Abstract
STUDY QUESTION
Is it feasible to disseminate testicular tissue cryopreservation with a standardized protocol through a coordinated network of centers and provide centralized ...processing/freezing for centers that do not have those capabilities?
SUMMARY ANSWER
Centralized processing and freezing of testicular tissue from multiple sites is feasible and accelerates recruitment, providing the statistical power to make inferences that may inform fertility preservation practice.
WHAT IS KNOWN ALREADY
Several centers in the USA and abroad are preserving testicular biopsies for patients who cannot preserve sperm in anticipation that cell- or tissue-based therapies can be used in the future to generate sperm and offspring.
STUDY DESIGN, SIZE, DURATION
Testicular tissue samples from 189 patients were cryopreserved between January 2011 and November 2018. Medical diagnosis, previous chemotherapy exposure, tissue weight, and presence of germ cells were recorded.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Human testicular tissue samples were obtained from patients undergoing treatments likely to cause infertility. Twenty five percent of the patient’s tissue was donated to research and 75% was stored for patient’s future use. The tissue was weighed, and research tissue was fixed for histological analysis with Periodic acid-Schiff hematoxylin staining and/or immunofluorescence staining for DEAD-box helicase 4, and/or undifferentiated embryonic cell transcription factor 1.
MAIN RESULTS AND THE ROLE OF CHANCE
The average age of fertility preservation patients was 7.9 (SD = 5) years and ranged from 5 months to 34 years. The average amount of tissue collected was 411.3 (SD = 837.3) mg and ranged from 14.4 mg—6880.2 mg. Malignancies (n = 118) were the most common indication for testicular tissue freezing, followed by blood disorders (n = 45) and other conditions (n = 26). Thirty nine percent (n = 74) of patients had initiated their chemotherapy prior to undergoing testicular biopsy. Of the 189 patients recruited to date, 137 have been analyzed for the presence of germ cells and germ cells were confirmed in 132.
LIMITATIONS, REASONS FOR CAUTION
This is a descriptive study of testicular tissues obtained from patients who were at risk of infertility. The function of spermatogonia in those biopsies could not be tested by transplantation due limited sample size.
WIDER IMPLICATIONS OF THE FINDINGS
Patients and/or guardians are willing to pursue an experimental fertility preservation procedure when no alternatives are available. Our coordinated network of centers found that many patients request fertility preservation after initiating gonadotoxic therapies. This study demonstrates that undifferentiated stem and progenitor spermatogonia may be recovered from the testicular tissues of patients who are in the early stages of their treatment and have not yet received an ablative dose of therapy. The function of those spermatogonia was not tested.
STUDY FUNDING/COMPETING INTEREST(S)
Support for the research was from the Eunice Kennedy Shriver National Institute for Child Health and Human Development grants HD061289 and HD092084, the Scaife Foundation, the Richard King Mellon Foundation, the Departments of Ob/Gyn & Reproductive Sciences and Urology of the University of Pittsburgh Medical Center, United States-Israel Binational Science Foundation (BSF), and the Kahn Foundation. The authors declare that they do not have competing financial interests.
Aging men display reduced reproductive health; however, testis aging is poorly understood at the molecular and genomic levels. Here, we utilized single-cell RNA-seq to profile over 44,000 cells from ...both young and older men and examined age-related changes in germline development and in the testicular somatic cells. Age-related changes in spermatogonial stem cells appeared modest, whereas age-related dysregulation of spermatogenesis and somatic cells ranged from moderate to severe. Altered pathways included signaling and inflammation in multiple cell types, metabolic signaling in Sertoli cells, hedgehog signaling and testosterone production in Leydig cells, cell death and growth in testicular peritubular cells, and possible developmental regression in both Leydig and peritubular cells. Remarkably, the extent of dysregulation correlated with body mass index in older but not in younger men. Collectively, we reveal candidate molecular mechanisms underlying the complex testicular changes conferred by aging and their possible exacerbation by concurrent chronic conditions such as obesity.
