Mario Poljak

A substantial proportion of cervical intraepithelial neoplasia grade 2 (CIN2) spontaneously regress, supporting the use of active surveillance over immediate treatment. Identification of prognostic factors associated with higher rates of spontaneous regression could enable better selection of patients in whom such approach would be safe. We evaluated the prognostic value of FAM19A4/miR124‐2 methylation status, HPV genotype, and baseline cytology for predicting spontaneous regression of CIN2. Women aged 18–40 years with CIN2 were prospectively enroled and followed for 2 years. At baseline, HPV genotyping was performed together with FAM19A4/miR124‐2 methylation analysis. Among 119 enroled patients, the rate of spontaneous regression of CIN2 was 61.3% (73/119). Regression of CIN2 was significantly associated with HPV genotype ( χ ² = 19.374; p = 0.002) and negative methylation result ( χ ² = 10.945; p = 0.012). Negative methylation in combination with low‐grade cytology and non‐HPV16 genotype was associated with regression rates of 71.4% and 70.4%, respectively. Spontaneous regression of CIN2 is more frequent in women with non‐HPV16 infection and negative FAM19A4/miR124‐2 methylation. A combination of these markers improves identification of women with lower risk of disease progression and supports safer implementation of active surveillance and conservative management. Clinical Trial Registration: The study was registered at ClinicalTrials.gov as NCT05624827.

Human papillomavirus (HPV) assays vary regarding the minimum amount of virus they detect. We investigated analytical thresholds of HPV detection and cervical screening sensitivity and specificity. One hundred cervical intraepithelial neoplasia grade 2 or worse (CIN2+) cases and 200 matched population‐based controls were obtained at the Swedish National HPV Reference Laboratory and analyzed by 10 laboratories across 10 countries. Cumulative sensitivity (weighted according to the global HPV type distribution in invasive cervical cancer (ICC)) and specificity were estimated at varying analytical detection thresholds. Consensus results found HPV in 99/100 CIN2+ cases and 52/200 controls. HPV16 prevalence declined in HPV‐vaccinated birth cohorts, among both cases and controls. Line plots of 1‐specificity and ICC‐weighted sensitivity found optimal analytical detection thresholds as 3 International Units (IU)/µl for HPV16/18, 25 IU/µl for HPV31/33/35/45/52/58 and 100 genome equivalents (GE)/µl for HPV 39/51/56/59 resulting in 92.00% cumulative specificity and 90.08% ICC‐weighted sensitivity. Thresholds defined using virus amounts per 10 ⁴ human cells gave similar results. Comparator assay testing using manufacturer‐defined thresholds achieved high ICC‐weighted sensitivity (96.61%) but low specificity (82.50%). This international collaborative study has identified HPV analytical detection thresholds optimizing the sensitivity and specificity of cervical screening.

Adenomyosis is a chronic gynecological condition affecting a substantial portion of women of reproductive age. With symptoms including abnormal uterine bleeding, chronic pelvic pain (CPP), dysmenorrhea, and infertility, poor response to symptomatic treatment and unfavorable outcomes of assisted reproductive technologies, it remains a significant diagnostic and therapeutic challenge. Here, we analyzed microbial compositions of the reproductive tract of women with adenomyosis ( n = 33) in comparison with healthy controls ( n = 31). Vaginal, cervical, and endometrial samples were collected using minimally invasive transcervical sampling techniques on the 22nd day of the menstrual cycle, during the so-called window of implantation. 16S rRNA was amplified and recorded using the next generation sequencing. Bioinformatic and statistical analysis focused on quantitative taxonomical characterization of the specimens’ microbiomes. Vaginal bacterial microbiome composition was consistent across the three anatomical sites. Compared with the control group, adenomyosis was associated with Lactobacillus iners, whereas Lactobacillus gasseri and Gardnerella vaginalis were negatively associated with adenomyosis and its clinical symptoms. G. vaginalis , typically considered a pathogen, was highlighted as an important dominant microbiome replacement for lactobacilli, more so in healthy women than in women with adenomyosis. Anaerococcus prevotii , Peptoniphilus grossensis, and Peptrostreptococcus anaerobius also showed weak correlation to adenomyosis. Differences in taxa abundance were detected in association with adenomyosis clinical symptoms. L. iners was associated with dysmenorrhea, heavy menstrual bleeding (HMB), as well as CPP. Prevotella disiens, Prevotella timonesis, and Dialister micraerophilus were associated with dysmenorrhea and Peptoniphilus grossensis with HMB, respectively. L. gasseri and L. jensenii appeared to anticorrelate with these symptoms. IMPORTANCE: Adenomyosis poorly responds to treatment and assisted reproductive technologies. Here, we report a comprehensive 16S rRNA-based analysis of vaginal, cervical, and endometrial samples, obtained minimally invasively (transcervically) in a cohort of Caucasian women during the receptive phase of endometrium. Results revealed the least invasive option, vaginal microbiome sampling, reliably predicts the microbiome compositions of cervix and endometrium. We showed substantial variation in microbial composition of adenomyosis patients. L. iners , a species with specific functional traits, was consistently associated with adenomyosis presence and related symptoms. This finding suggests microbiome remodeling as a viable novel therapeutic option for adenomyosis. Furthermore, our findings indicate that the pathogenic role of G. vaginalis may be context-dependent. Ongoing genomic and ecological profiling is essential to clarify Gardnerella's dual commensal-pathogenic nature. Previous adenomyosis studies have mostly focused on the vaginal microbiome, whereas the endometrial microbiome has rarely been studied and never in the time of window of implantation.

