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Study Question: Are infertile men with reduced semen quality at risk of a further decrease in testicular function?Summary Answer: Infertile men with severely reduced semen quality risk further deterioration of semen quality 15 years after treatment for infertility, and a lower baseline sperm concentration was associated with a more pronounced increase in LH and decrease in testosterone/LH ratio at follow-up.What Is Known Already: Male factors account for up to 50% of human infertility. The most common finding is spermatogenic failure (SgF) yet the life course of semen quality and testosterone production in such men has not been described.Study Design, Size, Duration: A follow-up study of men with SgF was performed 15 years after the initial infertility assessment between January 1995 and December 2000.Participants/materials, Setting, Methods: Hospital records were used to identify potential participants in the study. A total of 137 men with primary male infertility due to SgF and 70 controls with good semen quality from couples with female factor infertility who attended a tertiary referral centre were included: the participation rate was 31% and 26%, respectively. The men provided semen samples and underwent a physical examination. Blood samples were taken to measure levels of reproductive hormones (FSH, LH, testosterone, sex hormone-binding globulin, estradiol and inhibin B). Current results were compared with results from the initial assessments.Main Results and the Role Of Chance: At the time of follow up the SgF men had significantly lower Leydig cell capacity than the control group as well as much lower semen quality. For the SgF men, between baseline sampling and follow up, the median sperm concentration decreased from 1.9 to 0.6 mill/ml and total sperm count from 7.7 to 2.0 million (P = 0.019 and 0.012, respectively), and 10% developed azoospermia. Calculated free testosterone (cFT), but not total testosterone (tT) decreased in the SgF group by ~0.6% (95% CI 0.1-1.2%) per year. In the SgF group, LH increased by 1.6% (CI 0.9-2.3%) annually, and consequently tT/LH and cFT/LH ratios had decreased by 1.3% (CI 0.5-2.1) and 2.1% (CI 1.2-3.0%), respectively. The increase in LH and the decreases in tT/LH and cFT/LH ratios were more pronounced in men with lower baseline sperm concentrations.Limitations, Reasons For Caution: We consider the case group as representative of infertile men not in need of testosterone treatment at baseline investigation, but do not have information on those that chose not to participate in the follow-up study. There were alterations in some hormone analysis methods during the follow-up period that may introduce uncertainty in interpretation of long-term changes in hormone levels despite rigorous quality control. The validity of the control group suffers from a lack of hormone values at baseline. Also, at follow-up, for practical reasons only one semen sample could be obtained, which makes the effect estimate more uncertain and there is a risk of non-differential misclassification.Wider Implications Of the Findings: Without being able to predict individual outcomes, it is prudent to consider sperm cryopreservation or advise not to postpone fertility treatment when men present with infertility due to impaired semen quality. Whether partly compensated Leydig cell insufficiency in men with SgF will eventually develop into overt testosterone deficiency cannot be determined from our study.Study Funding/competing Interest(s): Aase and Einar Danielsen (Grant no. 10-001053), Nordic Research Committee (Grant no. 5109), The Kirsten and Freddie Johansen Fund, and Rigshospitalet's Research Fund (grant no. R24-A812). There are no competing interests. [ABSTRACT FROM AUTHOR]

Background: Developmental arrest of fetal germ cells may lead to neoplastic transformation and formation of germ cell tumours via carcinoma in situ (CIS) cells. Normal fetal germ cell development requires complete erasure and re-establishment of DNA methylation. In contrast to normal spermatogonia, the genome of CIS cells remains unmethylated in the adult testis. We here investigated the possible active and passive pathways that can sustain the CIS genome hypomethylated in the adult testis.Methods: The levels of 5-methyl-cytosine (5mC) and 5-hydroxy-methyl-cytosine (5hmC) in DNA from micro-dissected CIS cells were assessed by quantitative measurements. The expression of TET1, TET2, APOBEC1, MBD4, APEX1, PARP1, DNMT1, DNMT3A, DNMT3B and DNMT3L in adult testis specimens with CIS and in human fetal testis was investigated by immunohistochemistry and immunofluorescence.Results: DNA from micro-dissected CIS cells contained very low levels of 5hmC produced by ten eleven translocation (TET) enzymes. CIS cells and fetal germ cells expressed the suggested initiator of active demethylation, APOBEC1, and the base excision repair proteins MBD4, APEX1 and PARP1, whereas TETs - the alternative initiators were absent. Both maintenance and de novo methyltransferases were detected in CIS cells.Conclusion: The data are consistent with the presence of an active DNA de-methylation pathway in CIS cells. The hypomethylated genome of CIS cells may contribute to phenotypic plasticity and invasive capabilities of this testicular cancer precursor. [ABSTRACT FROM AUTHOR]

