Circulating levels and the bioactivity of miR-30b increase during pubertal progression in boys.

Background: Puberty marks the transition from childhood to adulthood and is initiated by activation of a pulsatile GnRH secretion from the hypothalamus. MKRN3 functions as a pre-pubertal break on the GnRH pulse generator and hypothalamic expression and circulating levels of MKRN3 decrease peri-pubertally. In rodents, microRNA miR-30b seems to directly target hypothalamic MKRN3 expression - and in boys, circulating levels of miR-30b-5p increase when puberty is pharmacologically induced. Similarly, miR-200b-3p and miR-155-5p have been suggested to inhibit expression of other proteins potentially involved in the regulation of GnRH secretion. Here we measure circulating levels of these three miRNAs as boys progress through puberty. Materials and Methods: Forty-six boys from the longitudinal part of the Copenhagen Puberty Study were included. All boys underwent successive clinical examinations including estimation of testis size by palpation. miR-30b-5p, miR-200b-3p, and miR-155-5p were measured in serum by RT-qPCR using a kit sensitive to the phosphorylation status of the miRNAs. Thirty-nine boys had miRNA levels measured in three consecutive samples (pre-, peri-, and post-pubertally) and seven boys had miR-30b-5p levels measured in ten consecutive samples during the pubertal transition. Results: When circulating levels of miR-30b-5p in pre- and peri-pubertal samples were compared with post-pubertal levels, we observed a significant increase of 2.3 and 2.2-fold (p-value<6.0×10-4), respectively, and a larger fraction of miR-30b-5p appeared to be phosphorylated post-pubertally indicating an increase in its bioactivity. We also observed a negative correlation between circulating levels of miR-30b-5p and MKRN3. The inter-individual variation in circulating miR-30b levels was substantial and we could not define a clinical threshold for miR-30b-5p suggestive of imminent puberty. Also, miR-155-5p showed significantly increasing levels from the peri- to the post-pubertal stage (p=3.0×10-3), whereas miR-200b-3p did not consistently increase. Conclusion: Both circulating levels of miR-30b-5p and its bioactivity increase during the pubertal transition in boys supporting its role in the activation of the HPG axis at the onset of physiologically normal puberty.

The seminal plasma microbiome of men with testicular germ cell tumours described by small RNA sequencing.

BACKGROUND: It has been estimated that microorganisms are involved in the pathogenesis of approximately 20% of all cancers. Testicular germ cell tumours (TGCTs) are the most common type of malignancy in young men and arise from the precursor cell, germ cell neoplasia in situ (GCNIS). The microbiome of seminal plasma and testicular tissue has not been thoroughly investigated in regard to TGCTs. OBJECTIVES: To investigate the differences in the seminal plasma microbiome between men with TGCT or GCNIS-only compared with controls. MATERIALS AND METHODS: The study population consisted of patients with GCNIS-only (n = 5), TGCT (n = 18), and controls (n = 25) with different levels of sperm counts in the ejaculate. RNA was isolated from the seminal plasma and sequenced. Reads not mapping to the human genome were aligned against a set of 2784 bacterial/archaeal and 4336 viral genomes using the Kraken pipeline. RESULTS: We identified reads from 2172 species and most counts were from Alteromonas mediterranea, Falconid herpesvirus 1, and Stigmatella aurantiaca. Six species (Acaryochloris marina, Halovirus HGTV-1, Thermaerobacter marianensis, Thioalkalivibrio sp. K90mix, Burkholderia sp. YI23, and Desulfurivibrio alkaliphilus) were found in significantly (q-value < 0.05) higher levels in the seminal plasma of TGCT and GCNIS-only patients compared with controls. In contrast, Streptomyces phage VWB was found at significantly higher levels among controls compared with TGCT and GCNIS-only patients combined. DISCUSSION: Often the microbiome is analysed by shotgun or 16S ribosomal sequencing whereas our present data build on small RNA sequencing. This allowed us to identify more viruses and phages compared with previous studies but also makes the results difficult to directly compare. CONCLUSION: Our study is the first to report identification of the microbiome species in seminal plasma of men with TGCT and GCNIS-only, which potentially could be involved in the pathogenesis of TGCTs. Further studies are, however, needed to confirm our findings.