Background/Objectives: To assess overall and type-specific HPV vaccine effectiveness in central and eastern Europe (CEE), the age-stratified prevalence of cervical HPV infection was determined among Slovenian women aged 20 to 64 attending a cervical cancer screening program 14 years after implementation of a national HPV vaccination program, which was then compared with 2009–2010 pre-vaccination data using the same methodological approach. Methods: Cervical samples of 4419 women were tested in 2023–2025 using the clinically validated Alinity m HR HPV Assay, and individual HPV types were determined by the Allplex HPV HR Detection assay. Results were compared with 2009–2010 pre-vaccination data generated using the same assay on an age-range matched cohort of women. Results: The overall prevalence of the 14 Alinity-targeted HPV types was 10.0% in 2023–2025 versus 13.3% in 2009–2010 (p < 0.001). HPV16 prevalence declined from 3.5% to 1.5% (p < 0.001), and HPV18 prevalence from 1.1% to 0.5% (p = 0.005). In women aged 20 to 24 with 40% uptake of quadrivalent HPV vaccine, overall HPV prevalence dropped from 25.3% to 12.8% (p < 0.001). No single case of HPV16/HPV18 infection was detected among vaccinated women. Conclusions: The first large-scale, systematic, and methodologically consistent study of HPV vaccine effectiveness in CEE showed a substantial reduction in high-risk HPV prevalence after implementation of the national program, with the greatest decline among women aged 20 to 24, who harbored the highest HPV burden in the pre-vaccination era. These locally acquired data will considerably inform public health strategies on cervical cancer elimination in CEE.

Recurrent respiratory papillomatosis (RRP) is caused by human papillomaviruses (HPV) 6 and 11, but the role of their genomic variants in the disease's clinical course is unclear. This study investigated whether long‐term persistence of a particular HPV genotype, subtype or genomic variant influences the RRP clinical course. HPV genotyping was performed in paired baseline and follow‐up RRP laryngeal tissue specimens of 59 patients. HPV6 and HPV11 genomic variants were determined in paired tissue specimens taken at least 10 years apart in 20 selected patients. HPV was identified in 58/59 patients, most commonly HPV6 (40/58), followed by HPV11 (17/58). The most prevalent HPV genomic variant was HPV11 A2. HPV6 A and HPV6 B1 were most frequent in aggressive RRP. In all patients, identical HPV genomic variants were identified in both paired specimens. RRP results from a long‐term infection with the same HPV genomic variant that can be identified decades after disease onset. We report the longest duration of genetically confirmed persistent HPV infection in peer‐reviewed literature, during a 44‐year interval in a patient with HPB6 B1. This study suggests that infection with a particular HPV genotype, subtype, or genomic variant does not significantly influence the clinical course of RRP.