Tortoiseshell coat color is normally restricted to female cats due to X-linkage of the gene that encodes the orange coat color. Tortoiseshell male cats do, however, occur at a low frequency among tortoiseshell cats because of chromosome aberrations similar to the Klinefelter syndrome in man: the extra X chromosome of a 39,XXY karyotype introduces the possibility of an orange and a non-orange allele which produce the mixture of orange and non-orange coat spotting known as tortoiseshell. We analyzed the chromosome complement of a fibroblast culture and did histological examinations of testicular tissue from a tortoiseshell male cat referred to us. Chromosome analysis using RBA-banding consistently revealed a 39,XXY karyotype. Histological examinations of testis biopsies from this cat showed degeneration of the tubules, hyperplasia of the interstitial tissue, and complete loss of germ cells. Immunostaining using anti-vimentin and anti-VASA (DDX4) showed that only Sertoli cells and no germ cells were observed in the testicular tubules. As no sign of spermatogenesis was detected, we conclude that this is a classic case of a sterile, male tortoiseshell cat with a 39,XXY chromosome complement. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The carcinoma in situ (CIS) cell is the common precursor of nearly all testicular germ cell tumours (TGCT). In a previous study, we examined the gene expression profile of CIS cells and found many features common to embryonic stem cells indicating that initiation of neoplastic transformation into CIS occurs early during foetal life. Progression into an overt tumour, however, typically first happens after puberty, where CIS cells transform into either a seminoma (SEM) or a nonseminoma (N-SEM). Here, we have compared the genome-wide gene expression of CIS cells to that of testicular SEM and a sample containing a mixture of N-SEM components, and analyse the data together with the previously published data on CIS. Genes showing expression in the SEM or N-SEM were selected, in order to identify gene expression markers associated with the progression of CIS cells. The identified markers were verified by reverse transcriptase-polymerase chain reaction and in situ hybridisation in a range of different TGCT samples. Verification showed some interpatient variation, but combined analysis of a range of the identified markers may discriminate TGCT samples as SEMs or N-SEMs. Of particular interest, we found that both DNMT3B (DNA (cytosine-5-)-methyltransferase 3 beta) and DNMT3L (DNA (cytosine-5-)-methyltransferase 3 like) were overexpressed in the N-SEMs, indicating the epigenetic differences between N-SEMs and classical SEM.British Journal of Cancer (2005) 92, 1934-1941. doi:10.1038/sj.bjc.6602560 www.bjcancer.com Published online 26 April 2005 [ABSTRACT FROM AUTHOR]

An introduction is presented in which editor discusses the various articles within the issue on topics including genetic variants of testicular cancer, germ cell tumors in children and association between testicular cancer and congenital malformations.

Testicular germ cell cancer manifests mainly in young adults as a seminoma or non-seminoma. The solid tumors are preceded by the presence of a non-invasive precursor cell, the carcinoma in situ cell (CIS), which shows great similarity to fetal germ cells. It is therefore hypothesized that the CIS cell is a fetal germ cell that has been arrested during development due to testicular dysgenesis. CIS cells retain a fetal and open chromatin structure, and recently several epigenetic modifiers have been suggested to be involved in testicular dysgenesis in mice. We here review the possible involvement of epigenetic modifiers with a focus on jumonji C enzymes in the development of testicular dysgenesis and germ cell cancer in men.

Abstract Background Irradiation or chemotherapy that suspend normal spermatogenesis is commonly used to treat various cancers. Fortunately, spermatogenesis in many cases can be restored after such treatments but knowledge is limited about the re-initiation process. Earlier studies have described the cellular changes that happen during recovery from irradiation by means of histology. We have earlier generated gene expression profiles during induction of spermatogenesis in mouse postnatal developing testes and found a correlation between profiles and the expressing cell types. The aim of the present work was to utilize the link between expression profile and cell types to follow the cellular changes that occur during post-irradiation recovery of spermatogenesis in order to describe recovery by means of gene expression. Methods Adult mouse testes were subjected to irradiation with 1 Gy or a fractionated radiation of two times 1 Gy. Testes were sampled every third or fourth day to follow the recovery of spermatogenesis and gene expression profiles generated by means of differential display RT-PCR. In situ hybridization was in addition performed to verify cell-type specific gene expression patterns. Results Irradiation of mice testis created a gap in spermatogenesis, which was initiated by loss of A1 to B-spermatogonia and lasted for approximately 10 days. Irradiation with 2 times 1 Gy showed a more pronounced effect on germ cell elimination than with 1 Gy, but spermatogenesis was in both cases completely reconstituted 42 days after irradiation. Comparison of expression profiles indicated that the cellular reconstitution appeared equivalent to what is observed during induction of normal spermatogenesis. Conclusion The data indicates that recovery of spermatogenesis can be monitored by means of gene expression, which could aid in designing radiation treatment regimes for cancer patients leading to better restoration of spermatogenesis.