PIWI-interacting RNAs and human testicular function.

Small noncoding RNAs (sncRNAs) are pieces of RNA with a length below 200 bp and represent a diverse group of RNAs having many different biological functions. The best described subtype is the microRNAs which primarily function in posttranscriptional gene regulation and appear essential for most physiological processes. Of particular interest for the germline is the PIWI-interacting RNAs (piRNAs) which are a class of sncRNA of 21-35 bp in length that are almost exclusively found in germ cells. Recently, it has become clear that piRNAs are essential for testicular function, and in this perspective, we outline the current knowledge of piRNAs in humans. Although piRNAs appear unique to germ cells, they have also been described in various somatic cancers and biofluids. Here, we discuss the potential function of piRNAs in somatic tissues and whether detection in biofluids may be used as a biomarker for testicular function. This article is categorized under: Reproductive System Diseases > Genetics/Genomics/Epigenetics Reproductive System Diseases > Molecular and Cellular Physiology.

Variant PNLDC1, Defective piRNA Processing, and Azoospermia.

BACKGROUND: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility. METHODS: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing. RESULTS: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1: the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations. CONCLUSIONS: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).

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.

Impact of psychological stress measured in three different scales on testis function: A cross-sectional study of 1362 young men.

BACKGROUND: Studies have reported associations between psychological stress and semen quality, but most have been performed on selected populations using different stress measures. Thus, it is uncertain which stress scale best quantifies the effects of stress on testicular function. OBJECTIVE: To study the association between three different measures of stress and testicular function in young men. MATERIAL AND METHODS: In total, 1362 men (median age 19 years) delivered semen and blood samples. They also answered a questionnaire including information from three stress scales: Stress Symptoms, Stressful Life Events and Perceived Stress. Various statistical analyses for associations between stress and testicular function (semen quality and reproductive hormones) were performed. RESULTS: Perceived Stress was negatively associated with sperm concentration, total count and motility and positively associated with serum FSH. Men with the highest scores (>30 points) had 38% (95% CI 3-84%) lower sperm concentration, 42% (95% CI 5-91%) lower total count and 22% (95% CI 2-32%) lower proportion of motile spermatozoa than men with the lowest scores (0-10 points). For the stress symptoms score, men with highest scores (>95th percentile vs. lower) had lower sperm concentration, total sperm count, motility and serum Inhibin-B/FSH-ratio. Although men with highest stress levels were characterized by an unhealthier lifestyle, adjusting for lifestyle factors did not attenuate results suggesting that the associations between stress and testicular function were not mediated by lifestyle. Stressful Life Events were not associated with testicular function. DISCUSSION AND CONCLUSION: The linear association between Perceived Stress and semen parameters and lack of dose-response association for the other two stress scales indicated that perceived stress was the most sensitive marker of stress affecting semen quality in young men. The lack of associations between Stressful Life Events and testis function confirmed that the perception of stressful events rather than the stressful event per se matters.

Evaluation of Circulating miRNA Biomarkers of Testicular Germ Cell Tumors during Therapy and Follow-up-A Copenhagen Experience.

New microRNA-based serum biomarkers (miRNA-367-3p, -371a-3p, -372-3p, and -373-3p) have shown great potential for the detection of testicular germ cell tumors (TGCTs), but few studies have investigated the clinical utility and performance of these tests in treatment monitoring. In this study, circulating miRNA levels were measured, together with serum tumor markers alpha-fetoprotein (AFP), ß-subunit of human chorionic gonadotropin (ß-HCG) and lactate dehydrogenase (LDH) in 406 consecutive blood samples obtained during the treatment and follow-up of 52 TGCT patients at the Copenhagen University Hospital. After testing three different methods of RNA isolation from peripheral blood and PCR quantification in a subset of samples (n = 15), the best performing setup of targeted isolation of miRNAs inside and outside exosomes was selected to analyze all samples. At primary diagnosis, the miRNAs significantly outperformed the serum tumor markers, with a sensitivity and specificity of 78% and 100% (based on 40 patients), respectively. The picture was not as clear when patient trajectories were investigated, with both positive and negative signals for miRNAs and serum tumor markers. To establish whether measuring miRNAs adds value beyond the primary diagnosis, large prospective clinical trials comparing miRNAs and classical tumor markers during the treatment and follow-up of TGCT patients are needed.

