Germ Cell Research Articles

Cut-offs for relapse detection in men with stage I testicular germ cell tumors during active surveillance within a prospective multicentre cohort study using either raw or housekeeper normalized miR-371a-3p serum levels

PurposeMiR-371a-3p represents a novel liquid biomarker that detects all histologies of germ-cell tumors (GCT) except teratoma. However, it is currently unclear whether miR-371a-3p results obtained directly from RT-PCR (raw Cq) or normalized for housekeeper genes and transformed into the relative quantity (RQ) value should be used and at what cut-off level. The purpose of this research was to evaluate, which values should be used, and a potential cut-off level for relapse-detection to inform subsequent studies. Materials and MethodsWe applied a CE-certified qRT-PCR test to measure miR-371a-3p at each follow-up visit during active surveillance in 34 men with stage I testicular GCT. MiR-371a-3p levels were calculated by the ΔΔ method. ResultsAbout 18 Patients had pure seminoma and 16 had mixed or nonseminomatous testicular GCT. Recurrences were detected in 10 patients and were correctly identified by both raw and housekeeper-normalized miR-371a-3p serum levels. The raw Cq, with a cut-off value of <28, resulted in only 1 false positive (3%), whereas RQ, with a cut-off value of >15, produced 6 false positive results (17%). Most of these false positive results normalized in subsequent measurements. The RQ approach detected recurrence in 1 patient 6 months earlier than the raw Cq approach. ConclusionOur preliminary data suggest that this CE-certified assay, using previously suggested cut-off values, is a promising method for detecting disease recurrence, provided a confirmatory second test is conducted to identify false positive results. To avoid unnecessary scans or overtreatment, we are currently validating this assay and cut-offs in a prospective cohort study.

Melatonin protects spermatogenic cells against DNA damage and necroptosis induced by atrazine

Atrazine (ATZ), a widely used triazine herbicide, has been shown to disrupt reproductive development in organisms. Melatonin (MLT) is a natural hormone and has been shown to have strong antioxidant properties. Due to its lipophilicity, it can cross biological barriers freely and act on germ cells directly. However, the mechanism through which melatonin affects atrazine-induced damage to male sperm cells remains unclear. This study aimed to investigate the effects of ATZ on spermatocyte development and to elucidate MLT's role in preventing ATZ-induced spermatogenesis failure. Pubertal mice were randomly divided into four groups: blank control group (Con), 5 mg/kg melatonin group (MLT), 170 mg/kg atrazine group (ATZ), and ATZ + MLT group. GC-1 cell culture was employed to access the in vitro effects of MLT and ATZ on spermatogonia. The results indicate that atrazine affected protein and metabolite composition, and reduced sperm viability, sperm motility (VAP, VSL and VCL) and levels of proteins related to spermatogenesis function in the mice testis. Melatonin alleviated the development of cellular DNA damage and necroptosis caused by atrazine both in vivo and in vitro. Moreover, we proposed that it was GC-1 cells developing necroptosis, but not other cell types in the testis. In conclusion, this study suggests that atrazine disrupts the development process, causing DNA damage in spermatozoa during spermatogenesis. Additionally, ATZ-induced necroptosis specifically targets spermatogenic cells. Notably, melatonin alleviates atrazine-induced necroptosis and DNA damage in spermatogenic cells. This study provides new insights into potential therapeutic strategies for atrazine-induced male infertility.

Roles of Cep215/Cdk5rap2 in establishing testicular architecture for mouse male germ cell development.

Cep215/Cdk5rap2 is a centrosome protein crucial for directing microtubule organization during cell division and morphology. Cep215 is a causal gene of autosomal recessive primary microcephaly type 3, characterized by a small brain size and a thin cerebral cortex. Despite previous attempts with Cep215 knockout (KO) mice to elucidate its developmental roles, interpreting their phenotypes remained challenging due to potential interference from alternative variants. Here, we generated KO mice completely lacking the Cep215 gene and investigated its specific contributions to male germ cell development. In the absence of Cep215, testis size decreased significantly, accompanied by a reduction in male germ cell numbers. Histological analyses unveiled the arrested development of male germ cells around the zygotene stage of meiosis. Concurrently, the formation of the blood-testis barrier (BTB) was impaired in Cep215 KO testes. These findings suggest that BTB failure contributes, at least partially, to male germ cell defects observed in Cep215 KO mice. We propose that the deletion of Cep215 may disrupt microtubule organization in Sertoli cells with a delay in spermatogonial stem cell mitosis, thereby impeding proper BTB formation.