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.). [ABSTRACT FROM AUTHOR]

Whether DNA methylation changes follow human physical trauma is uncertain. We aimed to investigate if severe trauma was associated with DNA methylation changes. In a prospective, observational, clinical study, we included severely injured adults and adults undergoing elective surgery (controls). Blood was obtained from trauma patients (n = 60) immediately- and 30-45 days post-trauma, and from surgical patients (n = 57) pre-, post-, and 30-45 days post-surgery. Epigenome-wide DNA methylation profiling was performed and analyzed for significant differentially methylated CpGs and -regions (DMRs) within and between groups. Within the trauma group we identified 10,126 significant differentially methylated CpGs and 1169 DMRs. No significant differential methylation was found in the surgical group. In the trauma group, differentially methylated sites were enriched in genes and pathways involved in blood coagulation and inflammatory response. Severe trauma was associated with profound alterations in the DNA methylome of circulating leucocytes, and differential methylation was located in trauma-relevant genes., (© 2024. The Author(s).)

Childhood obesity is a significant global health issue with complex and multifactorial origins, often beginning before conception and influenced by both maternal and paternal health. The increased prevalence of pre-pregnancy obesity and gestational diabetes mellitus in women of reproductive age contributes to a heightened risk of metabolic dysfunction in offspring. Current clinical practices often implement lifestyle interventions after the first trimester, and have limited success, implying that they miss a critical window for effective metabolic adjustments. This review examines the limitations of lifestyle interventions during pregnancy in improving perinatal outcomes and highlights the importance of initiating such interventions before conception to positively impact parental health and fetal development. A re-evaluation of strategies is needed to enhance the metabolic health of prospective parents as a preventive measure against childhood obesity., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Research Question: Are the prospective reproductive outcomes in couples experiencing recurrent pregnancy loss (RPL) related to the sperm DNA fragmentation index (DFI), as measured by sperm chromatin structure assay, sperm morphology and sperm concentration at referral?, Design: This prospective cohort study included 95 couples seen between 1 April 2018 and 1 December 2019 at the tertiary Copenhagen RPL Unit, Copenhagen University Hospital, Rigshospitalet and Hvidovre Hospital, Denmark. The couples had experienced three or more unexplained consecutive pregnancy losses or two late pregnancy losses (>12 weeks gestation). Follow-up was 12-31 months., Results: Eighty-one of 95 (85.3%) couples achieved pregnancy after referral. In the first pregnancy after referral, 46 (56.8%) couples achieved a live birth, and 35 (43.2%) couples experienced another pregnancy loss. There was no significant difference in baseline DFI between couples that experienced pregnancy loss [median 11.7, interquartile range (IQR) 9.1-17.3] and couples that achieved a live birth (median 12.5, IQR 9.3-16.5; P = 0.971). Improving sperm morphology increased the odds of a live birth after referral (adjusted OR 1.26, 95% CI 1.05-1.52; P = 0.014). DFI and sperm concentration were not associated with the outcome of the first pregnancy after referral. Overall, 35.9% of the men had DFI ≥15 at inclusion. Couples that failed to achieve pregnancy had a higher median DFI of 17.7 (IQR 7.7-27.2) compared with the rest of the cohort (median 12.0, IQR 9.3-16.5; P = 0.041)., Conclusions: At referral, sperm DFI, morphology and concentration cannot be used to identify RPL couples at risk of another pregnancy loss. Increased baseline DFI was associated with difficulty achieving another pregnancy, and improving sperm morphology was associated with increased odds of a live birth., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Klinefelter syndrome (47,XXY) causes infertility with a testicular histology comprising two types of Sertoli cell-only tubules, representing mature and immature-like Sertoli cells, and occasionally focal spermatogenesis. Here, we show that the immature-like Sertoli cells highly expressed XIST and had two X-chromosomes, while the mature Sertoli cells lacked XIST expression and had only one X-chromosome. Sertoli cells supporting focal spermatogenesis also lacked XIST expression and the additional X-chromosome, while the spermatogonia expressed XIST despite having only one X-chromosome. XIST was expressed in Sertoli cells until puberty, where a gradual loss was observed. Our results suggest that a micro-mosaic loss of the additional X-chromosome is needed for Sertoli cells to mature and to allow focal spermatogenesis., (© 2024. The Author(s).)

