Ibuprofen alters human testicular physiology to produce a state of compensated hypogonadism.

Concern has been raised over increased male reproductive disorders in the Western world, and the disruption of male endocrinology has been suggested to play a central role. Several studies have shown that mild analgesics exposure during fetal life is associated with antiandrogenic effects and congenital malformations, but the effects on the adult man remain largely unknown. Through a clinical trial with young men exposed to ibuprofen, we show that the analgesic resulted in the clinical condition named "compensated hypogonadism," a condition prevalent among elderly men and associated with reproductive and physical disorders. In the men, luteinizing hormone (LH) and ibuprofen plasma levels were positively correlated, and the testosterone/LH ratio decreased. Using adult testis explants exposed or not exposed to ibuprofen, we demonstrate that the endocrine capabilities from testicular Leydig and Sertoli cells, including testosterone production, were suppressed through transcriptional repression. This effect was also observed in a human steroidogenic cell line. Our data demonstrate that ibuprofen alters the endocrine system via selective transcriptional repression in the human testes, thereby inducing compensated hypogonadism.

Transcriptome profiling of fetal Klinefelter testis tissue reveals a possible involvement of long non-coding RNAs in gonocyte maturation.

In humans, the most common sex chromosomal disorder is Klinefelter syndrome (KS), caused by the presence of one or more extra X-chromosomes. KS patients display a varying adult phenotype but usually present with azoospermia due to testicular degeneration, which accelerates at puberty. The timing of the germ cell loss and whether it is caused by dysgenetic fetal development of the testes is not known. We investigated eight fetal KS testes and found a marked reduction in MAGE-A4-positive pre-spermatogonia compared with testes from 15 age-matched controls, indicating a failure of the gonocytes to differentiate into pre-spermatogonia. Transcriptome analysis by RNA-sequencing of formalin-fixed, paraffin-embedded testes originating from four fetal KS and five age-matched controls revealed 211 differentially expressed transcripts in the fetal KS testis. We found a significant enrichment of upregulated X-chromosomal transcripts and validated the expression of the pseudoautosomal region 1 (PAR1) gene, AKAP17A. Moreover, we found enrichment of long non-coding RNAs in the KS testes (e.g. LINC01569 and RP11-485F13.1). In conclusion, our data indicate that the testicular phenotype observed among adult men with KS is initiated already in fetal life by failure of the gonocyte differentiation into pre-spermatogonia, which could be due to aberrant expression of long non-coding RNAs.

Whole grain-rich diet reduces body weight and systemic low-grade inflammation without inducing major changes of the gut microbiome: a randomised cross-over trial.

OBJECTIVE: To investigate whether a whole grain diet alters the gut microbiome and insulin sensitivity, as well as biomarkers of metabolic health and gut functionality. DESIGN: 60 Danish adults at risk of developing metabolic syndrome were included in a randomised cross-over trial with two 8-week dietary intervention periods comprising whole grain diet and refined grain diet, separated by a washout period of ≥6 weeks. The response to the interventions on the gut microbiome composition and insulin sensitivity as well on measures of glucose and lipid metabolism, gut functionality, inflammatory markers, anthropometry and urine metabolomics were assessed. RESULTS: 50 participants completed both periods with a whole grain intake of 179±50 g/day and 13±10 g/day in the whole grain and refined grain period, respectively. Compliance was confirmed by a difference in plasma alkylresorcinols (p<0.0001). Compared with refined grain, whole grain did not significantly alter glucose homeostasis and did not induce major changes in the faecal microbiome. Also, breath hydrogen levels, plasma short-chain fatty acids, intestinal integrity and intestinal transit time were not affected. The whole grain diet did, however, compared with the refined grain diet, decrease body weight (p<0.0001), serum inflammatory markers, interleukin (IL)-6 (p=0.009) and C-reactive protein (p=0.003). The reduction in body weight was consistent with a reduction in energy intake, and IL-6 reduction was associated with the amount of whole grain consumed, in particular with intake of rye. CONCLUSION: Compared with refined grain diet, whole grain diet did not alter insulin sensitivity and gut microbiome but reduced body weight and systemic low-grade inflammation. TRIAL REGISTRATION NUMBER: NCT01731366; Results.