Molecular testing for human papillomaviruses (HPV) is gradually replacing cytology in cervical cancer screening. In this longitudinal population‐based cohort study, 4140 women 20 to 64 years old attending organized screening were tested at baseline by five different screening methods and followed for 9 years. To assess long‐term safety, the cumulative risks of CIN2+/CIN3+ were estimated after a negative baseline result obtained by conventional cytology and four clinically validated HPV assays: Hybrid Capture 2 (hc2), RealTime High Risk HPV assay (RealTime), cobas 4800 HPV Test (cobas_4800), and Alinity m HR HPV (Alinity). HPV‐negative women at baseline had a substantially lower risk for CIN2+ compared to those with normal baseline cytology: 0.84% (95% CI, 0.46–1.22), 0.90% (95% CI, 0.51–1.29), 0.78% (95% CI, 0.42–1.15), and 0.75% (95% CI, 0.39–1.11) for hc2, RealTime, cobas_4800, and Alinity, respectively, compared to 2.46% (95% CI, 1.88–3.03) for cytology. No differences were observed between HPV assays in longitudinal sensitivity (range: 86.21%–90.36%) and negative predictive values (range: 99.54%–99.70%) for CIN2+ in women ≥30 years, but were significantly different from cytology (p < .05). The 9‐year cumulative risk of CIN2+ differed significantly between HPV genotypes, reaching 32.1% (95% CI, 14.5–46.1) for HPV16, 24.9% (95% CI, 4.7–40.8) for HPV18/45, 27.2% (95% CI, 14.6–37.8) for HPV31/33/35/52/58, and 8.1% (95% CI, 0.0–16.7) for HPV39/51/56/59. Four clinically validated HPV assays showed comparable safety and better assurance against precancerous lesions than cytology, but some important differences were identified in the performance characteristics of HPV assays impacting the referral rate. Information about the HPV genotype is valuable for guiding further clinical action in HPV‐based screening programs.

While HC2 and GP5+/6+ PCR‐EIA were pivotal in test validation of new HPV assays, they represent the first generation of comparator tests based upon technologies that are not in widespread use anymore. In the current guideline, criteria for second‐generation comparator tests are presented that include more detailed resolution of HPV genotypes. Second‐generation comparator tests should preferentially target only the 12 genotypes classified as carcinogenic (IARC‐group I), and show consistent non‐inferior sensitivity for CIN2+ and CIN3+ and specificity for ≤CIN1 compared to one of the first‐generations comparators, in at least three validation studies using benchmarks of 0.95 for relative sensitivity and 0.98 for relative specificity. Validation should take into account used storage media and other sample handling procedures. Meta‐analyses were conducted to identify the assays that fulfill these stringent criteria. Four tests fulfilled the new criteria: (1) RealTime High‐Risk HPV Test (Abbott), (2) Cobas‐4800 HPV test (Roche Molecular System), (3) Onclarity HPV Assay (BD Diagnostics), and (4) Anyplex II HPV HR Detection (Seegene), each evaluated in three to six studies. Whereas the four assays target 14 carcinogenic genotypes, the first two identify separately HPV16 and 18, the third assay identifies five types separately and the fourth identifies all the types separately.

Enterovirus (EV) infections have various symptoms and severe complications, including death. To determine EV prevalence and EV types in Slovenia, data on over 25 000 EV RNA tests for diagnostics and surveillance from 2014 to 2023 were analyzed. Altogether, 3733 cerebrospinal fluid (CSF) and 21 297 respiratory (sentinel and clinical) samples were tested for EV RNA. EV typing was performed on all residual EV‐positive CSF samples and on subset of respiratory specimens. Altogether, 1238 samples tested positive for EV RNA: 238 (6.4%) CSF and 1000 (4.7%) respiratory samples. EV‐positive patients were predominantly male (p < 0.001). Many EV‐positive CSF samples were from infants under 3 months (33.1%), whereas most EV‐positive respiratory samples were from children 1 to 2 years old (49.2%). Echovirus 30 (E‐30) was most frequent in CSF (33.0%), followed by CV‐B5 (13.8%) and E‐6 (13.8%). CV‐A6 was most frequent in respiratory samples (16.0%), followed by EV‐D68 (7.6%) and CV‐A5 (7.4%). EV types in CSF and respiratory samples show diverse dynamics, with some outbreaks indicated. A significant difference was found in the EV detection rate between CSF and respiratory samples by age. Various EV types were characterized, showing that some EV types are more neurotropic or cause more severe infections.