Testicular germ cell tumours (TGCTs) derived from immature (type I) and pluripotent germ cell neoplasia in situ (GCNIS, type II) are characterised by remarkable phenotypic heterogeneity and plasticity. In contrast, the rare spermatocytic tumour (SpT, type III), derived from mature spermatogonia, is considered a homogenous and benign tumour but may occasionally present as an anaplastic or an aggressive sarcomatoid tumour. While various oncogenic processes had been proposed, the precise mechanism driving malignant progression remained elusive until the molecular characterisation of a series of atypical SpTs described in a recent issue of The Journal of Pathology. The emerging picture suggests the presence of two distinct trajectories for SpTs, involving either RAS/mitogen-activated protein kinase pathway mutations or a ploidy shift with secondary TP53 mutations and/or gain of chromosome 12p, the latter known as pathognomonic for type II GCNIS-derived TGCTs. Here, we discuss the implications of these findings, seen from the perspective of germ cell biology and the unique features of different TGCTs. The evolving phenotype of SpTs, induced by genomic and epigenetic changes, illustrates that the concept of plasticity applies to all germ cell tumours, making them inherently heterogenous and capable of significant transformation during progression. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Background: Development of the gonads during fetal life is complex and vital for adult reproductive health. Cell and animal studies have shown an alarming effect of mild analgesics on germ cells in both males and females. More than 50% of pregnant women use mild analgesics during pregnancy, which potentially could compromise the reproductive health of the next generation., Objectives: We present a research protocol designed to evaluate the effect of prenatal exposure to mild analgesics and endocrine-disrupting chemicals on gonadal function in the offspring., Population: Healthy, singleton pregnant women and their partners., Design: The COPANA cohort is a prospective, observational pregnancy and birth cohort., Methods: Participants were enrolled during the first trimester of pregnancy. Information on the use of mild analgesics was collected retrospectively 3 months prior to pregnancy and prospectively every 2 weeks throughout the study. We collected extensive data on lifestyle and reproductive health. Biospecimens were collected in the first trimester (maternal and paternal urine- and blood samples), in the third trimester in conjunction with a study-specific ultrasound scan (maternal urine sample), and approximately 3 months post-partum during the infant minipuberty period (maternal and infant urine- and blood samples). A comprehensive evaluation of reproductive function in the infants during the minipuberty phase was performed, including an ultrasound scan of the testis or ovaries and uterus., Preliminary Results: In total, 685 pregnant women and their partners were included between March 2020 and January 2022. A total of 589 infants (287 males) and their parents completed the follow-up during the minipuberty phase (December 2020-November 2022)., Conclusions: The Copenhagen Analgesic Study holds the potential to provide novel and comprehensive insights into the impact of early and late prenatal exposure to mild analgesics and other endocrine-disrupting chemicals on future reproductive function in the offspring., (© 2024 The Authors. Paediatric and Perinatal Epidemiology published by John Wiley & Sons Ltd.)