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•A transcriptional single-cell atlas of testes from older men•Age-related changes in SSCs are modest•Age-related dysregulation of spermatogenesis and somatic cells are pronounced•Dysregulation correlated with BMI in older but not younger men
Guo, Cairns, and colleagues revealed aging-related alternations of human germ cells and testicular somatic cells through single-cell transcriptomic profiling, providing candidate molecular mechanisms underlying the complex testicular changes conferred by aging and their possible exacerbation by concurrent chronic conditions such as obesity.
Abstract
STUDY QUESTION
Do spermatogonia, including spermatogonial stem cells (SSCs), undergo metabolic changes during prepubertal development?
SUMMARY ANSWER
Here, we show that the metabolic ...phenotype of prepubertal human spermatogonia is distinct from that of adult spermatogonia and that SSC development is characterized by distinct metabolic transitions from oxidative phosphorylation (OXPHOS) to anaerobic metabolism.
WHAT IS KNOWN ALREADY
Maintenance of both mouse and human adult SSCs relies on glycolysis, while embryonic SSC precursors, primordial germ cells (PGCs), exhibit an elevated dependence on OXPHOS. Neonatal porcine SSC precursors reportedly initiate a transition to an adult SSC metabolic phenotype at 2 months of development. However, when and if such a metabolic transition occurs in humans is ambiguous.
STUDY DESIGN, SIZE, DURATION
To address our research questions: (i) we performed a meta-analysis of publicly available and newly generated (current study) single-cell RNA sequencing (scRNA-Seq) datasets in order to establish a roadmap of SSC metabolic development from embryonic stages (embryonic week 6) to adulthood in humans (25 years of age) with a total of ten groups; (ii) in parallel, we analyzed single-cell RNA sequencing datasets of isolated pup (n = 3) and adult (n = 2) murine spermatogonia to determine whether a similar metabolic switch occurs; and (iii) we characterized the mechanisms that regulate these metabolic transitions during SSC maturation by conducting quantitative proteomic analysis using two different ages of prepubertal pig spermatogonia as a model, each with four independently collected cell populations.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Single testicular cells collected from 1-year, 2-year and 7-year-old human males and sorted spermatogonia isolated from 6- to 8-day (n = 3) and 4-month (n = 2) old mice were subjected to scRNA-Seq. The human sequences were individually processed and then merged with the publicly available datasets for a meta-analysis using Seurat V4 package. We then performed a pairwise differential gene expression analysis between groups of age, followed by pathways enrichment analysis using gene set enrichment analysis (cutoff of false discovery rate < 0.05). The sequences from mice were subjected to a similar workflow as described for humans. Early (1-week-old) and late (8-week-old) prepubertal pig spermatogonia were analyzed to reveal underlying cellular mechanisms of the metabolic shift using immunohistochemistry, western blot, qRT-PCR, quantitative proteomics, and culture experiments.
MAIN RESULTS AND THE ROLE OF CHANCE
Human PGCs and prepubertal human spermatogonia show an enrichment of OXPHOS-associated genes, which is downregulated at the onset of puberty (P < 0.0001). Furthermore, we demonstrate that similar metabolic changes between pup and adult spermatogonia are detectable in the mouse (P < 0.0001). In humans, the metabolic transition at puberty is also preceded by a drastic change in SSC shape at 11 years of age (P < 0.0001). Using a pig model, we reveal that this metabolic shift could be regulated by an insulin growth factor-1 dependent signaling pathway via mammalian target of rapamycin and proteasome inhibition.
LARGE SCALE DATA
New single-cell RNA sequencing datasets obtained from this study are freely available through NCBI GEO with accession number GSE196819.
LIMITATIONS, REASONS FOR CAUTION
Human prepubertal tissue samples are scarce, which led to the investigation of a low number of samples per age. Gene enrichment analysis gives only an indication about the functional state of the cells. Due to limited numbers of prepubertal human spermatogonia, porcine spermatogonia were used for further proteomic and in vitro analyses.
