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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Small non-coding RNA sequencing reveals global dysregulation of piwi-interacting RNA (piRNA) expression in gonadal malignant germ cell tumours. Since the discovery of ribosomal and transfer RNA (rRNA and tRNA) in the 1950s, it has been known that RNA molecules can serve other functions than translating genomic sequences into proteins in the form of messenger RNAs (mRNAs). It is a great pleasure to introduce the readers of Andrology to this special issue devoted to the involvement of small RNAs in male-specific aspects of physiology and pathology. 40 Chen Q, Yan W, Duan E. Epigenetic inheritance of acquired traits through sperm RNAs and sperm RNA modifications. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

Background: Spermatogenesis depends on stimulation by follicle‐stimulating hormone (FSH) which binds to FSH receptors (FSHR) on testicular Sertoli cells. Three FSH‐related single‐nucleotide polymorphisms (SNPs), FSHB ‐211G>T (rs10835638), FSHR ‐29G>A (rs1394205) and FSHR 2039A>G (rs6166) affect FSH action, and have been suggested to affect testicular function, but the evidence is uncertain. Objective: To describe the associations between the three SNPs and testicular function in a large and well‐characterised cohort of men from the general population. Materials and methods: A cross‐sectional study of 2020 Danish men unselected regarding testicular function. Outcome variables were semen parameters, reproductive hormones and testis size. Genotyping was done by competitive allele‐specific quantitative PCR. Differences in genotype frequencies were tested by chi‐square test and associations between genotypes and outcomes were assessed by multivariate linear regressions. Results: The SNPs affected serum FSH; carriers of the variant affecting FSH secretion (FSHB ‐211G>T) had lower FSH levels while carriers of variants affecting receptor expression (FSHR ‐29G>A) and receptor sensitivity (FSHR 2039A>G) had higher FSH levels. Carriers of FSHB ‐211G>T had lower calculated free testosterone/LH ratio. Although both FSHB ‐211G>T and FSHR 2039A>G were associated with smaller testis size, no clear association was detected in relation to any semen parameters, except a lower total number of morphologically normal spermatozoa in the heterozygous carriers of the FSHB ‐211G>T Discussion and Conclusion: The studied polymorphisms have only minor modulating influence on testis size and function in healthy men. We detected subtle effects of the three SNPs on FSH levels, but also effects of FSHB ‐211G>T on calculated free testosterone/LH ratio, compatible with altered Leydig cell function. Thus, the role of these FSH‐related polymorphisms is complex and modest in men with normal testicular function, but the possible importance of FSH polymorphisms in men with impaired testicular function should be evaluated in future studies in more detail. [ABSTRACT FROM AUTHOR]

Context: Transient thelarche (TT), that is, the appearance, regression and subsequent reappearance of breast buds, is a frequent phenomenon, but little is known about pubertal transition in these girls. Objective: To describe pubertal progression, growth, genotypes, reproductive hormones and growth factors in girls with TT compared to those who do not present TT (non‐TT). Design: Retrospective analysis of a longitudinal population‐based study. Patients or Other Participants: Girls (n = 508) of the Chilean Growth and Obesity cohort. Measurements: Pubertal progression, reproductive hormones, follicle stimulating hormone (FSH) beta subunit/FSH receptor gene single nucleotide polymorphisms and growth. Results: Thirty‐seven girls (7.3%) were presented TT. These girls entered puberty by pubarche more frequently (51%) than girls with normal progression (non‐TT; n = 471; 23%, P =.005). Girls with TT who were under 8 years old had lower androgens, anti‐Müllerian hormone (AMH), luteinizing hormone (LH) and oestradiol (all P <.05) than older girls with TT. At the time of Tanner breast stage 2 (B2), girls with TT had higher androgens, LH, FSH, IGF1, LH, insulin and oestradiol (P <.01) than at the time of TT. TT girls were older at B2 (10.3 ± 1.1 vs. 9.2 ± 1.2 years, P <.001) and menarche (12.3 ± 0.8 vs. 12.0 ± 1.0 years, P =.040) than their counterparts (non‐TT). No differences in anthropometric variables or FSHB/FSHR genotypes were detected. Conclusion: Transient thelarche is a frequent phenomenon that does not appear to be mediated by hypothalamic‐pituitary‐gonadal axis activation or by adiposity. Hormonal differences between earlier TT and later TT suggest that their mechanisms are different. [ABSTRACT FROM AUTHOR]