Klinefelter syndrome comorbidities linked to increased X chromosome gene dosage and altered protein interactome activity.

Klinefelter syndrome (KS) (47,XXY) is the most common male sex chromosome aneuploidy. Diagnosis and clinical supervision remain a challenge due to varying phenotypic presentation and insufficient characterization of the syndrome. Here we combine health data-driven epidemiology and molecular level systems biology to improve the understanding of KS and the molecular interplay influencing its comorbidities. In total, 78 overrepresented KS comorbidities were identified using in- and out-patient registry data from the entire Danish population covering 6.8 million individuals. The comorbidities extracted included both clinically well-known (e.g. infertility and osteoporosis) and still less established KS comorbidities (e.g. pituitary gland hypofunction and dental caries). Several systems biology approaches were applied to identify key molecular players underlying KS comorbidities: Identification of co-expressed modules as well as central hubs and gene dosage perturbed protein complexes in a KS comorbidity network build from known disease proteins and their protein-protein interactions. The systems biology approaches together pointed to novel aspects of KS disease phenotypes including perturbed Jak-STAT pathway, dysregulated genes important for disturbed immune system (IL4), energy balance (POMC and LEP) and erythropoietin signalling in KS. We present an extended epidemiological study that links KS comorbidities to the molecular level and identify potential causal players in the disease biology underlying the identified comorbidities.

Comparison of global gene expression profiles of microdissected human foetal Leydig cells with their normal and hyperplastic adult equivalents.

STUDY QUESTION: Do human adult Leydig cells (ALCs) within hyperplastic micronodules display characteristics of foetal LCs (FLCs)? SUMMARY ANSWER: The gene expression profiles of FLCs and all ALC subgroups were clearly different, but there were no significant differences in expressed genes between the normally clustered and hyperplastic ALCs. WHAT IS KNOWN ALREADY: LCs are the primary androgen producing cells in males throughout development and appear in chronologically distinct populations; FLCs, neonatal LCs and ALCs. ALCs are responsible for progression through puberty and for maintenance of reproductive functions in adulthood. In patients with reproductive problems, such as infertility or testicular cancer, and especially in men with high gonadotrophin levels, LC function is often impaired, and LCs may cluster abnormally into hyperplastic micronodules (defined as clusters of >15 LCs in a cross-section). STUDY DESIGN, SIZE, DURATION: A genome-wide microarray study of LCs microdissected from human foetal and adult tissue samples (n = 12). Additional tissue specimens (n = 15) were used for validation of the mRNA expression data at the protein level. PARTICIPANTS/MATERIALS, SETTING, METHODS: Frozen human tissue samples were used for the microarray study, including morphologically normal foetal (gestational week 10-11) testis samples, and adult testis specimens with normal LC distribution, LC micronodules or LC micronodules adjacent to hCG-producing testicular germ cell tumours. Transcriptome profiling was performed on Agilent whole human genome microarray 4 × 44 K chips. Microarray data pre-processing and statistical analysis were performed using the limma R/Bioconductor package in the R software, and differentially expressed genes were further analysed for gene set enrichment using the DAVID Bioinformatics software. Selected genes were studied at the protein level by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: The transcriptomes of FLCs and ALCs differed significantly from each other, whereas the profiles of the normally clustered and hyperplastic ALCs were similar despite morphological heterogeneity. The study revealed several genes not known previously to be expressed in LCs during early development, including sulfotransferase family 2A member 1 (SULT2A1), WNT1-inducible signalling pathway protein 2 (WISP2), hydroxyprostaglandin dehydrogenase (HPGD) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), whose expression changes were validated at the protein level. LARGE SCALE DATA: The transcriptomic data are deposited in ArrayExpress (accession code E-MTAB-5453). LIMITATIONS, REASONS FOR CAUTION: The small number of biological replicates and the necessity of RNA amplification due to the scarcity of human tissues, especially foetal specimens, are the main limitations of the study. Heterogeneous subpopulations of LCs within micronodules were not discriminated during microdissection and might have affected the expression profiling. The study was constrained by the lack of availability of truly normal controls. Testis samples used as 'controls' displayed complete spermatogenesis and were from patients with germ cell neoplasia but with undetectable hCG and normal hormone levels. WIDER IMPLICATIONS OF THE FINDINGS: The changes in LC morphology and function observed in patients with reproductive disorders possibly reflect subtle changes in the expression of many genes rather than regulatory changes of single genes or pathways. The study provides new insights into the development and maturation of human LCs by the identification of a number of potential functional markers for FLC and ALC. STUDY FUNDING AND COMPETING INTEREST(S): The study was supported by research grants from the Danish Cancer Society, the Capital Region's Research Fund for Health Research, Rigshospitalet's research funds, the Villum Kann Rasmussen Foundation, the Danish Innovation Fund, ReproUnion, Kirsten and Freddy Johansen's foundation and the Novo Nordisk Foundation. None of the funding agencies had any influence on the study. The authors declare no conflicts of interest.

