Monocytes expressing activin A and CCR2 exacerbate chronic testicular inflammation by promoting immune cell infiltration.

STUDY QUESTION: Does the chemokine/chemokine receptor axis, involved in immune cell trafficking, contribute to the pathology of testicular inflammation and how does activin A modulate this network? SUMMARY ANSWER: Testicular chemokines and their receptors (especially those essential for trafficking of monocytes) are elevated in orchitis, and activin A modulates the expression of the chemokine/chemokine receptor network to promote monocyte/macrophage and T cell infiltration into the testes, causing extensive tissue damage. WHAT IS KNOWN ALREADY: The levels of CC motif chemokine receptor (CCR)2 and its ligand CC motif chemokine ligand (CCL)2 are increased in experimental autoimmune orchitis (EAO) compared with healthy testes, and mice deficient in CCR2 are protected from EAO-induced tissue damage. Activin A induces CCR2 expression in macrophages, promoting their migration. Moreover, there is a positive correlation between testicular activin A concentration and the severity of autoimmune orchitis. Inhibition of activin A activity by overexpression of follistatin (FST) reduces EAO-induced testicular damage. STUDY DESIGN, SIZE, DURATION: EAO was induced in 10-12-week-old male C57BL/6J (wild-type; WT) and B6.129P2-Ccr2tm1Mae/tm1Mae (Ccr2-/-) mice (n = 6). Adjuvant (n = 6) and untreated (n = 6) age-matched control mice were also included. Testes were collected at 50 days after the first immunization with testicular homogenate in complete Freund's adjuvant. In another experimental setup, WT mice were injected with a non-replicative recombinant adeno-associated viral vector carrying a FST315-expressing gene cassette (rAAV-FST315; n = 7-9) or an empty control vector (n = 5) 30 days prior to EAO induction. Appropriate adjuvant (n = 4-5) and untreated (n = 4-6) controls were also examined. Furthermore, human testicular biopsies exhibiting focal leukocytic infiltration and impaired spermatogenesis (n = 17) were investigated. Biopsies showing intact spermatogenesis were included as controls (n = 9). Bone-marrow-derived macrophages (BMDMs) generated from WT mice were treated with activin A (50 ng/ml) for 6 days. Activin-A-treated or untreated BMDMs were then co-cultured with purified mouse splenic T cells for two days to assess chemokine and cytokine production. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of chemokines in total testicular RNA collected from mice. Immunofluorescence staining was used to detect activin A, F4/80, and CD3 expression in mouse testes. The expression of chemokine/chemokine-receptor-encoding genes was examined in human testicular biopsies by qRT-PCR. Correlations between chemokine expression levels and either the immune cell infiltration density or the mean spermatogenesis score were analyzed. Immunofluorescence staining was used to evaluate the expression of CD68 and CCR2 in human testicular biopsies. RNA isolated from murine BMDMs was used to characterize these cells in terms of their chemokine/chemokine receptor expression levels. Conditioned media from co-cultures of BMDMs and T cells were collected to determine chemokine levels and the production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interferon (IFN)-γ by T cells. MAIN RESULTS AND THE ROLE OF CHANCE: Induction of EAO in the testes of WT mice increased the expression of chemokine receptors such as Ccr1 (P < 0.001), Ccr2 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.0001), CXC motif chemokine receptor (Cxcr)3 (P < 0.01), and CX3C motif chemokine receptor (Cx3cr)1 (P < 0.001), as well as that of most of their ligands. Ccr2 deficiency reversed some of the changes associated with EAO by reducing the expression of Ccr1 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.01), Cxcr3 (P < 0.001), and Cx3cr1 (P < 0.0001). Importantly, the biopsies showing impaired spermatogenesis and concomitant focal leukocytic infiltration exhibited higher expression of CCL2 (P < 0.01), CCR1 (P < 0.05), CCR2 (P < 0.001), and CCR5 (P < 0.001) than control biopsies with no signs of inflammation and intact spermatogenesis. The gene expression of CCR2 and its ligand CCL2 correlated positively with the immune cell infiltration density (P < 0.05) and negatively with the mean spermatogenesis score (P < 0.001). Moreover, CD68+ macrophages expressing CCR2 were present in human testes with leukocytic infiltration with evidence of tubular damage. Treatment of BMDMs, as surrogates for testicular macrophages, with activin A increased their expression of Ccr1, Ccr2, and Ccr5 while reducing their expression of Ccl2, Ccl3, Ccl4, Ccl6, Ccl7 Ccl8, and Ccl12. These findings were validated in vivo, by showing that inhibiting activin A activity by overexpressing FST in EAO mice decreased the expression of Ccr2 (P < 0.05) and Ccr5 (P < 0.001) in the testes. Interestingly, co-culturing activin-A-treated BMDMs and T cells reduced the levels of CCL2 (P < 0.05), CCL3/4 (P < 0.01), and CCL12 (P < 0.05) in the medium and attenuated the production of TNF (P < 0.05) by T cells. The majority of cells secreting activin A in EAO testes were identified as macrophages. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: BMDMs were used as surrogates for testicular macrophages. Hence, results obtained from the in vitro experiments might not be fully representative of the situation in the testes in vivo. Moreover, since total RNA was extracted from the testicular tissue to examine chemokine expression, the contributions of individual cell types as producers of specific chemokines may have been overlooked. WIDER IMPLICATIONS OF THE FINDINGS: Our data indicate that macrophages are implicated in the development and progression of testicular inflammation by expressing CCR2 and activin A, which ultimately remodel the chemokine/chemokine receptor network and recruit other immune cells to the site of inflammation. Consequently, inhibition of CCR2 or activin A could serve as a potential therapeutic strategy for reducing testicular inflammation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the International Research Training Group in 'Molecular pathogenesis on male reproductive disorders', a collaboration between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK1871/1-2) funded by the Deutsche Forschungsgemeinschaft and Monash University, a National Health and Medical Research Council of Australia Ideas Grant (1184867), and the Victorian Government's Operational Infrastructure Support Programme. The authors declare no competing financial interests.