Hollaender award 2023: Adventures in applied genetic toxicology.

This work describes career "adventures" into applied research in environmental mutagenesis. Surprising and interesting results, as well as publications in Nature and Science, may counter an assumption that applied research is not as exciting or impactful as basic research. The narrative is described in terms of "mentors," whose advice had a lasting impact and resonated in many ways. Adventures included forays into nitrosamine mutagenicity, nanomaterial assessment, photoactivated DNA damaging agents, p53 gene mutagenicity, germ cell mutagenic risk, bacterial mutagenicity assays, genotoxicity of cell phone radiation, Covid diagnostic PCR assays, personalized cancer prevention, and >25 years in regulatory safety assessment at FDA. FDA work included review of genotoxicity data, experiments in the lab, international standards generation, and collaboration with others to foster better analyses of DNA damaging agents, generally in relation to cancer risk. As demonstrated by accidental adventures that led to scientific as well as life-altering personal realizations, many of the most critical happenings in science and in life turn out to be "random," unexpected, events. Finally, with this work and that of my lifelong tripmate William Lijinsky as models, it is suggested that a "non-hypothesis driven," open-ended approach to research can be path-breaking and forefront.

Similarity of Human Mitochondrial DNA Nucleotide Substitution Spectra Reconstructed Over One and Many Generations

Using phylogenetic analysis of mitochondrial whole genome nucleotide sequences (mtDNA), allowing the study of genetic changes over many generations, a spectrum of nucleotide substitutions (along the L-strand of mtDNA) was reconstructed in European populations. The spectra of mtDNA nucleotide substitutions observed in a heteroplasmic state (at ≥1% and ≥5% levels) in first generation children were also analyzed. It was found that the spectra of nucleotide substitutions reconstructed over one and many generations do not differ practically in the main parameters: the distribution of pyrimidine and purine substitutions (with predominance of transitions TC), the ratio of the number of transitions and transversions. Analysis of the phylogenetic tree of mtDNA haplotypes in Europeans clearly revealed the influence of negative (purifying) selection on mitochondrial gene pools. It is suggested that the selective processes guiding the mtDNA evolution in one and many generations are of a similar nature, i.e., caused by negative selection. The problem of how mutations occur and spread in mitochondria of germ line cells is discussed.

ARID1A governs the silencing of sex-linked transcription during male meiosis in the mouse

We present evidence implicating the BAF (BRG1/BRM Associated Factor) chromatin remodeler in meiotic sex chromosome inactivation (MSCI). By immunofluorescence (IF), the putative BAF DNA binding subunit, ARID1A (AT-rich Interaction Domain 1 a), appeared enriched on the male sex chromosomes during diplonema of meiosis I. Germ cells showing a Cre-induced loss of ARID1A arrested in pachynema and failed to repress sex-linked genes, indicating a defective MSCI. Mutant sex chromosomes displayed an abnormal presence of elongating RNA polymerase II coupled with an overall increase in chromatin accessibility detectable by ATAC-seq. We identified a role for ARID1A in promoting the preferential enrichment of the histone variant, H3.3, on the sex chromosomes, a known hallmark of MSCI. Without ARID1A, the sex chromosomes appeared depleted of H3.3 at levels resembling autosomes. Higher resolution analyses by CUT&amp;RUN revealed shifts in sex-linked H3.3 associations from discrete intergenic sites and broader gene-body domains to promoters in response to the loss of ARID1A. Several sex-linked sites displayed ectopic H3.3 occupancy that did not co-localize with DMC1 (DNA meiotic recombinase 1). This observation suggests a requirement for ARID1A in DMC1 localization to the asynapsed sex chromatids. We conclude that ARID1A-directed H3.3 localization influences meiotic sex chromosome gene regulation and DNA repair.