The most prominent RNA modification - N6-methyladenosine (m6A) - affects gene regulation and cancer progression. The extent and effect of m6A on long non-coding RNAs (lncRNAs) is, however, still not clear. The most established method for m6A detection is methylated RNA immunoprecipitation and sequencing (MeRIP-seq). However, Oxford Nanopore Technologies recently developed direct RNA-seq (dRNA-seq) method, allowing m6A identification at higher resolution and in its native form. We performed whole transcriptome sequencing of the glioblastoma cell line U87-MG with both MeRIP-seq and dRNA-seq. For MeRIP-seq, m6A peaks were identified using nf-core/chipseq, and for dRNA-seq - EpiNano pipeline. MeRIP-seq analysis revealed 5086 lncRNAs transcripts, while dRNA-seq identified 336 lncRNAs transcripts from which 556 and 198 were found to be m6A modified, respectively. While 24 lncRNAs with m6A overlapped between two methods. Gliovis database analysis revealed that the expression of the major part of identified overlapping lncRNAs was associated with glioma grade or patient survival prognosis. We found that the frequency of m6A occurrence in lncRNAs varied more than 9-fold throughout the provided list of 24 modified lncRNAs. The highest m6A frequency was detected in MIR1915HG, THAP9-AS1, MALAT1, NORAD1 , and NEAT1 ( 49-88nt), while MIR99AHG, SNHG3, LOXL1-AS1, ILF3-DT showed the lowest m6A frequency (445-261nt). Taken together, (1) we provide a high accuracy list of 24 m6A modified lncRNAs of U87-MG cells; (2) we conclude that MeRIP-seq is more suitable for an initial m6A screening study, due to its higher lncRNA coverage, whereas dRNA-seq is most useful when more in-depth analysis of m6A quantity and precise location is of interest. Abbreviations: (dRNA-seq) direct RNA-seq, (GBM) glioblastoma, (LGG) low-grade glioma, (lncRNAs) long non-coding RNAs, (m6A) N6-methyladenosine, (MeRIP-seq) methylated RNA immunoprecipitation and sequencing, (ncRNA) non-coding RNA, (ONT) Oxford Nanopore Technologi; Lietuvos Mokslo Taryba.

A major part of the human Y chromosome consists of palindromes with multiple copies of genes primarily expressed in testis, many of which have been claimed to affect male fertility. Here we examine copy number variation in these palindromes based on whole genome sequence data from 11,527 Icelandic men. Using a subset of 7947 men grouped into 1449 patrilineal genealogies, we infer 57 large scale de novo copy number mutations affecting palindrome 1. This corresponds to a mutation rate of 2.34 × 10 -3 mutations per meiosis, which is 4.1 times larger than our phylogenetic estimate of the mutation rate (5.72 × 10 -4 ), suggesting that de novo mutations on the Y are lost faster than expected under neutral evolution. Although simulations indicate a selection coefficient of 1.8% against non-reference copy number carriers, we do not observe differences in fertility among sequenced men associated with their copy number genotype, but we lack statistical power to detect differences resulting from weak negative selection. We also perform association testing of a diverse set of 341 traits to palindromic copy number without any significant associations. We conclude that large-scale palindrome copy number variation on the Y chromosome has little impact on human phenotype diversity., (© 2023. The Author(s).)

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., (© 2022 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mørup, Stakaitis, Main, Golubickaite, Hagen, Juul and Almstrup.)

The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals 1-6 , probably owing to the evolutionary pressure on males to be reproductively successful 7 . However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals., (© 2022. The Author(s).)

Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human "knockouts", and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification., (© 2022. The Author(s).)

Objective: Self-reported psychological stress has been associated with decreased semen quality. Cortisol levels in scalp hair (hair cortisol concentration, HCC) has emerged as a potential objective marker of psychological stress. Thus, we investigated if HCC was associated with markers of testicular function. Furthermore, we examined whether three common single nucleotide polymorphisms in the glucocorticoid-receptor gene (NR3C1, chromosome 5), potentially affecting receptor sensitivity, were associated with HCC and could influence the studied association between HCC and testicular function., Design: Cross-sectional study., Methods: We analysed HCC, serum-levels of reproductive hormones, semen parameters, and the three NR3C1-polymorphisms; BclI (rs41423247), Tth111I (rs10052957), and 9β (rs6198), in a population of 696 men from the general population., Results: HCC was not associated with testicular function, and adjustment for the three NR3C1-polymorphisms did not alter the results. However, HCC increased significantly with the number of Tth111I minor-alleles (T) and decreased significantly with the number of 9β minor-alleles (G)., Conclusion: Given previously shown associations between stress and semen quality, and that no association between HCC and self-reported stress was observed in the current study, we speculate that negative reproductive effects of stress may not be mediated directly by cortisol. This study demonstrates associations between HCC and glucocorticoid receptor gene variants indicating that these SNPs may influence systemic glucocorticoid levels, but the potential health effects of such alterations are yet unknown., Competing Interests: Declaration of Competing Interest There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© 2022 The Authors. WIREs Mechanisms of Disease published by Wiley Periodicals LLC.)