Synthetic endocrine disrupting chemicals ( EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm-specific CatSper channel and, thereby, evoke an intracellular Ca2+ increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low-dose mixtures to elevate Ca2+ levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization. [ABSTRACT FROM AUTHOR]

Testicular germ cell tumours ( TGCT) of young adults arise from the intratubular precursor, carcinoma in situ ( CIS). CIS cells are thought to be developmentally arrested and transformed fetal germ cells that survive through childhood and gain invasive capacity after puberty. Given that germ cell neoplasms arise frequently in undervirilized and dysgenetic gonads and the striking physiological difference between meiotic entry in ovaries (fetal life) versus testes (at puberty), this study aimed to investigate whether errors in regulation of meiosis may be implicated in the pathogenesis of CIS or its invasive progression to TGCT. The main focus was on a key sex differentiation and meiosis regulator, DMRT1, which has also been linked to TGCT risk in recent genetic association studies. Expression patterns of DMRT1 and other meiosis regulators ( SCP3, DMC1, STRA8, CYP26B1, NANOS2, NANOS3) were investigated in pre- and post-pubertal CIS samples and TGCT by quantitative RT-PCR and immunohistochemistry. The results demonstrated that meiosis markers and meiosis inhibitors were simultaneously expressed in CIS cells, in both pre- and post-pubertal testis samples. DMRT1 was present in a restricted subset of CIS cells, which was relatively greater in pre-pubertal (27%) compared to adult (2.6%) samples. In contrast to the majority of CIS cells, DMRT1-positive CIS cells in adult testes were not proliferating. DMRT1 and most of the other meiosis regulators were absent or expressed at low levels in invasive TGCT, except in spermatocytic seminoma (not derived from CIS). In conclusion, this study indicates that meiosis signalling is dysregulated in CIS cells and that a key regulator of the mitosis-meiosis switch, DMRT1, is expressed in 'early-stage' CIS cells but is down-regulated with further invasive transformation. Whether this mixed meiosis signalling in CIS cells is caused by insufficient virilization of the fetal somatic niche or a partial post-pubertal maturation remains uncertain and requires further study. [ABSTRACT FROM AUTHOR]

Klinefelter syndrome (KS) is the most frequent sex chromosome disorder in males, but the phenotype varies greatly and is therefore highly under-diagnosed. We aimed at describing the phenotypic characteristics throughout life from clinical follow-up of our large cohort of patients with KS. A retrospective observational study of 166 males with nonmosaic 47,XXY KS aged 0.3-80.3 years. Data on phenotype, growth, body composition, bone mineral density, sex hormones, lipids, glycosylated haemoglobin (HbA1C) and prostate-specific antigen were recorded. In addition, histological examination of testicular biopsies from 29 patients was performed. Patients with Klinefelter were taller already in childhood. All patients had smaller testicular volume and elevated luteinizing hormone (LH) and follicle-stimulating hormone levels in adulthood. Cryptorchidism was reported in 14%, gynaecomastia in 44%, and 36% required speech therapy or educational support. The abnormal biochemical parameters became evident after onset of puberty and correlated with histological findings of a gradual deterioration of seminiferous tubules and massive Leydig cell hyperplasia in adults. Our patients presented with a wide spectrum of the classical Klinefelter symptoms. In adulthood, two features were consistently present in every patient: small testes and high LH/testosterone ratio, often despite normal testosterone levels. Such biochemical parameters combined with small testes should lead to a suspicion of KS. [ABSTRACT FROM AUTHOR]

The carcinoma in situ (CIS) stage is the common precursor of testicular germ cell tumours (TGCTs) that arise in young adults. Within the past decade genome wide gene expression tools have been developed and have greatly advanced the insight into the biology of TGCTs. Two independent data sets on global gene expression in testicular CIS have been previously published. We have merged the two data sets on CIS samples ( n = 6) and identified the shared gene expression signature in relation to expression in normal testis. Among the top-20 highest expressed genes, one-third was transcription factors and the list included some ‘novel’ CIS markers (i.e. DOCK11 and ANXA3). Genes related to biological terms ‘nucleic acid binding’ and ‘translational activity’ (e.g. transcription factors and ribosomal proteins, respectively) were consistently and significantly over-represented. Some of the significantly over-expressed genes in CIS cells were selected for validation by RT-PCR ( IFI16, DOCK11, and ANXA3), immunohistochemistry (HLXB9), or in situ hybridization ( IFI16). High-level analysis utilizing the Ingenuity pathway analysis tool indicated that networks relating to ‘gene expression in cancer’ and ‘embryonic development’ were significantly altered and could collectively affect cellular pathways like the WNT signalling cascade, which thus may be disrupted in testicular CIS. The merged CIS data from two different microarray platforms, to our knowledge, provide the most precise CIS gene expression signature to date. [ABSTRACT FROM AUTHOR]