From Egg to Adult: A Developmental Table of the Ant Monomorium pharaonis.

Ants are one of the most ecologically and evolutionarily successful groups of animals and exhibit a remarkable degree of phenotypic diversity. This success is largely attributed to the fact that all ants are eusocial and live in colonies with a reproductive division of labor between morphologically distinct queen and worker castes. Yet, despite over a century of studies on caste determination and evolution in ants, we lack a complete ontogenetic series from egg to adult for any ant species. We, therefore, present a developmental table for the Pharaoh ant Monomorium pharaonis, a species whose colonies simultaneously produce reproductive queens and completely sterile workers. In total, M. pharaonis embryonic, larval, and pupal development lasts 45 days. During embryogenesis, the majority of developmental events are conserved between M. pharaonis and the fruit fly Drosophila melanogaster. We discovered, however, two types of same-stage embryos before gastrulation: (1) embryos with internalized germ cells; and (2) embryos with germ cells outside of the blastoderm at the posterior pole. Moreover, we also found two-types of embryos following germ band extension: (1) embryos with primordial germ cells that will develop into reproductive queens; and (2) embryos with no germ cells that will develop into completely sterile workers. Together, these data show that queen and worker castes are already determined and differentiated by early embryogenesis. During larval development, we confirmed that reproductive and worker larvae proceed through three larval instars. Using anatomical and developmental markers, we can further discern the development of gyne (unmated queen) larvae, male larvae, and worker larvae as early as the 1st instar. Overall, we hope that the ontogenetic series we present here will serve as a blueprint for the generation of future ant developmental tables.

Transient receptor potential a1b regulates primordial germ cell numbers and sex differentiation in developing zebrafish.

Temperature is a leading environmental factor determining the sex ratio of some animal populations, such as fish, amphibians, and reptiles. However, the underlying mechanism by which temperature affects gender is still poorly understood. Transient receptor potential a1b (Trpa1b) belongs to the ion channel family of transient receptor potentials and exhibits dual thermosensitivity to heat and cold. In this study, we have unveiled a novel function of the trpa1b gene. Zebrafish generated through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 with Trpa1b-null manifest a male-biased sex ratio. The quantity of primordial germ cells (PGCs) in zebrafish is closely linked to gender determination and gonadal development. Yet the role of the trpa1b gene in zebrafish reproductive development remains unexplored in the literature. Our investigation revealed a significant reduction in PGCs in Trpa1b mutant zebrafish compared to their wild-type counterparts 24-h postfertilization (hpf). Transcriptome sequencing of tissues near the reproductive crest of embryos at 1.25 days postfertilization (dpf) revealed differential changes in PGC-related marker genes and genes related to sperm cell development and differentiation. The relative expression of ddx4 and sycp3 genes was significantly downregulated, whereas amh was significantly upregulated at 20 dpf in trpa1b-/- zebrafish. The results of this study provide valuable insights and references for studying the molecular mechanism of sex determination in zebrafish. Undoubtedly, these results will further enhance our understanding of gender differentiation and gonadal development in fish and other vertebrates.

Primary liver yolk sac tumor with lung metastasis in adolescence: Rare case report

Introduction: Germ cell tumors (GCTs) often arise within the gonads but occasionally appear within the extragonadal areas. Yolk sac tumors (YSTs), a type of GCT, are most often found in infants and adolescents due to puberty. However, primary liver YST cases are rare, with only one other reported case in the adolescent population. Case Report: The current case examines a 15-year-old female patient experiencing significant weight loss, respiratory issues, and fevers. After her blood work displayed high alpha-fetoprotein (AFP) levels, a biopsy revealed a stage four primary liver YST with lung metastasis. The infrequency and primary location of the tumor, combined with her age, create challenges in diagnosis and treatment. Due to its rarity, the reporting of this case may aid in diagnosis when encountered. We highlight and present our experience of management and prognosis of primary-hepatic YST when encountered in adolescents. Conclusion: At two years follow-up, the patient showed no evidence of recurrence or metastasis.