Objective: To evaluate whether optimized and standardized diagnostic procedures would improve detection of germ cell neoplasia in situ (GCNIS) in the contralateral testis of patients with testicular germ cell tumour (TGCT) and decrease the rate of metachronous tumours, which in a nationwide Danish study was estimated to be 1.9%., Patients and Methods: This was a retrospective analysis of outcomes in 655 patients with TGCT who underwent contralateral biopsies (1996-2007) compared with those in 459 non-biopsied TGCT controls (1984-1988). The biopsies were performed using a standardized procedure with immunohistochemical GCNIS markers and assessed by experienced evaluators. Initial histopathology reports were reviewed, and pathology and survival data were retrieved from national Danish registers. In 604/608 patients diagnosed as GCNIS-negative (four were lost to follow-up), the cumulative incidence of metachronous TGCT was estimated in a competing risk setting using the Grey method. All cases of metachronous TGCT were re-examined using immunohistochemistry., Results: Germ cell neoplasia in situ was found in 47/655 biopsied patients (7.2%, 95% confidence interval [CI] 5.4-9.5%). During the follow-up period (median 17.3 years) five of the 604 GCNIS-negative patients developed a TGCT. In 1/5 false-negative biopsies, GCNIS was found on histological revision using immunohistochemistry and 2/5 biopsies were inadequate because of too small size. The estimated cumulative incidence rate of second tumour after 20 years of follow-up was 0.95% (95% CI 0.10%-1.8%) compared with 2.9% (95% CI 1.3%-4.4%) among the non-biopsied TGCT patients (P = 0.012). The estimates should be viewed with caution due to the small number of patients with metachronous TGCT., Conclusions: Optimized diagnostic procedures improved the detection rate of GCNIS in patients with TGCT and minimized their risk of developing metachronous bilateral cancer. Urologists should be aware of the importance of careful tissue excision (to avoid mechanical compression) and the need of adequate biopsy size. Performing contralateral biopsies is beneficial for patients' care and should be offered as a part of their management., (© 2022 The Authors. BJU International published by John Wiley & Sons Ltd on behalf of BJU International.)

Infertility affects around 7% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with SPGF and detected rare bi-allelic loss-of-function variants in FKBP6 in five additional persons. All six individuals had no or extremely few sperm in the ejaculate, which were not suitable for medically assisted reproduction. Evaluation of testicular tissue revealed an arrest at the stage of round spermatids. Lack of FKBP6 expression in the testis was confirmed by RT-qPCR and immunofluorescence staining. In mice, Fkbp6 is essential for spermatogenesis and has been described as being involved in piRNA biogenesis and formation of the synaptonemal complex (SC). We did not detect FKBP6 as part of the SC in normal human spermatocytes, but small RNA sequencing revealed that loss of FKBP6 severely impacted piRNA levels, supporting a role for FKBP6 in piRNA biogenesis in humans. In contrast to findings in piRNA-pathway mouse models, we did not detect an increase in LINE-1 expression in men with pathogenic FKBP6 variants. Based on our findings, FKBP6 reaches a "strong" level of evidence for being associated with male infertility according to the ClinGen criteria, making it directly applicable for clinical diagnostics. This will improve patient care by providing a causal diagnosis and will help to predict chances for successful surgical sperm retrieval., Competing Interests: Declaration of interests C.N.J. has an Investigator-led grant from Logixx Pharma Ltd, UK., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)

Background: Couples increasingly experience infertility and seek help from assisted reproductive techniques to become pregnant. However, 5%-15% of the couples that are selected for in vitro fertilisation (IVF) experience a total fertilisation failure (TFF), where no zygotes develop despite oocytes and semen parameters appear to be normal. We hypothesise that TFF during IVF could be related to improper membrane fusion of gametes., Objective: To investigate the membrane integrity and fusion proteins in spermatozoa from men in couples experiencing TFF., Materials and Methods: A total of 33 infertile couples, 17 of which experienced TFF during IVF and 16 matched control couples with normal IVF fertilisation rates, were selected and the men re-called to deliver an additional semen sample. Proteins involved in gamete membrane fusion on spermatozoa (IZUMO1, SPESP1 and Syncytin-1) as well as O-glycosylation patterns (Tn and GALNT3), were investigated by immunofluorescence. The DNA fragmentation index, acrosomal integrity and viability of spermatozoa were determined by flow and image cytometry., Results: No significant changes in the expression of GALNT3, Tn and Syncytin-1 were observed between the TFF and control groups. The fraction of spermatozoa expressing SPESP1, the median IZUMO1 staining intensity, and the percentage of viable acrosome-intact spermatozoa were significantly lower in the TFF group compared to controls. Furthermore, following progesterone-induced acrosomal exocytosis, a significant difference in the fraction of spermatozoa expressing SPESP1 and the median IZUMO1 staining intensity were observed between the control and TFF group., Discussion and Conclusion: Our results indicate that acrosomal exocytosis, IZUMO1 and SPESP1 expression in spermatozoa could play a crucial role in achieving fertilisation during IVF. However, the size of our cohort was quite small, and our results need to be validated with quantitative methods in larger cohorts., (© 2022 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

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., (©2022 American Association for Cancer Research.)

