Comprehensive assessment of liquid-based cytology and molecular markers (HPV, microRNA, STD) from a single specimen in the diagnosis of cervical intraepithelial neoplasia

Objective. To demonstrate the feasibility of a comprehensive assessment of molecular and cytological parameters from a single specimen (preserved in a stabilizing liquid medium), and to identify patterns of association between these parameters and the degree of cervical intraepithelial neoplasia.

Materials and methods. A retrospective multicenter study was conducted involving 79 female patients of reproductive age (decreed cohorts aged 21, 24, and 27 years old) who underwent cytological screening (PAP test) in medical institutions in Gomel and Gomel Region in February–March 2026. The biomaterial was analyzed using liquid-based cytology (LBC) (the Bethesda System, TBS 2014). From the residual volume of the same specimen, genotyping for 22 HPV types (real-time PCR), testing for sexually transmitted diseases (STD) (Ureaplasma spp., Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium), and quantitative analysis of the expression of six marker microRNAs (miR-21, miR-29b, miR-145, miR-451a, miR -1246, miR -1290) were performed, with calculation of the diagnostic parameter miR-CERVIX. Statistical analysis included the Kruskal-Wallis test, Spearman’s correlation analysis, and Fisher’s exact test.

Results. A statistically significant dependence of microRNA expression levels on the degree of cytological changes was established (p<0.001). The median miR-CERVIX values were: for NILM – 0.33 [0.15; 0.46], for LSIL – 0.61 [0.56; 0.64], and for HSIL – 0.69 [0.57; 0.77]. These values were significantly higher when comparing NILM with the LSIL and HSIL groups (p<0.001). In the HSIL group, a high-oncogenic risk HPV was detected in 100% of cases, with HPV type 16 dominating (either isolated or in combination). HPV multi-infection was more frequent in the LSIL and HSIL groups. Correlation analysis confirmed a statistically significant dependence (p<0.001) of microRNA expression levels on the degree of cytological changes (NILM, LSIL, HSIL).

High variability of the miR-CERVIX index was detected in the NILM group (0.01–0.74), indicating subclinical molecular changes and justifying extended work-up in patients with values ≥0.60. Furthermore, the miR-CERVIX index allows personalization of management strategy in the LSIL group: values <0.60 are associated with a favorable outcome, whereas values ≥0.75 indicate a risk of progression.

Conclusion. The comprehensive assessment of the liquid-based cytology and molecular markers from a single sample prevents preanalytical errors, ensures high data comparability, and optimizes the clinical pathway, thereby eliminating the need for additional visits for specimen collection. The miR-CERVIX index is a sensitive marker of the CIN degree. It enables differential risk stratification within the NILM and LSIL groups, whereas in the HSIL group it serves as an objective confirmation of the molecular aggressiveness of the lesion. A comprehensive approach including the assessment of HPV status and microRNA profile forms a methodological basis for optimizing diagnostic triage and requires further prospective validation with histological confirmation.

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