The vaginal microbiome is stable in prepubertal and sexually mature Ellegaard Göttingen Minipigs throughout an estrous cycle.

Although the pig has been introduced as an advanced animal model of genital tract infections in women, almost no knowledge exists on the porcine vaginal microbiota, especially in barrier-raised Göttingen Minipigs. In women, the vaginal microbiota plays a crucial role for a healthy vaginal environment and the fate of sexually transmitted infections such as Chlamydia trachomatis infections. Therefore, knowledge on the vaginal microbiota is urgently needed for the minipig model. The aim of this study was to characterize the microbiota of the anterior vagina by 16 s rRNA gene sequencing in prepubertal and sexually mature Göttingen Minipigs during an estrous cycle. The dominating phyla in the vaginal microbiota consisted of Firmicutes, Proteobacteria, Actinobacteria, Bacteriodetes and Tenericutes. The most abundant bacterial families were Enterobacteriaceae, unclassified families from Gammaproteobacteria, Clostridiales Family XI Incertae Sedis, Paenibacillaceae, Lactobacillaceae, Ruminococcaceae and Syntrophaceae. We found a higher abundance of Lactobacillaceae in the prepubertal Göttingen Minipigs compared to sexually mature non-pregnant Göttingen Minipigs. However, correlation tests and diversity parameters revealed a very stable vaginal microbiota in the Göttingen Minipigs, both before and after sexual maturity and on different days throughout an estrous cycle. The vaginal microbiota in Göttingen Minipigs was not dominated by lactobacilli, as it is in women and according to our results the minipig vaginal microbiota is very stable, in opposite to women. These differences should be considered when using the minipig as a model of the genital tract in women.

Transcriptome profiling of mice testes following low dose irradiation.

BACKGROUND: Radiotherapy is used routinely to treat testicular cancer. Testicular cells vary in radio-sensitivity and the aim of this study was to investigate cellular and molecular changes caused by low dose irradiation of mice testis and to identify transcripts from different cell types in the adult testis. METHODS: Transcriptome profiling was performed on total RNA from testes sampled at various time points (n = 17) after 1 Gy of irradiation. Transcripts displaying large overall expression changes during the time series, but small expression changes between neighbouring time points were selected for further analysis. These transcripts were separated into clusters and their cellular origin was determined. Immunohistochemistry and in silico quantification was further used to study cellular changes post-irradiation (pi). RESULTS: We identified a subset of transcripts (n = 988) where changes in expression pi can be explained by changes in cellularity. We separated the transcripts into five unique clusters that we associated with spermatogonia, spermatocytes, early spermatids, late spermatids and somatic cells, respectively. Transcripts in the somatic cell cluster showed large changes in expression pi, mainly caused by changes in cellularity. Further investigations revealed that the low dose irradiation seemed to cause Leydig cell hyperplasia, which contributed to the detected expression changes in the somatic cell cluster. CONCLUSIONS: The five clusters represent gene expression in distinct cell types of the adult testis. We observed large expression changes in the somatic cell profile, which mainly could be attributed to changes in cellularity, but hyperplasia of Leydig cells may also play a role. We speculate that the possible hyperplasia may be caused by lower testosterone production and inadequate inhibin signalling due to missing germ cells.