Study Question: What is the load, distribution and added clinical value of secondary findings (SFs) identified in exome sequencing (ES) of patients with non-obstructive azoospermia (NOA)?, Summary Answer: One in 28 NOA cases carried an identifiable, medically actionable SF., What Is Known Already: In addition to molecular diagnostics, ES allows assessment of clinically actionable disease-related gene variants that are not connected to the patient's primary diagnosis, but the knowledge of which may allow the prevention, delay or amelioration of late-onset monogenic conditions. Data on SFs in specific clinical patient groups, including reproductive failure, are currently limited., Study Design, Size, Duration: The study group was a retrospective cohort of patients with NOA recruited in 10 clinics across six countries and formed in the framework of the international GEMINI (The GEnetics of Male INfertility Initiative) study., Participants/materials, Setting, Methods: ES data of 836 patients with NOA were exploited to analyze SFs in 85 genes recommended by the American College of Medical Genetics and Genomics (ACMG), Geisinger's MyCode, and Clinical Genome Resource. The identified 6374 exonic variants were annotated with ANNOVAR and filtered for allele frequency, retaining 1381 rare or novel missense and loss-of-function variants. After automatic assessment of pathogenicity with ClinVar and InterVar, 87 variants were manually curated. The final list of confident disease-causing SFs was communicated to the corresponding GEMINI centers. When patient consent had been given, available family health history and non-andrological medical data were retrospectively assessed., Main Results and the Role of Chance: We found a 3.6% total frequency of SFs, 3.3% from the 59 ACMG SF v2.0 genes. One in 70 patients carried SFs in genes linked to familial cancer syndromes, whereas 1 in 60 cases was predisposed to congenital heart disease or other cardiovascular conditions. Retrospective assessment confirmed clinico-molecular diagnoses in several cases. Notably, 37% (11/30) of patients with SFs carried variants in genes linked to male infertility in mice, suggesting that some SFs may have a co-contributing role in spermatogenic impairment. Further studies are needed to determine whether these observations represent chance findings or the profile of SFs in NOA patients is indeed different from the general population., Limitations, Reasons for Caution: One limitation of our cohort was the low proportion of non-Caucasian ethnicities (9%). Additionally, as comprehensive clinical data were not available retrospectively for all men with SFs, we were not able to confirm a clinico-molecular diagnosis and assess the penetrance of the specific variants., Wider Implications of the Findings: For the first time, this study analyzed medically actionable SFs in men with spermatogenic failure. With the evolving process to incorporate ES into routine andrology practice for molecular diagnostic purposes, additional assessment of SFs can inform about future significant health concerns for infertility patients. Timely detection of SFs and respective genetic counseling will broaden options for disease prevention and early treatment, as well as inform choices and opportunities regarding family planning. A notable fraction of SFs was detected in genes implicated in maintaining genome integrity, essential in both mitosis and meiosis. Thus, potential genetic pleiotropy may exist between certain adult-onset monogenic diseases and NOA., Study Funding/competing Interest(s): This work was supported by the Estonian Research Council grants IUT34-12 and PRG1021 (M.L. and M.P.); National Institutes of Health of the United States of America grant R01HD078641 (D.F.C., K.I.A. and P.N.S.); National Institutes of Health of the United States of America grant P50HD096723 (D.F.C. and P.N.S.); National Health and Medical Research Council of Australia grant APP1120356 (M.K.O'B., D.F.C. and K.I.A.); Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação grant POCI-01-0145-FEDER-007274 (A.M.L., F.C. and J.G.) and FCT: IF/01262/2014 (A.M.L.). J.G. was partially funded by FCT/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), through the Centre for Toxicogenomics and Human Health-ToxOmics (grants UID/BIM/00009/2016 and UIDB/00009/2020). M.L.E. is a consultant for, and holds stock in, Roman, Sandstone, Dadi, Hannah, Underdog and has received funding from NIH/NICHD. Co-authors L.K., K.L., L.N., K.I.A., P.N.S., J.G., F.C., D.M.-M., K.A., K.A.J., M.K.O'B., A.M.L., D.F.C., M.P. and M.L. declare no conflict of interest., Trial Registration Number: N/A., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A severe decline in child births has occurred over the past half century, which will lead to considerable population declines, particularly in industrialized regions. A crucial question is whether this decline can be explained by economic and behavioural factors alone, as suggested by demographic reports, or to what degree biological factors are also involved. Here, we discuss data suggesting that human reproductive health is deteriorating in industrialized regions. Widespread infertility and the need for assisted reproduction due to poor semen quality and/or oocyte failure are now major health issues. Other indicators of declining reproductive health include a worldwide increasing incidence in testicular cancer among young men and alterations in twinning frequency. There is also evidence of a parallel decline in rates of legal abortions, revealing a deterioration in total conception rates. Subtle alterations in fertility rates were already visible around 1900, and most industrialized regions now have rates below levels required to sustain their populations. We hypothesize that these reproductive health problems are partially linked to increasing human exposures to chemicals originating directly or indirectly from fossil fuels. If the current infertility epidemic is indeed linked to such exposures, decisive regulatory action underpinned by unconventional, interdisciplinary research collaborations will be needed to reverse the trends., (© 2021. Springer Nature Limited.)

