BackgroundIt is well-established that spermatogenesis, semen quality, and reproductive hormones are interlinked. It is, however, less well-described how various specific testicular histopathologies are linked to reproductive hormones and semen quality.ObjectivesTo describe the detailed relationship between specific testicular histopathologies and the serum concentrations of reproductive hormones and semen quality.Materials and methodsDescriptive histological patterns on testicular biopsies from 4245 patients referred for andrological workup in our clinic between 1990 and 2022 were grouped according to a published histological coding system: (1) complete spermatogenesis (completeSPG, n = 3171), (2) reduced spermatogenesis (reducedSPG, n = 657), (3) heterogeneous (hetArrest, n = 226), and (4) homogeneous (homArrest, n = 191) spermatogenic arrest at the spermatocyte or spermatid stage. As a proxy for the number of spermatogonia, spermatocytes, and spermatids, immunohistochemical staining for MAGE-A4, PIWIL1, and TNP1 were quantified on a representative set of biopsies (n = 100). Serum concentrations of FSH, LH, T, SHBG, and inhibin B (n = 1813) and semen parameters (n = 833) were available.ResultsCompared with the completeSPG group, the number of spermatogonia was only reduced in the hetArrest group, while the number of spermatocytes and spermatids were lower in all groups. All groups had significantly higher FSH and LH and lower T, free T, and inhibin B concentrations when compared with the completeSPG group, except for the homArrest group, where inhibin B was unaffected. The hetArrest group had the lowest number of germ cells, the most pronounced change in reproductive hormones, and the lowest sperm counts. We found a strong correlation between the number of germ cells present and the corresponding serum concentrations of FSH, LH, T, and inhibin B.Discussion and ConclusionsA histopathological pattern of heterogeneous spermatogenic arrest is associated with a more severe phenotype than a pattern of homogeneous arrest, and the group with reduced spermatogenesis showed the mildest phenotype.

BackgroundPuberty 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 MethodsForty-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.ResultsWhen 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.ConclusionBoth 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.

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.

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

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.

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.

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-β 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.

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), β-subunit of human chorionic gonadotropin (β-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.

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.

STUDY QUESTIONHow are temporal trends in lifestyle factors, including exposure to maternal smoking in utero, associated to semen quality in young men from the general population?SUMMARY ANSWERExposure to maternal smoking was associated with lower sperm counts but no overall increase in sperm counts was observed during the study period despite a decrease in this exposure.WHAT IS KNOWN ALREADYMeta-analyses suggest a continuous decline in semen quality but few studies have investigated temporal trends in unselected populations recruited and analysed with the same protocol over a long period and none have studied simultaneous trends in lifestyle factors.STUDY DESIGN, SIZE, DURATIONCross-sectional population-based study including ~300 participants per year (total number = 6386) between 1996 and 2016.PARTICIPANTS/MATERIALS, SETTING, METHODSThe study is based on men from the Greater Copenhagen area, Denmark, with a median age of 19 years, and unselected with regard to fertility status and semen quality. The men delivered a semen sample, had a blood sample drawn and a physical examination performed and answered a comprehensive questionnaire, including information on lifestyle and the mother's pregnancy. Temporal trends in semen quality and lifestyle were illustrated graphically, and trends in semen parameters and the impact of prenatal and current lifestyle factors were explored in multiple regression analyses.MAIN RESULTS AND THE ROLE OF CHANCEThroughout the study period, 35% of the men had low semen quality. Overall, there were no persistent temporal trends in semen quality, testicular volume or levels of follicle-stimulating hormone over the 21 years studied. The men's alcohol intake was lowest between 2011 and 2016, whereas BMI, use of medication and smoking showed no clear temporal trends. Parental age increased, and exposure in utero to maternal smoking declined from 40% among men investigated in 1996-2000 to 18% among men investigated in 2011-2016. Exposure to maternal smoking was associated with lower sperm counts but no overall increase in sperm counts was observed despite the decrease in this exposure.LIMITATIONS, REASONS FOR CAUTIONInformation of current and prenatal lifestyle was obtained by self-report, and the men delivered only one semen sample each.WIDER IMPLICATIONS OF THE FINDINGSThe significant decline in in utero exposure to maternal smoking, which was not reflected in an overall improvement of semen quality at the population level, suggest that other unknown adverse factors may maintain the low semen quality among Danish men.STUDY FUNDING/COMPETING INTEREST(S)The study has received financial support from the ReproUnion; the Research fund of Rigshospitalet, Copenhagen University Hospital; the European Union (Contract numbers BMH4-CT96-0314,QLK4-CT-1999-01422, QLK4-CT-2002-00603, FP7/2007-2013, DEER Grant agreement no. 212844); the Danish Ministry of Health; the Danish Environmental Protection Agency; A.P. Møller and wife Chastine McKinney Møllers foundation; and Svend Andersens Foundation. None of the funders had any role in the study design, collection, analysis or interpretation of data, writing of the paper or publication decisions.TRIAL REGISTRATION NUMBERN/A.

Chronic angiogenesis is a hallmark of most tumors and takes place in a hostile tumor microenvironment (TME) characterized by hypoxia, low nutrient and glucose levels, elevated lactate and low pH. Despite this, most studies addressing angiogenic signaling use hypoxia as a proxy for tumor conditions. Here, we compared the effects of hypoxia and TME conditions on regulation of the Na+/H+ exchanger NHE1, Ser/Thr kinases Akt1–3, and downstream effectors in endothelial cells.

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.

Perceived stress has been associated with decreased semen quality but the mechanisms have not been elucidated. It is not known whether cortisol, the major stress hormone in humans, can act directly via receptors in the testis, and whether variants in the gene encoding the glucocorticoid receptor (NR3C1) can possibly modulate the effect. To address these questions, we investigated the expression of the glucocorticoid receptor in human testicular tissue, including adult and fetal samples (n = 20) by immunohistochemical staining, and in silico analysis of publicly available datasets. In the adult testis NR3C1 protein was detected in peritubular cells, a subset of Leydig cells, Sertoli cells (weak), and spermatogonia, but not in spermatids. The NR3C1 expression pattern in fetal testis samples differed by a notably stronger reaction in Sertoli cells, lack of staining in gonocytes but the presence in a subset of pro-spermatogonia, and the almost absent reaction in nascent peritubular cells. In parallel, we explored the association between adult testicular function and three single nucleotide NR3C1 polymorphisms (BcII [rs41423247], 9β [rs6198], and Tth111I [rs10052957]) affecting glucocorticoid sensitivity. Testicular function was determined by semen analysis and reproductive hormone profiling in 893 men from the general population. The NR3C1 SNP BclI was associated with semen quality in an over-dominant manner with heterozygotes having better semen parameters compared to both homozygote constellations, and with sperm motility showing the strongest association. This association was supported by a higher inhibin B and inhibin B/FSH ratio, as well as a lower FSH in BclI heterozygotes. The SNPs 9β and Tth111I were not associated with semen parameters. Although the clinical impact of the findings is limited, the results substantiate a suggested link between stress and testicular function. Hence this investigation should be regarded as a discovery study generating hypotheses for future studies.

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.