A genome-wide association study of men with symptoms of testicular dysgenesis syndrome and its network biology interpretation.

BACKGROUND: Testicular dysgenesis syndrome (TDS) is a common disease that links testicular germ cell cancer, cryptorchidism and some cases of hypospadias and male infertility with impaired development of the testis. The incidence of these disorders has increased over the last few decades, and testicular cancer now affects 1% of the Danish and Norwegian male population. METHODS: To identify genetic variants that span the four TDS phenotypes, the authors performed a genome-wide association study (GWAS) using Affymetrix Human SNP Array 6.0 to screen 488 patients with symptoms of TDS and 439 selected controls with excellent reproductive health. Furthermore, they developed a novel integrative method that combines GWAS data with other TDS-relevant data types and identified additional TDS markers. The most significant findings were replicated in an independent cohort of 671 Nordic men. RESULTS: Markers located in the region of TGFBR3 and BMP7 showed association with all TDS phenotypes in both the discovery and replication cohorts. An immunohistochemistry investigation confirmed the presence of transforming growth factor ß receptor type III (TGFBR3) in peritubular and Leydig cells, in both fetal and adult testis. Single-nucleotide polymorphisms in the KITLG gene showed significant associations, but only with testicular cancer. CONCLUSIONS: The association of single-nucleotide polymorphisms in the TGFBR3 and BMP7 genes, which belong to the transforming growth factor ß signalling pathway, suggests a role for this pathway in the pathogenesis of TDS. Integrating data from multiple layers can highlight findings in GWAS that are biologically relevant despite having border significance at currently accepted statistical levels.

Predicting Hearing Loss in Testicular Cancer Patients after Cisplatin-Based Chemotherapy.

Testicular cancer is predominantly curable, but the long-term side effects of chemotherapy have a severe impact on life quality. In this research study, we focus on hearing loss as a part of overall chemotherapy-induced ototoxicity. This is a unique approach where we combine clinical data from the acclaimed nationwide Danish Testicular Cancer (DaTeCa)-Late database. Clinical and genetic data on 433 patients were collected from hospital files in October 2014. Hearing loss was classified according to the FACT/GOG-Ntx-11 version 4 self-reported Ntx6. Machine learning models combining a genome-wide association study within a nested cross-validated logistic regression were applied to identify patients at high risk of hearing loss. The model comprising clinical and genetic data identified 67% of the patients with hearing loss; however, this was with a false discovery rate of 49%. For the non-affected patients, the model identified 66% of the patients with a false omission rate of 19%. An area under the receiver operating characteristic (ROC-AUC) curve of 0.73 (95% CI, 0.71-0.74) was obtained, and the model suggests genes SOD2 and MGST3 as important in improving prediction over the clinical-only model with a ROC-AUC of 0.66 (95% CI, 0.65-0.66). Such prediction models may be used to allow earlier detection and prevention of hearing loss. We suggest a possible biological mechanism for cisplatin-induced hearing loss development. On confirmation in larger studies, such models can help balance treatment in clinical practice.

IgG and IgM cooperate in coating of intestinal bacteria in IgA deficiency.

Immunoglobulin A (IgA) is acknowledged to play a role in the defence of the mucosal barrier by coating microorganisms. Surprisingly, IgA-deficient humans exhibit few infection-related complications, raising the question if the more specific IgG may help IgM in compensating for the lack of IgA. Here we employ a cohort of IgA-deficient humans, each paired with IgA-sufficient household members, to investigate multi-Ig bacterial coating. In IgA-deficient humans, IgM alone, and together with IgG, recapitulate coating of most bacterial families, despite an overall 3.6-fold lower Ig-coating. Bacterial IgG coating is dominated by IgG1 and IgG4. Single-IgG2 bacterial coating is sparse and linked to enhanced Escherichia coli load and TNF-α. Although single-IgG2 coating is 1.6-fold more prevalent in IgA deficiency than in healthy controls, it is 2-fold less prevalent than in inflammatory bowel disease. Altogether we demonstrate that IgG assists IgM in coating of most bacterial families in the absence of IgA and identify single-IgG2 bacterial coating as an inflammatory marker.

Using electronic patient records to discover disease correlations and stratify patient cohorts.