Context: IGF-I is important for postnatal growth and may be of diagnostic value in infants suspected of pituitary disease; however, little is known about the impact of IGF-I and its determinants on infant growth. Importantly, detailed reference ranges for IGF-I and IGF binding protein-3 (IGFBP-3) concentrations during infancy are lacking., Objective: To evaluate the rapid changes in weight and length as well as their determinants in healthy infants, and to establish age- and sex-specific reference curves for IGF-I and IGFBP-3 in children aged 0 to 1 years., Design: Prospective longitudinal study., Setting: Cohort study., Participants: A total of 233 healthy children (114 girls) with repeated blood samples during the first year of life., Main Outcome Measure(s): Serum concentrations of IGF-I and IGFBP-3, length velocity, weight velocity, and PAPPA2 (rs1325598) genotype., Results: Individual trajectories of length and weight velocities were sex specific. We provide detailed reference curves based on longitudinal data for IGF-I and IGFBP-3 during infancy. In both girls and boys, IGF-I decreased during infancy, whereas IGFBP-3 remained stable. IGF-I and IGFBP-3, but not PAPPA2 genotype, were positively associated with weight gain, but not with longitudinal growth. When stratified by sex, the association between weight gain and IGF-I only remained significant in girls., Conclusions: Interestingly, we found a significant association between IGF-I and infant weight gain in girls, but not with longitudinal growth in the first year of life. Our findings highlight the role of IGF-I as an important anabolic hormone that is not limited to linear growth., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Infertility affects 15%-25% of all couples during their reproductive life span. It is a significant societal and public health problem with potential psychological, social, and economic consequences. Furthermore, infertility has been linked to adverse long-term health outcomes. Despite the advanced diagnostic and therapeutic techniques available, approximately 30% of infertile couples do not obtain a live birth after fertility treatment. For these couples, there are no further options to increase their chances of a successful pregnancy and live birth., Objectives: Three overall questions will be studied: (1) What are the risk factors and natural life courses of infertility, early embryonic loss, and adverse pregnancy outcomes? (2) Can we develop new diagnostic and prognostic biomarkers for fecundity and treatment success? And (3) what are the health characteristics of women and men in infertile couples at the time of fertility treatment and during long-term follow-up?, Material and Methods: ReproUnion Biobank and Infertility Cohort (RUBIC) is established as an add-on to the routine fertility management at Copenhagen University Hospital Departments in the Capital Region of Denmark and Reproductive Medicine Centre at Skåne University Hospital in Sweden. The aim is to include a total of 5000 couples equally distributed between Denmark and Sweden. The first patients were enrolled in June 2020. All eligible infertile couples are prospectively asked to participate in the project. Participants complete an extensive questionnaire and undergo a physical examination and collection of biospecimens (blood, urine, hair, saliva, rectal swabs, feces, semen, endometrial biopsies, and vaginal swabs). After the cohort is established, the couples will be linked to the Danish and Swedish national registers to obtain information on parental, perinatal, childhood, and adult life histories, including disease and medication history. This will enable us to understand the causes of infertility and identify novel therapeutic options for this important societal problem., (© 2021 American Society of Andrology and European Academy of Andrology.)