Activin A and CCR2 regulate macrophage function in testicular fibrosis caused by experimental autoimmune orchitis.

Experimental autoimmune-orchitis (EAO), a rodent model of chronic testicular inflammation and fibrosis, replicates pathogenic changes seen in some cases of human spermatogenic disturbances. During EAO, increased levels of pro-inflammatory and pro-fibrotic mediators such as TNF, CCL2, and activin A are accompanied by infiltration of leukocytes into the testicular parenchyma. Activin A levels correlate with EAO severity, while elevated CCL2 acting through its receptor CCR2 mediates leukocyte trafficking and recruits macrophages. CCR2 + CXCR4 + macrophages producing extracellular matrix proteins contribute widely to fibrogenesis. Furthermore, testicular macrophages (TMs) play a critical role in organ homeostasis. Therefore, we aimed to investigate the role of the activin A/CCL2-CCR2/macrophage axis in the development of testicular fibrosis. Following EAO induction, we observed lower levels of organ damage, collagen deposition, and leukocyte infiltration (including fibronectin+, collagen I+ and CXCR4+ TMs) in Ccr2-/- mice than in WT mice. Furthermore, levels of Il-10, Ccl2, and the activin A subunit Inhba mRNAs were lower in Ccr2-/- EAO testes. Notably, fibronectin+ TMs were also present in biopsies from patients with impaired spermatogenesis and fibrotic alterations. Overexpression of the activin A antagonist follistatin reduced tissue damage and collagen I+ TM accumulation in WT EAO testes, while treating macrophages with activin A in vitro increased the expression of Ccr2, Fn1, Cxcr4, and Mmp2 and enhanced migration along a CCL2 gradient; these effects were abolished by follistatin. Taken together, our data indicate that CCR2 and activin A promote fibrosis during testicular inflammation by regulating macrophage function. Inhibition of CCR2 or activin A protects against damage progression, offering a promising avenue for therapeutic intervention.

Pathohistological features of the aging human lacrimal gland.

AIM: To assess sex-related differences in the pathohistological features of the human lacrimal gland and to investigate age-related and sex-related differences in stereologically measured volume density of the secretory tissue, connective tissue, and fat. METHODS: We performed an observational analysis of acinar atrophy, periacinar fibrosis, periductal fibrosis, ductal dilation, ductal proliferation, fatty infiltration, and lymphocyte infiltration of hematoxylin and eosin-stained lacrimal gland samples from 81 cornea donors. Stereological analysis of the volume density of the secretory tissue, connective tissue, and fat was performed on samples from 66 donors. RESULTS: Up to 69% of all samples showed degenerative changes. Female samples had a higher frequency of all observed degenerative changes, except ductal dilation. While acinar atrophy was significantly more prevalent in women, ductal dilation was significantly more prevalent in men. Stereological analysis indicated lower portions of acini and higher portions of connective tissue and fat, as well as a more pronounced age-related progression of degenerative changes in female samples. CONCLUSION: Female lacrimal glands are more susceptible to degeneration, and this susceptibility could play an important role in the higher incidence of dry eye disease in older women. A further stereological analysis using more samples from younger age groups is needed to elucidate age-related and sex-related differences in the structure of the human lacrimal gland and their impact on dry eye disease.