Testicular germ cell tumours have a favourable prognosis if detected early, but are potentially lethal in a subset of patients. Multi-modality treatment is often necessary, thus the preferable time of diagnosis is at the pre-invasive, but unfortunately often asymptomatic precursor stage of carcinoma in situ (CIS). This review describes current possible approaches for the detection of CIS. At present, an open testicular biopsy is the only definitive way of establishing the presence of CIS. The tissue section should be of an adequate size, be properly fixed, and evaluation be supported by at least one solid immunohistochemical marker, for example PLAP, OCT-3/4 or AP-2γ. Determination of who should be offered testicular biopsies is based on clinical and ultrasonic examination along with the evaluation of risk factors. A surgical biopsy is an invasive procedure with potential complications, although rare. Therefore, a noninvasive and equally reliable method is needed. Testicular ultrasound is risk-free, painless and at present the only noninvasive method of aid for andrologists when CIS is suspected. The presence of testicular microlithiasis is, in some cases, indicative of pre-malignant changes, especially in males with additional risk factors. Promising results have recently been obtained with a novel noninvasive detection method based on immunocytological AP-2γ-staining of CIS cells in semen. This method could be a supporting method in andrology centres where careful follow-up is possible. In conclusion, one difficulty is to determine in which males CIS should be suspected; secondly, there does not as yet exist an optimal noninvasive method of diagnosis that is more acceptable than an open surgical biopsy. [ABSTRACT FROM AUTHOR]

Since the discovery of testicular carcinoma in situ (CIS) – the precursor cell for the vast majority of germ cell tumours – it has been proposed that CIS cells could be derived from transformed primordial germ cells or gonocytes. Here, we review recent discoveries not only substantiating that initial hypothesis but also indicating that CIS cells have a striking phenotypic similarity to embryonic stem cells (ESC). Many cancers have been proposed to originate from tissue-specific stem cells [so-called ‘cancer stem cells’ (CSC)] and we argue that CIS may be a very good example of a CSC, but with exceptional features due to the retention of embryonic pluripotency. In addition, considering the fact that pre-invasive CIS cells are transformed from early fetal cells, possibly due to environmentally induced alterations of the niche, we discuss potential risks linked to the uncontrolled therapeutic use of ESC. [ABSTRACT FROM AUTHOR]

Aim: Klinefelter syndrome (KS) is the most frequent sex chromosome disorder in males, but the phenotype varies greatly and is therefore highly under-diagnosed. We aimed at describing the phenotypic characteristics throughout life from clinical follow-up of our large cohort of patients with KS., Methods: A retrospective observational study of 166 males with nonmosaic 47,XXY KS aged 0.3-80.3 years. Data on phenotype, growth, body composition, bone mineral density, sex hormones, lipids, glycosylated haemoglobin (HbA1C) and prostate-specific antigen were recorded. In addition, histological examination of testicular biopsies from 29 patients was performed., Results: Patients with Klinefelter were taller already in childhood. All patients had smaller testicular volume and elevated luteinizing hormone (LH) and follicle-stimulating hormone levels in adulthood. Cryptorchidism was reported in 14%, gynaecomastia in 44%, and 36% required speech therapy or educational support. The abnormal biochemical parameters became evident after onset of puberty and correlated with histological findings of a gradual deterioration of seminiferous tubules and massive Leydig cell hyperplasia in adults., Conclusion: Our patients presented with a wide spectrum of the classical Klinefelter symptoms. In adulthood, two features were consistently present in every patient: small testes and high LH/testosterone ratio, often despite normal testosterone levels. Such biochemical parameters combined with small testes should lead to a suspicion of KS., (© 2011 The Author(s)/Acta Paediatrica © 2011 Foundation Acta Paediatrica.)