamous intraepithelial lesions (LSIL); atypical squamous cells, cannot exclude HSIL (ASCH); atypical squamous cells of undetermined significance (ASCUS); and normal cytology or negative for intraepithelial lesion or malignancy (NILM). 20

DNA Extraction
A 1-mL aliquot of a 5-mL LBC sample was centrifuged (Sorvall Biofuge Primo R, Thermo-Fisher Scientific Inc., Waltham, MA, USA) for 30 min. at 13,000 rpm. The pellet was then resuspended in 200µL phosphate-buffered saline after removing the supernatant. DNA isolation was conducted with a High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany), based on the corporation's protocols, with 50µL of final elution volume. 21 Purified DNA was kept at -20 o C for ≤1 week prior to being used for PCR.

DNA Positive Control
A positive control was obtained from the

PCR for Detecting U. urealyticum, U.parvum, C.trachomatis, and M.hominis
Conventional duplex PCR was used to identify U.urealyticum, U.parvum, C.trachomatis, and M.hominis. Duplex PCR was carried out twice using primers according to previous studies, with modifications. 24

Statistical Analysis
Data were inputted into Microsoft Excel 2019 v16.0 (Microsoft, Redmond, WA, USA) and subsequently analyzed using IBM SPSS Statistics for Windows (version 20.0; IBM Corp., Armonk, NY, USA). A comprehensive descriptive analysis was conducted to generate summary statistics, including mean, median, standard deviation, and

DISCUSSION
HPV infection has been reported as the primary etiological agent of precancerous and cancerous lesions on the cervix. 2,5 Several studies have demonstrated that other risk factors also play a role in cervical precancerous and cancerous lesion development, such as early onset of sexual activity, high parity, smoking, hormonal contraception, immunosuppression, and STIs other than HPV. 9,10,28 According to the current study, there was no association between early onset sexual activity, high parity, smoking, contraception, or immunosuppression, and cervical precancerous lesions (Table 1). Pap smear results showed that 24 of 70 patients had abnormal cervical cytology, classified as ASCUS (37.5%), ASCH (20.89%), LSIL (8.33%), HSIL (29.17%), or cervical carcinoma (4.2%). Of the 24 patients with abnormal cervical cytology, 11 (45.8%) were positive for HPV. Abnormal cervical cytology with negative HPV was predominantly found in atypical squamous cell types (ASCUS and ASCH), whereas abnormal cervical cytology with positive HPV was predominantly found in HSIL 45.5% (5,11). The current study revealed a significant correlation between HPV infection and cervical dysplasia (p = 0.007; Table 2).HPV is a sexually transmitted infectious agent that plays an important role in cervical carcinogenesis. Approximately 90% of cervical cancer cases are dominated by HPV infection. 4,5,8,29 Primary viral E6 and E7 oncoproteins are produced as the malignancy progresses by integrating the HPV genome into host cells. Their overexpression aids in the development of cervical carcinoma by impairing the function of the tumor suppressor p53 and degrading the retinoblastoma protein, respectively. 30 A limitation of the current study was that HPV genotyping was not performed; therefore, there was no differentiation of HR and LR-HPV infection.
Although HPV is the primary causative agent of precancerous and cancerous lesions of the cervix, other STIs have been discovered to play a role in the pathogenesis, although the specific mechanisms remain unclear. 3,10,11 C.trachomatis and M. hominis increase the susceptibility of cervical cells to HPV infection, which could be implicated in cervical dysplasia. 3,12 The current study showed that there was a significant correlation between abnormal cervical cytology and M.hominis (p = 0.037), while C.trachomatis, U.urealyticum, and U.parvum showed no significant association. The results for C.trachomatis were consistent with those of another study. 31 By contrast, another study showed a significant association between U. urealyticum and U. parvum with abnormal cervical cytology. 18,32,33 M.hominis was found predominantly in cervical dysplasia (6 out of 9 positive patients) and predominantly in HSIL. Cervical dysplasia development appears to be influenced by M.hominis by initiating inflammation of cells and might be involved in increasing cervical cell susceptibility to HPV infection. 34,35 Furthermore, establishing persistent infections could promote abnormal cell growth by altering host cell life cycles, elevating the inflammatory cytokines linked to DNA breakdown, and affecting apoptotic pathways. [34][35][36] The direct relationship between HPV and M.hominis in cervical cytology is shown in Figure 2, which also shows a significant association between U.urealyticum and U.parvum with HPV infection, with p-values of 0.012 and 0.022, respectively, whereas other organisms showed no association (Table 3).U.urealyticum and U.parvum are commensal and pathogenic bacteria found in the human urogenital tract. 11,13,18 U.urealyticum and U.parvum play a role in HPV infection, implying an association with cervical dysplasia. 12,34,37 Previous studies have shown similar results to those of the current study. 11,12,32,37 Ureaplasma infections are implicated in increased susceptibility of inflamed cervical cells to HPV infection and persistence. 3,32 U.urealyticum and U.parvum infection could suppress cell-mediated immunity by affecting the balance between cellular and humoral immune responses, which could affect the protection of cells against HPV infection. 3 Furthermore, U.urealyticum stimulates the expression of HPV oncoprotein E6 mRNA by 4.8 fold in SiHa cells. 13 This suggested that U.urealyticum plays a role in the pathogenesis of cervical precancerous and cancerous lesions by increasing the expression of oncoprotein E6. Figure 2 shows an indirect relationship among U.urealyticum, U.parvum, and cervical cytology through HPV infection.

CONCLUSION
This study demonstrated an association among U.urealyticum, U.parvum, and HPV infection. U.urealyticum and U.parvum presumably play roles in HPV infection and persistence, which can lead to cervical dysplasia. In addition, M.hominis was associated with abnormal cervical cytology. However, further studies need to be conducted with larger populations to reveal the role of bacterial coinfection with high-risk HPV and to serotype U.urealyticum and U.parvum.