Expression of Androgen and Estrogen Receptors in the Human Lacrimal Gland.

Lacrimal gland dysfunction causes dry eye disease (DED) due to decreased tear production. Aqueous-deficient DED is more prevalent in women, suggesting that sexual dimorphism of the human lacrimal gland could be a potential cause. Sex steroid hormones are a key factor in the development of sexual dimorphism. This study aimed to quantify estrogen receptor (ER) and androgen receptor (AR) expression in the human lacrimal gland and compare it between sexes. RNA was isolated from 35 human lacrimal gland tissue samples collected from 19 cornea donors. AR, ERα, and ERß mRNA was identified in all samples, and their expression was quantified using qPCR. Immunohistochemical staining was performed on selected samples to evaluate protein expression of the receptors. ERα mRNA expression was significantly higher than the expression of AR and ERß. No difference in sex steroid hormone (SSH) receptor mRNA expression was observed between sexes, and no correlation was observed with age. If ERα protein expression is found to be concordant with mRNA expression, it should be investigated further as a potential target for hormone therapy of DED. Further research is needed to elucidate the role of sex steroid hormone receptors in sex-related differences of lacrimal gland structure and disease.

Distribution of human papillomavirus genotypes in women with high-grade cervical intraepithelial lesions and cervical carcinoma and analysis of human papillomavirus-16 genomic variants.

AIM: To analyze the distribution of high-risk human papillomavirus (HR-HPV) genotypes and the diversity of HPV-16 genomic variants in Croatian women with high-grade squamous intraepithelial lesions (HSIL) and cervical carcinoma. METHODS: Tissue biopsy specimens were obtained from 324 women with histopathologically confirmed HSIL or cervical carcinoma, 5 women with low-grade SIL, and 49 women with negative histopathology. HR-HPV DNA was detected with Ampliquality HPV-type nucleic-acid hybridization assay, which identifies 29 different HPV genotypes. HPV-16 genomic variants were analyzed by an in-house sequencing. RESULTS: The most common HPV type in women with HSIL was HPV-16, detected in 127/219 (57.9%) specimens. HPV-16 was also the dominant type in squamous cell cervical carcinoma (46/69 or 66.7%) and in adenocarcinoma (18/36 or 50.0%). Out of 378 patients, 360 had HR-HPV (282 single infections and 79 multiple infections), 3 (0.8%) patients had low-risk HPV, and 15 (4%) tested negative. HPV-16 variants were determined in 130 HPV-16 positive specimens, including 74 HSIL and 46 carcinoma specimens. In HSIL specimens, 41 distinct variants were found, 98.6% belonging to the European branch and 1.4% belonging to the African branch. In cervical carcinoma specimens, 95% isolates grouped in 41 variants belonging to the European branch, one isolate (2.5%) belonged to the North American, and one (2.5%) to the Asian-American branch. CONCLUSION: HPV-16, mainly belonging to the European branch, was the most frequent HPV genotype in women from Croatia with histologically confirmed HSIL and cervical cancer.

The comparison of Th1, Th2, Th9, Th17 and Th22 cytokine profiles in acute and chronic HIV-1 infection.

The aim of this study was to compare cytokine expression on both gene and protein levels in acute and chronic phase of HIV type 1 (HIV-1) infection. Thirty four patients were enrolled for cytokine expression analysis on protein level in acute and chronic stage of HIV-1 infection. Using PCR array technology, expression of 84 cytokine genes was measured in 3 patients in acute and 3 patients in chronic stage of HIV-1 infection. Bead-based cytometry was used to quantify levels of Th1/Th2/Th9/Th17/Th22 cytokines. The results showed statistically significant increase of 13 cytokine gene expression (cd40lg, csf2, ifna5, il12b, il1b, il20, lta, osm, spp1, tgfa, tnfsf 11, 14 and 8) and downregulation of the il12a expression in chronic HIV type 1 infection. Concentrations of IL-10, IL-4 and TNF-α were increased in the acute HIV type 1 infection when compared to control group. During chronic HIV type 1 infection there was an increase of IL-10, TNF-α, IL-2, IL-6, IL-13 and IL-22 levels when compared to control group. Comparison of cytokine expression between two stages of infection showed a significant decrease in IL-9 concentration. This study showed changes in cytokine profiles on both gene and protein levels in different stages of HIV-infection.