Enhanced cell survival in prepubertal testicular tissue cryopreserved with membrane lipids and antioxidants rich cryopreservation medium.

The present study explores the advantages of enriching the freezing medium with membrane lipids and antioxidants in improving the outcome of prepubertal testicular tissue cryopreservation. For the study, testicular tissue from Swiss albino mice of prepubertal age group (2weeks) was cryopreserved by slow freezing method either in control freezing medium (CFM; containing DMSO and FBS in DMEM/F12) or test freezing medium (TFM; containing soy lecithin, phosphatidylserine, phosphatidylethanolamine, cholesterol, vitamin C, sodium selenite, DMSO and FBS in DMEM/F12 medium) and stored in liquid nitrogen for at least one week. The tissues were thawed and enzymatically digested to assess viability, DNA damage, and oxidative stress in the testicular cells. The results indicate that TFM significantly mitigated freeze-thaw-induced cell death, DNA damage, and lipid peroxidation compared to tissue cryopreserved in CFM. Further, a decrease in Cyt C, Caspase-3, and an increase in Gpx4 mRNA transcripts were observed in tissues frozen with TFM. Spermatogonial germ cells (SGCs) collected from tissues frozen with TFM exhibited higher cell survival and superior DNA integrity compared to those frozen in CFM. Proteomic analysis revealed that SGCs experienced a lower degree of freeze-thaw-induced damage when cryopreserved in TFM, as evident from an increase in the level of proteins involved in mitigating the heat stress response, transcriptional and translational machinery. These results emphasize the beneficial role of membrane lipids and antioxidants in enhancing the cryosurvival of prepubertal testicular tissue offering a significant stride towards improving the clinical outcome of prepubertal testicular tissue cryopreservation.

Spinal Metastases in Non-Seminomatous Germ Cell Testicular Tumors: Prognosis and Integrated Therapeutic Approaches—A Systematic Review with an Institutional Case Illustration

(1) Background: Testicular cancer, although accounting for only 0.5% to 1% of all solid male cancers, is the most common malignancy in males aged 15 to 35 years. Non-seminomatous germ cell tumors (NSGCT) represent nearly half of all testicular germ cell tumors and are associated with a more aggressive clinical course. Spinal metastases, while rare, pose significant challenges due to their potential to cause spinal cord compression, neurological deficits, and severe pain. This systematic review aims to evaluate prognosis and treatment approaches for spinal metastases in NSGCT, with a focus on multidisciplinary care and treatment outcomes. (2) Methods: A systematic review was conducted following PRISMA guidelines. PubMed, Scopus, and Embase were searched on 18 September 2024, using the Boolean search strategy [(Nonseminomatous germ cell tumor (NSGCT) AND (spinal OR vertebral metastases)]. Case reports, case series, and cohort studies providing detailed patient data were included. Data on patient demographics, tumor histology, metastatic site, treatments, and outcomes were extracted for analysis. (3) Results: A total of 164 cases of NSGCT with spinal metastases were analyzed, with patients aged 23 to 40 years (median: 31.5 years). The lumbar spine was involved in all cases, and spinal cord compression occurred in 59.8% of patients, often causing severe neurological symptoms such as cauda equina syndrome. Chemotherapy, primarily cisplatin-based, was administered in all cases, while surgical interventions, including laminectomy and vertebrectomy, were performed in cases of spinal compression and instability. Complete remission occurred in only 2.4% of patients. Progressive improvement was observed in 56.7% of cases, while 20.1% of patients died. Outcomes varied, highlighting the importance of individualized, multidisciplinary care to manage both systemic and localized disease. (4) Conclusions: Spinal metastases in NSGCT represent a complex clinical scenario, requiring a combination of chemotherapy, surgery, and in some cases, radiotherapy. Chemotherapy remains essential, but surgery is critical for addressing spinal compression and instability. A multidisciplinary approach is vital for optimizing outcomes, as prognosis is variable, with some patients achieving improvement while others face progressive disease or death. Further research is needed to refine the role of radiotherapy and improve long-term treatment strategies for this rare complication.