Electronic patient records remain a rather unexplored, but potentially rich data source for discovering correlations between diseases. We describe a general approach for gathering phenotypic descriptions of patients from medical records in a systematic and non-cohort dependent manner. By extracting phenotype information from the free-text in such records we demonstrate that we can extend the information contained in the structured record data, and use it for producing fine-grained patient stratification and disease co-occurrence statistics. The approach uses a dictionary based on the International Classification of Disease ontology and is therefore in principle language independent. As a use case we show how records from a Danish psychiatric hospital lead to the identification of disease correlations, which subsequently can be mapped to systems biology frameworks.

Corrigendum: Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias.

[This corrects the article DOI: 10.3389/fmicb.2017.01934.].

Association Study between Polymorphisms in DNA Methylation-Related Genes and Testicular Germ Cell Tumor Risk.

BACKGROUND: Testicular germ cell tumors (TGCT), histologically classified as seminomas and nonseminomas, are believed to arise from primordial gonocytes, with the maturation process blocked when they are subjected to DNA methylation reprogramming. SNPs in DNA methylation machinery and folate-dependent one-carbon metabolism genes have been postulated to influence the proper establishment of DNA methylation. METHODS: In this pathway-focused investigation, we evaluated the association between 273 selected tag SNPs from 28 DNA methylation-related genes and TGCT risk. We carried out association analysis at individual SNP and gene-based level using summary statistics from the Genome Wide Association Study meta-analysis recently conducted by the international Testicular Cancer Consortium on 10,156 TGCT cases and 179,683 controls. RESULTS: In individual SNP analyses, seven SNPs, four mapping within MTHFR, were associated with TGCT risk after correction for multiple testing (q ≤ 0.05). Queries of public databases showed that three of these SNPs were associated with MTHFR changes in enzymatic activity (rs1801133) or expression level in testis tissue (rs12121543, rs1476413). Gene-based analyses revealed MTHFR (q = 8.4 × 10-4), methyl-CpG-binding protein 2 (MECP2; q = 2 × 10-3), and ZBTB4 (q = 0.03) as the top TGCT-associated genes. Stratifying by tumor histology, four MTHFR SNPs were associated with seminoma. In gene-based analysis MTHFR was associated with risk of seminoma (q = 2.8 × 10-4), but not with nonseminomatous tumors (q = 0.22). CONCLUSIONS: Genetic variants within MTHFR, potentially having an impact on the DNA methylation pattern, are associated with TGCT risk. IMPACT: This finding suggests that TGCT pathogenesis could be associated with the folate cycle status, and this relation could be partly due to hereditary factors.

Transcriptomic changes upon epoxiconazole exposure in a human stem cell-based model of developmental toxicity.

Conazole fungicides such as epoxiconazole are mostly used on cereals of crops to inhibit fungal growth through direct inhibition of sterol 14α-demethylase (CYP51A1). However, this enzyme is highly conserved and in humans it is part of the steroid hormone biosynthesis pathway. Endocrine disrupting effects of epoxiconazole have been shown in rodents and have been substantiated by in vitro data, however, the underlying molecular mechanisms are not clear. We took advantage of a human stem cell based in vitro model for developmental toxicity to study the molecular effects of epoxiconazole. This model is based on 3D cultures of embryoid bodies and differentiation into cardiomyocytes, which mimics the early stages of embryonic development. We have previously shown that epoxiconazole impairs differentiation of these embryoid bodies and therefore has the potential to affect human embryonic development. We employed global transcriptome analysis using RNA sequencing and found that the steroid biosynthesis pathway including CYP51A1, the human sterol 14α-demethylase, was highly deregulated by epoxiconazole in our model. We confirmed that most genes of the steroid biosynthesis pathway were upregulated, including CYP51A1, suggesting a compensatory mechanism at the gene expression level. Our data suggest that epoxiconazole acts mainly by decreasing cholesterol biosynthesis in the cells. We conclude that epoxiconazole bears the potential to harm human embryonic development through inhibition of the steroid biosynthesis pathway. As this may be a common feature of compounds that target sterol 14α-demethylase, we add evidence to the assumption that conazole fungicides may be human developmental toxicants.