Chlamydophila pneumoniae and Mycoplasma pneumoniae were not identified in sinus mucosa of patients with chronic rhinosinusitis.

Chronic rhinosinusitis is a symptomatic inflammation of the mucosa of the nose and paranasal sinuses lasting for at least 12 weeks. Atypical bacteria Chlamydophila pneumoniae and Mycoplasma pneumoniae are important causes of human respiratory tract infection. Also, they were identified in bronchial respiratory epithelium of patients with chronic obstructive pulmonary disease or asthma. Having in mind the unified airway concept, it is also possible that these bacteria can cause persistent infection of sinus mucosa in patients with chronic rhinosinusitis. Sixty consecutive patients with chronic rhinosinusitis who underwent the functional endoscopic sinus surgery due to medical therapy failure were included in the study. During the operation, sinuses were irrigated with sterile 0.9% NaCl solution and this lavage was immediately aspirated. Aspirates were used for the detection of C. pneumoniae and M. pneumoniae DNA using real-time PCR. C. pneumoniae and M. pneumoniae DNA were not detected in samples analysed. Atypical bacteria C. pneumoniae and M. pneumoniae did not cause persistent infection of sinus mucosa in patients with chronic rhinosinusitis.

Dynamics of HSD17B3 expression in human fetal testis: implications for the role of Sertoli cells in fetal testosterone biosynthesis.

Introduction: Androgens play a pivotal role in shaping male sexual characteristics, with testosterone being an essential hormone in orchestrating various developmental processes. Testosterone biosynthesis involves a series of enzymatic reactions, among which the 17ß-hydroxysteroid dehydrogenase type 3 (HSD17B3) holds significance. While its role in adult Leydig cells is well established, its localization and importance during the fetal period remain less known, especially in humans. This study aims to delineate the dynamics of HSD17B3 expression in human fetal testes to clarify the contribution of specific cell types to testosterone biosynthesis. Methods: Using immunofluorescence staining, we investigated the expression pattern of HSD17B3 in human fetal and adult testicular tissues. Results and discussion: The findings of this study revealed a distinct temporal and cellular expression pattern of HSD17B3 protein in the fetal period. We detected its expression exclusively in Sertoli cells, the highest during the second trimester. This unique localization suggests the inclusion of fetal Sertoli cells in testosterone production during the critical masculinization-programming window. Furthermore, we demonstrated a shift in HSD17B3 expression from Sertoli cells to Leydig cells in adulthood, corroborating findings from rodent studies. This study sheds light on the intricate, still underexplored regulation of steroidogenesis during fetal development, whose disturbance might lead to testicular dysgenesis. Further research is warranted to elucidate the regulatory pathways governing the expression of HSD17B3 and its transition between Sertoli and Leydig cells, potentially paving the way for novel therapeutic interventions in disorders of sexual development.

Characterization of Human Immunodeficiency Virus-1 Transmission Clusters and Transmitted Drug-Resistant Mutations in Croatia from 2019 to 2022.

Molecular epidemiology of HIV-1 infection is challenging due to the highly diverse HIV-genome. We investigated the genetic diversity and prevalence of transmitted drug resistance (TDR) followed by phylogenetic analysis in 270 HIV-1 infected, treatment-naïve individuals from Croatia in the period 2019-2022. The results of this research confirmed a high overall prevalence of TDR of 16.7%. Resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside RTIs (NNRTIs), and protease inhibitors (PIs) was found in 9.6%, 7.4%, and 1.5% of persons, respectively. No resistance to integrase strand-transfer inhibitors (INSTIs) was found. Phylogenetic analysis revealed that 173/229 sequences (75.5%) were part of transmission clusters, and the largest identified was T215S, consisting of 45 sequences. Forward transmission was confirmed in several clusters. We compared deep sequencing (DS) with Sanger sequencing (SS) on 60 randomly selected samples and identified additional surveillance drug resistance mutations (SDRMs) in 49 of them. Our data highlight the need for baseline resistance testing in treatment-naïve persons. Although no major INSTIs were found, monitoring of SDRMs to INSTIs should be continued due to the extensive use of first- and second-generation INSTIs.