Mucinous Cystadenoma Arising in Post-Treatment Non-Seminomatous Germ Cell Tumor: A Rare Occurrence

Mucinous Cystadenoma Arising in Post-Treatment Non-Seminomatous Germ Cell Tumor: A Rare Occurrence

Undescended Testis and Right Inguinal Hernia in Adult: A Case Report

Cryptorchidism also known as undescended testis of at least one side is one of the causes of an inguinal hernia. This case report discusses an adult patient presented with a swelling in the right groin. On examination, the patient was found to have a right inguinal hernia and right undescended testis. Testis was intraabdominal lying near deep ring. Testicular tumor markers were normal. Right orchidectomy and right inguinal hernia repair with polypropylene mesh was done. Following surgery, postoperative course was uneventful. Histopathological examination revealed marked hypo spermatogenesis with no signs of testicular malignancy (No granuloma/parasite/intratubular germ cell neoplasia/malignancy). Keywords: undescended testis, cryptorchidism, inguinal hernia, testicular malignancy.

Ex Vivo Fluorescence Confocal Microscopy Meets Innovation and Revolutionary Technology, for “Real-Time” Histological Evaluation, in Pediatric Surgical Oncology

Background and Aim: Ex vivo fluorescence confocal microscopy (FCM) systems are innovative optical imaging tools that create virtual high-resolution histological images without any standard tissue processing, either freezing or fixing in formalin and embedding in paraffin. These systems have opened an era that would revolutionize pathological examination by providing rapid, real-time assessments across various pathology subspecialties, potentially replacing conventional methods that are tissue- and time-consuming. This study aimed to present the first utilization of FCM in pediatric surgical oncology, focusing on assessing the benefits, particularly in facilitating rapid and accurate diagnosis. Methods: This preliminary study comprised five consecutive patients undergoing surgical biopsy for disease characterization and surgical strategy selection. After biopsy, tissue samples were prepared and analyzed using FCM without sectioning. A pathologist who evaluated macroscopic and microscopic images, once obtained remotely, could promptly indicate any interventions that require timeliness. Samples were then evaluated with conventional methods. Results: All five lesions were deemed suitable for evaluation. Preliminary diagnoses utilizing FCM included atypical Spitz nevus (1), Wilm’s tumor (1), lymph node reactive hyperplasia (1), malignant germ cell tumor of the testis (1), and Hodgkin’s lymphoma (1). Final histopathological analyses revealed atypical Spitz nevus (1), Wilm’s tumor (1), hyperplastic lymphadenopathy with a prevalent marginal pattern (1), mixed nonseminomatous malignant germinal neoplasm consisting of embryonal carcinoma (90%) and yolk sac tumor (10%), and Hodgkin’s lymphoma nodular sclerosis variant (1). In the case of diagnosis of atypical Spitz nevus, the widening of the resection margins was performed in the same surgery. In the case of testicular neoplasm, radical orchiectomy was performed. A high level of agreement between FCM evaluation and definitive histological examination was observed for all parameters evaluated. Conclusions: FCM represents a significant advancement in pathological imaging technology, offering potential benefits in enhancing traditional tissue processing methods. This preliminary report marks the first application of FCM in pediatric surgical oncology. Our findings underscore the promising role of FCM as an adjunctive tool in pediatric oncology, facilitating prompt diagnosis and treatment initiation.

Cellular retinoic acid binding proteins regulate germ cell proliferation and sex determination in zebrafish.