Ocular surface microbiota in patients with aqueous tear-deficient dry eye.

PURPOSE: An altered ocular surface microbiota may contribute to the pathophysiology of dry eye disease. The aim of the study was to explore potential differences in microbiota diversity and composition in aqueous tear-deficient dry eye (with and without ocular graft-versus-host disease) compared with controls. METHODS: Swab samples from the inferior fornix of the conjunctiva were obtained from patients with aqueous tear-deficient dry eye with and without ocular graft-versus-host disease (n = 18, n = 21, respectively) and controls (n = 28). Isolated bacterial DNA from swabs were analyzed with 16S rRNA gene amplicon sequencing. RESULTS: Decreased microbiota diversity was observed in patients with aqueous tear-deficient dry eye (p ≤ 0.003) who also showed a difference in microbiota composition compared with controls (p = 0.001). Although several genera were less abundant in aqueous tear-deficient dry eye, a minimal core ocular surface microbiota comprising five genera was shared by >75% of the study participants: Enhydrobacter, Brevibacterium, Staphylococcus, Streptococcus and Cutibacterium. Pseudomonas was identified as a bacterial biomarker for controls and Bacilli for patients with aqueous tear-deficient dry eye. CONCLUSIONS: Ocular surface microbiota in patients with aqueous tear-deficient dry eye was characterized by an aberrant microbiota composition in comparison to controls, with decreased diversity and reduced relative abundances of several genera. Additionally, a few genera were present in most of the study population, indicating that a minimal core ocular surface microbiota may exist.

Ocular Surface Microbiota in Contact Lens Users and Contact-Lens-Associated Bacterial Keratitis.

Our objectives were to investigate whether the conjunctival microbiota is altered by contact lens wear and/or bacterial keratitis and to explore the hypothesis that commensals of conjunctival microbiota contribute to bacterial keratitis. Swab samples from both eyes were collected separately from the inferior fornix of the conjunctiva of non-contact-lens users (nparticipants = 28) and contact lens users (nparticipants = 26) and from patients with contact-lens-associated bacterial keratitis (nparticipants = 9). DNA from conjunctival swab samples was analyzed with 16S rRNA gene amplicon sequencing. Pathogens from the corneal infiltrates were identified by cultivation. In total, we identified 19 phyla and 283 genera; the four most abundant genera were Pseudomonas, Enhydrobacter, Staphylococcus, and Cutibacterium. Several pathogens related to bacterial keratitis were identified in the conjunctival microbiota of the whole study population, and the same bacteria were identified by both methods in the conjunctiva and cornea for four patients with contact-lens-associated bacterial keratitis. The overall conjunctival microbiota profile was not altered by contact lens wear or bacterial keratitis; thus, it does not appear to contribute to the development of bacterial keratitis in contact lens users. However, in some individuals, conjunctival microbiota may harbor opportunistic pathogens causing contact-lens-associated bacterial keratitis.

Small RNAs in Seminal Plasma as Novel Biomarkers for Germ Cell Tumors.

Circulating miRNAs secreted by testicular germ cell tumors (TGCT) show great potential as novel non-invasive biomarkers for diagnosis of TGCT. Seminal plasma (SP) represents a biofluid closer to the primary site. Here, we investigate whether small RNAs in SP can be used to diagnose men with TGCTs or the precursor lesions, germ cell neoplasia in situ (GCNIS). Small RNAs isolated from SP from men with TGCTs (n = 18), GCNIS-only (n = 5), and controls (n = 25) were sequenced. SP from men with TGCT/GCNIS (n = 37) and controls (n = 22) were used for validation by RT-qPCR. In general, piRNAs were found at lower levels in SP from men with TGCTs. Ten small RNAs were found at significantly (q-value < 0.05) different levels in SP from men with TGCT/GCNIS than controls. Random forests classification identified sets of small RNAs that could detect either TGCT/GCNIS or GCNIS-only with an area under the curve of 0.98 and 1 in ROC analyses, respectively. RT-qPCR validated hsa-miR-6782-5p to be present at 2.3-fold lower levels (p = 0.02) in the SP from men with TGCTs compared with controls. Small RNAs in SP show potential as novel biomarkers for diagnosing men with TGCT/GCNIS but validation in larger cohorts is needed.