Reinke crystals: Hallmarks of adult Leydig cells in humans.

BACKGROUND: Reinke crystals are structures pathognomonic for Leydig cells, which have the important function of testosterone production and are vital for male reproductive health. These crystalline inclusions are thought to be of protein origin; however, the molecular composition has not yet been resolved. OBJECTIVES: This review summarizes all available information regarding Reinke crystal's characteristics and aims to produce a comprehensive guide for research on this topic as well as to determine and discuss potential Reinke-protein candidates. METHODS: Pubmed was thoroughly searched for all publications regarding Reinke crystals and 137 publications were identified. All publications were surveyed and all relevant information was included in the review. RESULTS: Along with the cytoplasm, structures that resemble Reinke crystals were also observed in the nucleus, suggesting that their formation depends only on protein concentration. Variations in tissue processing protocols could impact Reinke crystal microscopic visualization, which is an important factor in diagnosing Leydig cell disorders such as Leydig cell tumors. Reinke crystals appear to be hallmarks of normally differentiated, adult, Leydig or Leydig-like cells in humans, while some abnormal and nonhuman Leydig cells contain Reinke-like paracrystalline inclusions or crystalloids. CONCLUSIONS: These characteristics point to some differentially expressed proteins, which could be involved in Reinke crystal formation. Differential Reinke crystal and paracrystalline inclusion presence could also be due to small changes in protein structure or the cell environment. Further research is needed to solve the ongoing mystery of the Reinke crystal, which would enhance our knowledge of Leydig cell contribution in the pathogenesis of various male reproductive disorders and improve their diagnosis and treatment.

Stereological properties of seminiferous tubules in infertile men with chromosomal and genetic abnormalities.

BACKGROUND: Male infertility is caused by genetic anomalies in 15-30% of cases. This study aimed to determine stereological properties of seminiferous tubules in infertile men with genetic anomalies, including Klinefelter Syndrome (KS), Y chromosome microdeletions (MYC) and CFTR gene mutations (CFTR); and to compare them to seminiferous tubules of men with obstructive azoospermia of non-genetic origin (control group). METHODS: The study was conducted on 28 human testis biopsy specimens obtained from 14 patients with MYC, 18 samples from 9 patients with KS, and 6 samples from 3 patients with CFTR. Whenever possible, a bilateral biopsy was included in the study. The control group had 33 samples from 18 patients (3 of them with a solitary testis). Qualitative and quantitative (stereological) analysis of seminiferous tubules (including the status of spermatogenesis, volume, surface area, length and number of tubules) were performed in all groups. RESULTS: Qualitative histological analysis revealed significant impairment of spermatogenesis in KS and MYC, whereas testicular parenchyma was fully maintained in CFTR and control groups. Spermatogenesis was most seriously impaired in KS. All stereological parameters were significantly lower in KS and MYC, compared to the CFTR and control groups. The total volume, surface and length of seminiferous tubules were significantly lower in KS compared with MYC. CONCLUSIONS: Stereological analysis is valuable in evaluating male infertility, whereas qualitative histological analysis can be helpful in assessing sperm presence in testicular tissue of patients with KS or MYK undergoing TESE.

Human papillomavirus genotype specificity of hybrid capture 2 low-risk probe cocktail.

A genotyping study of 285 Hybrid Capture 2 low-risk probe cocktail-positive specimens showed cross-reactivity with several untargeted human papillomavirus genotypes. Cross-reactivity was often clinically beneficial due to the detection of untargeted low-risk genotypes. A total of 8.4% of positive results, usually weak, were due to cross-reactivity with high-risk genotypes. Establishment of a gray zone is recommended.

Analysis of HIV-1 diversity, primary drug resistance and transmission networks in Croatia.