Cellular retinoic acid (RA) binding proteins (Crabps) solubilize intracellular RA and transport it to its nuclear receptors or cytoplasmic degradation enzymes. Despite their extreme conservation across chordates, genetic studies of Crabp function have revealed few essential functions. We have generated loss-of-function mutations in all four zebrafish Crabps and find essential roles for Crabp2s in gonad development and sex determination. Transgenic RA reporters show strong RA-responses in germ cells at the bipotential stage of gonad development. Double mutants lacking the functions of both Crabp2a and Crabp2b predominantly become male, which correlates with their smaller gonad size and reduced germ cell proliferation during gonad development at late larval and early juvenile stages. In contrast, mutants lacking the functions of both Crabp1a and Crabp1b have normal sex ratios. Exogenous RA treatments at bipotential gonad stages increase germ cell number, consistent with a direct role for RA in promoting germ cell proliferation. Our results suggest essential functions for Crabps in gonad development and sex determination.

Potential next generation markers of testicular germ cell tumors: miRNA-371a-3p.

Testicular germ cell tumors (TGCTs) account for approximately 98% of all testicular cancers, predominantly affecting young to middle-aged men. Early diagnosis and treatment result in a cure rate of over 95%. However, conventional serum tumor markers (STMs) such as alpha-fetoprotein (AFP), β-human chorionic gonadotropin (β-hCG), and lactate dehydrogenase (LDH), which are recommended by NCCN and EAU guidelines, have limited sensitivity, often below 60%, which diminishes their clinical utility. Recently, miRNA-371a-3p, an embryonic stem cell-associated microRNA, has been identified as being specifically expressed in TGCTs. This microRNA can be reliably detected in peripheral blood and fulfills all seven Lange-Winfield criterias for tumor markers. Notably, miRNA-371a-3p has demonstrated superior diagnostic, therapeutic, and follow-up capabilities compared to conventional STMs in TGCTs. Its potential to replace conventional STMs in clinical practice is already recognized in several clinical guidelines. A PubMed search using subject headings and free-text terms related to MicroRNA-371a-3p in TGCT management was conducted. Relevant references were also tracked, and key studies were reviewed based on predefined exclusion criteria. Out of 368 identified studies, 67 met inclusion criteria. These studies focused on MicroRNA-371a-3p's discovery, detection methods, diagnostic utility in TGCTs, and cost-effectiveness. First identified over a decade ago, microRNA-371a-3p is now established as a highly specific blood-based marker for TGCTs, valuable for diagnosis, monitoring, and follow-up, and more cost-effective than conventional STMs. MicroRNA-371a-3p is a promising, highly sensitive marker for TGCTs, offering better performance and cost efficiency than conventional STMs, likely to become the next-generation diagnostic tool for TGCTs.

Testicular histopathology and its association with germ cell numbers, serum concentrations of reproductive hormones, and semen quality.

It 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. To describe the detailed relationship between specific testicular histopathologies and the serum concentrations of reproductive hormones and semen quality. Descriptive 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. Compared 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. A 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.

Cyclophosphamide-induced testicular injury: the role of chrysin in mitigating iron overload and ferroptosis.

This study evaluated the beneficial effects of chrysin against cyclophosphamide (CP)-induced testicular toxicity in rats across several parameters, including hormones, oxidative stress, inflammation, apoptosis, and protein expression. Rats were pretreated with oral doses of chrysin at 25, 50, or 100 mg/kg daily for 7 days. On the 8th day, all groups except controls received CP (200 mg/kg) injection. Chrysin doses continued for 7 more days. Hormones, oxidative stress markers, inflammatory cytokines, apoptosis regulators, and iron regulatory proteins were assessed. CP decreased testosterone, inhibin B, GSH, and GPx4 and increased FSH, cholesterol, MDA, IL-6, and BAX. It also drastically reduced TfR1, liprin, and IREB2. Chrysin dose-dependently counteracted these effects. The highest 100 mg/kg chrysin dose increased testosterone, inhibin B, GSH, GPx4, BCL2, TfR1, liprin, and IREB2 while decreasing FSH, cholesterol, MDA, IL-6, and BAX close to control levels. There were also significant incremental benefits for testosterone, inhibin B, and other parameters with higher chrysin doses. Chrysin dose-dependently attenuated CP-induced hormonal dysfunction, oxidative stress, inflammation, apoptosis, and iron-regulatory protein suppression. The maximum dose showed the most optimal protective effects in restoring the testicular toxicity markers. These results validate the promising spermatoprotective properties of chrysin against chemotherapeutic germ cell damage.