Acetaminophen metabolism revisited using non-targeted analyses: Implications for human biomonitoring.

The analgesic paracetamol/acetaminophen (N-acetyl-4-aminophenol, APAP) is commonly used to relieve pain, fever and malaise. While sales have increased worldwide, a growing body of experimental and epidemiological evidence has suggested APAP as a possible risk factor for various health disorders in humans. To perform internal exposure-based risk assessment, the use of accurate and optimized biomonitoring methods is critical. However, retrospectively assessing pharmaceutical use of APAP in humans is challenging because of its short half-life. The objective of this study was to address the key issue of potential underestimation of APAP use using current standard analytical methods based on urinary analyses of free APAP and its phase II conjugates. The question we address is whether investigating additional metabolites than direct phase II conjugates could improve the monitoring of APAP. Using non-targeted analyses based on high-resolution mass spectrometry, we identified, in a controlled longitudinal exposure study with male volunteers, overlooked APAP metabolites with delayed formation and excretion rates. We postulate that these metabolites are formed via the thiomethyl shunt after the enterohepatic circulation as already observed in rodents. Importantly, these conjugated thiomethyl metabolites were (i) of comparable diagnostic sensitivity as the free APAP and its phase II conjugates detected by current methods; (ii) had delayed peak levels in blood and urine compared to other APAP metabolites and therefore potentially extend the window of exposure assessment; and (iii) provide relevant information regarding metabolic pathways of interest from a toxicological point of view. Including these metabolites in future APAP biomonitoring methods therefore provides an option to decrease potential underestimation of APAP use. Moreover, our data challenge the notion that the standard methods in biomonitoring based exclusively on the parent compound and its phase II metabolites are adequate for human biomonitoring of a non-persistent chemical such as APAP.

Genetically Inferred Telomere Length and Testicular Germ Cell Tumor Risk.

BACKGROUND: Studies evaluating the association between peripheral blood leukocyte telomere length (LTL) and testicular germ cell tumor (TGCT) risk have produced conflicting results. METHODS: Using available genotype data from the Testicular Cancer Consortium (TECAC), polygenic risk score and Mendelian randomization analyses of genetic variants previously associated with LTL were used to assess potential etiologic associations between telomere length and TGCT risk. RESULTS: Genetically inferred telomere length was not associated with TGCT risk among 2,049 cases and 6,921 controls with individual-level genotype data (OR, 1.02; 95% confidence interval, 0.97-1.07). Mendelian randomization analyses using summary statistic data further indicated no evidence for an association between telomere length and TGCT risk among all available TECAC participants (3,558 cases and 13,971 controls). CONCLUSIONS: Our analyses in the largest molecular genetic testicular cancer study to date provide no evidence for an association between genetically inferred peripheral blood LTL and TGCT risk. IMPACT: The lack of evidence for an overall association indicates that peripheral blood LTL is likely not a strong biomarker for TGCT risk.

OCT4 and downstream factors are expressed in human somatic urogenital epithelia and in culture of epididymal spheres.

The transcription factor OCT4 plays a crucial role in the earliest differentiation of the mammalian embryo and in self-renewal of embryonic stem cells. However, it remains controversial whether this gene is also expressed in somatic tissues. Here, we use a combination of RT-PCR on whole and microdissected tissues, in situ hybridization, immunohistochemistry and western blotting to show that OCT4 and SOX2 together with downstream targets, UTF1 and REX1/ZFP42, are expressed in the human male urogenital tract. We further support these results by the analysis of DNA methylation of a region in the OCT4 promoter. In culture, human primary epididymal cells formed spheres that continued to express the investigated genes for at least 20 days. Transcriptomic analysis of cultured cells showed up-regulation of CD29, CD44 and CD133 that are normally associated with sphere-forming cancer stem cells. Furthermore, stimulation with retinoic acid resulted in down-regulation of OCT4 expression, however, without multilineage differentiation. Our results show that OCT4 and associated genes are expressed in somatic epithelial cells from the urogenital tract and that these cells can form spheres, a general marker of stem cell behaviour.