Molecular epidemiology of HIV-1 infection in treatment-naive HIV-1 infected persons from Croatia was investigated. We included 403 persons, representing 92.4% of all HIV-positive individuals entering clinical care in Croatia in 2014-2017. Overall prevalence of transmitted drug resistance (TDR) was estimated at 16.4%. Resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside RTI (NNRTIs) and protease inhibitors (PIs) was found in 11.4%, 6.7% and 2.5% of persons, respectively. Triple-class resistance was determined in 2.2% of individuals. In addition, a single case (1.0%) of resistance to integrase strand-transfer inhibitors (InSTIs) was found. Deep sequencing was performed on 48 randomly selected samples and detected additional TDR mutations in 6 cases. Phylogenetic inference showed that 347/403 sequences (86.1%) were part of transmission clusters and identified forward transmission of resistance in Croatia, even that of triple-class resistance. The largest TDR cluster of 53 persons with T215S was estimated to originate in the year 1992. Our data show a continuing need for pre-treatment HIV resistance testing in Croatia. Even though a low prevalence of resistance to InSTI was observed, surveillance of TDR to InSTI should be continued.

The prevalence of transmitted drug resistance in newly diagnosed HIV-infected individuals in Croatia: the role of transmission clusters of men who have sex with men carrying the T215S surveillance drug resistance mutation.

The aim of this study was to determine the prevalence of transmitted drug resistance (TDR) in newly diagnosed and treatment-naive HIV-infected patients from Croatia and evaluate a possible contribution of transmission clusters to the spread of resistant virus. The study enrolled treatment-naive HIV-infected patients that entered clinical care at the Croatian Reference Center for HIV/AIDS between 2006 and 2008. The protease gene and a part of the reverse transcriptase gene of the HIV-1 genome were sequenced by using the Trugene HIV-1 Genotyping System. The prevalence of transmitted drug resistance was analyzed by using the surveillance drug resistance mutations (SDRM) list recommended by the WHO in 2009. We report findings for 118 of 180 eligible patients (65.6% coverage). SDRM were detected in 26 of 118 patients (22.0%) who were infected with subtype B and belonged mostly to the men having sex with men (MSM). The majority of patients with primary resistance carried SDRM associated with resistance to nucleoside analogues reverse transcriptase inhibitors (NRTIs, 23 of 118 patients, 19.5%). The most frequently found NRTI SDRM was T215S (17 of 118 patients, 14.4%). SDRM associated with resistance to nonnucleoside reverse transcriptase inhibitors were detected in three (2.5%) patients and primary resistance to protease inhibitors was not detected. Non-B subtypes were detected in 13/118 patients (11%). A total of 12 transmission pairs and eight distinct transmission clusters were identified with the largest cluster harboring sequences from 19 patients; among them all but two were carrying the T215S mutation. This study showed a high prevalence of TDR in newly diagnosed MSM from Croatia and is an important contribution concerning the relationship between local transmission clusters and the spread of resistant virus.

Digene HPV Genotyping RH Test RUO: comparative evaluation with INNO-LiPA HPV Genotyping Extra Test for detection of 18 high-risk and probable high-risk human papillomavirus genotypes.

BACKGROUND: Standardized and validated methods for the specific detection and identification of a spectrum of high-risk (hr) HPV genotypes will be necessary if HPV genotyping gains an important role in the clinical management of HPV-related precancerous lesions and cancers. OBJECTIVES: The first comparative evaluation of novel HPV genotyping Digene HPV Genotyping RH Test RUO (Qiagen, Hilden, Germany) with standard INNO-LiPA HPV Genotyping Extra CE assay (Innogenetics, Gent, Belgium). STUDY DESIGN: Seventy hr-HPV positive samples were tested in parallel with both genotyping assays. The results were interpreted taking into account 15 hr-HPV and 3 probable hr-HPV genotypes that can be identified by both assays (assay-common genotypes). RESULTS: Concordant results (a complete match of assay-common genotypes or negative using both assays) and compatible results (at least one genotype in common) were obtained in 42 (60.0%) and 28 (40.0%) samples, respectively. No discordant results for assay-common genotypes were obtained. Of 42 samples with compatible results, the presence of at least one assay-common genotype was detected in 37 samples, while no HPV was detected in two samples by both assays and only a single low-risk HPV was detected by INNO-LiPA in three samples. CONCLUSIONS: A novel Digene test is suitable for the detection of hr-HPV genotypes in clinical samples and it provides comparable results to the well established INNO-LiPA assay. Although INNO-LiPA identified significantly more samples with multiple HPV genotypes than the Digene test, the clinical benefit of such a difference is at present unclear.