Multi-Omics Approaches Uncovered Critical mRNA–miRNA–lncRNA Networks Regulating Multiple Birth Traits in Goat Ovaries

The goat breeding industry on the Tibetan Plateau faces strong selection pressure to enhance fertility. Consequently, there is an urgent need to develop goat lines with higher fertility and adaptability. The ovary, as a key organ determining reproductive performance, is regulated by a complex transcriptional network involving numerous protein-coding and non-coding genes. However, the molecular mechanisms of the key mRNA–miRNA–lncRNA regulatory network in goat ovaries remain largely unknown. This study focused on the histology and differential mRNA/miRNA/lncRNA between Chuanzhong black goat (CBG, high productivity, multiple births) and Tibetan goat (TG, strong adaptability, single birth) ovaries. Histomorphological analysis showed that the medulla proportion in CBG ovaries was significantly reduced compared to TG. RNA-Seq and small RNA-Seq analysis identified 1218 differentially expressed (DE) mRNAs, 100 DE miRNAs, and 326 DE lncRNAs, which were mainly enriched in ovarian steroidogenesis, oocyte meiosis, biosynthesis of amino acids and protein digestion, and absorption signaling pathways. Additionally, five key mRNA–miRNA–lncRNA interaction networks regulating goat reproductive performance were identified, including TCL1B–novel68_mature–ENSCHIT00000010023, AKAP6–novel475_mature–ENSCHIT00000003176, GLI2–novel68_mature–XR_001919123.1, ITGB5–novel65_star–TCONS_00013850, and VWA2–novel71_mature–XR_001919911.1. Further analyses showed that these networks mainly affected ovarian function and reproductive performance by regulating biological processes such as germ cell development and oocyte development, which also affected the plateau adaptive capacity of the ovary by participating in the individual immune and metabolic capacities. In conclusion, we identified numerous mRNA–miRNA–lncRNA interaction networks involved in regulating ovarian function and reproductive performance in goats. This discovery offers new insights into the molecular breeding of Tibetan Plateau goats and provides a theoretical foundation for developing new goat lines with high reproductive capacity and strong adaptability to the plateau environment.

High detection rate of circulating-tumor DNA from cerebrospinal fluid of children with central nervous system germ cell tumors

Central nervous system germ cell tumors (CNS-GCT) are malignant neoplasms that arise predominantly during adolescence and young adulthood. These tumors are typically sensitive to treatment, but resulting long-term health deficits are common. Additional clinical challenges include surgical risks associated with tumor biopsy, and need to determine treatment response for adapting radiotherapy protocols. The aim of this study was to establish the detectability of circulating-tumor DNA (ctDNA) from cerebrospinal fluid (CSF) of children with CNS-GCT as a potential biomarker. We obtained CSF from patients with CNS-GCT by lumbar puncture or intra-operatively. Cell-free DNA (cfDNA) was extracted and subjected to low-pass whole genome sequencing (LP-WGS). Copy-number alterations (CNAs) were inferred and served as a marker of measurable residual disease (MRD). Comparisons with imaging findings and tumor marker levels were made. A total of 29 CSF samples from 21 patients (16 with germinoma, 5 with non-germinomatous GCT) were sequenced. Twenty samples from 19 patients were collected at diagnosis, and 9 samples from 7 patients were collected during or after therapy. Among the diagnostic samples, CNAs were detected in samples from 17/19 patients (89%), which included 8 with marker-negative tumors. Specific clinical scenarios suggested that serial cfDNA analysis may carry utility in tracking treatment responses as well as clarifying indeterminate imaging findings. Our results provide evidence for the high-sensitivity in detecting ctDNA from CSF of CNS-GCT patients using LP-WGS, with potential utility for non-invasive diagnosis and disease monitoring in upcoming CNS-GCT studies.