Application of miRNAs in the diagnosis and monitoring of testicular germ cell tumours.

Testicular germ cell tumours (TGCTs) are the most frequent cancer type in young men and originate from the common precursor germ cell neoplasia in situ (GCNIS). For decades, clinical management of patients with TGCT has relied on classic serum tumour markers: α-fetoprotein, human chorionic gonadotropin subunit-ß and lactate dehydrogenase. In the past 10 years, microRNAs have been shown to outperform classic serum tumour markers in the diagnosis of primary tumours and in follow-up monitoring and prediction of relapse. miR-371a-3p is the most consistent marker and exhibits >90% diagnostic sensitivity and specificity in TGCT. However, miR-371a-3p cannot be used to diagnose GCNIS or mature teratoma. Future efforts must technically standardize the microRNA-based methods internationally and introduce miR-371a-3p as a molecular liquid biopsy-based marker for TGCTs in the clinic.

Polymorphisms in JMJD1C are associated with pubertal onset in boys and reproductive function in men.

JMJD1C, a member of the Jumonji-domain containing histone demethylases protein family, has been associated with levels of sex-hormone binding globulin (SHBG) and testosterone in men, and knock-out rodent models show age-dependent infertility. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) nearby JMJD1C are associated with pubertal onset in boys and with male reproduction. 671 peri-pubertal boys, 1,027 young men, 315 fertile men, and 252 infertile men were genotyped for two JMJD1C SNPs (rs7910927 and rs10822184). rs7910927 and rs10822184 showed high linkage. Boys with the rs7910927 TT genotype entered puberty 3.6 months earlier than their peers (p = 2.5 × 10-2). In young men, the number of T alleles was associated with decreased levels of SHBG, follicle-stimulating hormone (FSH), testosterone, and testosterone x luteinizing hormone, as well as increased levels of Inhibin B, Inhibin B/FSH ratio, and testis size. No significant associations with semen parameters were observed and the genotype distribution was comparable among fertile and infertile men. In conclusion, genetic variation in the vicinity of JMJD1C had a surprisingly large impact on the age at pubertal onset in boys as well as levels of reproductive hormones and testis size in men, emphasizing the relationship between JMJD1C and reproductive functions.

ErbB2 upregulates the Na+,HCO3(-)-cotransporter NBCn1/SLC4A7 in human breast cancer cells via Akt, ERK, Src, and Kruppel-like factor 4.

Misregulation of acid-base transport plays central roles in cancer development. We previously demonstrated the strong up-regulation of the Na(+),HCO3(-) cotransporter NBCn1 (SLC4A7) in MCF-7 breast cancer cells by a truncated, constitutively active ErbB2 (HER2) receptor, ΔNErbB2, and showed that NBCn1 expression and activity are increased in breast cancer tissue from patients. Here, we present the first in-depth characterization of an SLC4A7 promoter and identify its minimal ΔNErbB2-sensitive region. Inhibition or siRNA-mediated knockdown of PI3K, Akt1, ERK1/2, or Src decreased the NBCn1 protein level in ΔNErbB2-expressing MCF-7 cells by ~50, 60, 30 and 35%, respectively. Further, knockdown of the transcription factor Krüppel-like factor 4 (KLF4) reduced NBCn1 protein expression by ~40%, and KLF4 overexpression increased NBCn1 expression by 50-80%. In contrast, knockdown of the closely related transcription factor specificity protein 1 (Sp1) or transfection with dominant-negative Sp1 increased NBCn1 expression by ~35 and ~50%, respectively. NBCn1 expression was also increased by stimulation of full-length ErbB1, -2, and -3 receptors in SKBr3 cells (1.5- and 2-fold by NRG1 or EGF, respectively) or after their exogenous expression in MCF-7 cells. Finally, stimulation with NRG1 or EGF more than doubled acid extrusion capacity in SKBr3 cells. In conclusion, NBCn1 is strongly upregulated by ErbB receptor signaling in a manner involving opposite effects of KLF4 and Sp1, transcription factors with central roles in cancer development. ErbB-induced up-regulation of NBCn1-mediated acid extrusion may play important physiological and pathophysiological roles in the breast epithelium and other tissues with high ErbB receptor levels.