WIDER IMPLICATIONS OF THE FINDINGS
We show that prepubertal human spermatogonia exhibit high OXHPOS and switch to an adult-like metabolism only after 11 years of age. Prepubescent cancer survivors often suffer from infertility in adulthood. SSC transplantation could provide a powerful tool for the treatment of infertility; however, it requires high cell numbers. This work provides key insight into the dynamic metabolic requirements of human SSCs across development that would be critical in establishing ex vivo systems to support expansion and sustained function of SSCs toward clinical use.
STUDY FUNDING/COMPETING INTEREST(S)
This work was funded by the NIH/NICHD R01 HD091068 and NIH/ORIP R01 OD016575 to I.D. K.E.O. was supported by R01 HD100197. S.K.M. was supported by T32 HD087194 and F31 HD101323. The authors declare no conflict of interest.
Spermatogenesis is a highly regulated process that produces sperm to transmit genetic information to the next generation. Although extensively studied in mice, our current understanding of primate ...spermatogenesis is limited to populations defined by state-specific markers from rodent data. As between-species differences have been reported in the duration and differentiation hierarchy of this process, it remains unclear how molecular markers and cell states are conserved or have diverged from mice to man. To address this challenge, we employ single-cell RNA sequencing to identify transcriptional signatures of major germ and somatic cell types of the testes in human, macaque, and mice. This approach reveals similarities and differences in expression throughout spermatogenesis, including the stem/progenitor pool of spermatogonia, markers of differentiation, potential regulators of meiosis, RNA turnover during spermatid differentiation, and germ cell-soma communication. These datasets provide a rich foundation for future targeted mechanistic studies of primate germ cell development and in vitro gametogenesis.
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•A multispecies scRNA analysis identifies conserved and divergent cell states•Functional characterization of a TSPAN33+ SPG population by xenotransplantation•Receptor-ligand pairs show divergent germline-soma communications
Spermatogenesis has been studied extensively in rodents, but the conservation among species is not well understood. Shami et al. employ single-cell RNA sequencing to compare transcriptional signatures of thousands of testes cells from human, macaque, and mouse, creating a resource to advance understanding of mammalian reproduction and evolution.
Mammalian spermatogenesis is a complex, highly productive process generating millions of sperm per day. Spermatogonial stem cells (SSCs) are at the foundation of spermatogenesis and can either ...self-renew, producing more SSCs, or differentiate to initiate spermatogenesis and produce sperm. The biological potential of SSCs to produce and maintain spermatogenesis makes them a promising tool for the treatment of male infertility. However, translating knowledge from rodents to higher primates (monkeys and humans) is challenged by different vocabularies that are used to describe stem cells and spermatogenic lineage development in those species. Furthermore, while rodent SSCs are defined by their biological potential to produce and maintain spermatogenesis in a transplant assay, there is no equivalent routine and accessible bioassay to test monkey and human SSCs or replicate their functions in vitro. This chapter describes progress characterizing, isolating, culturing, and transplanting SSCs in higher primates.
The vaginal microbiota plays a pivotal role in reproductive, sexual, and perinatal health and disease. Unlike the well-established connections between diet, metabolism, and the intestinal microbiota, ...parallel mechanisms influencing the vaginal microbiota and pathogen colonization remain overlooked. In this study, we combine a mouse model of
strain COH1 group B
(GBS) vaginal colonization with a mouse model of pubertal-onset obesity to assess diet as a determinant of vaginal microbiota composition and its role in colonization resistance. We leveraged culture-dependent assessment of GBS clearance and culture-independent, sequencing-based reconstruction of the vaginal microbiota in relation to diet, obesity, glucose tolerance, and microbial dynamics across time scales. Our findings demonstrate that excessive body weight gain and glucose intolerance are not associated with vaginal GBS density or timing of clearance. Diets high in fat and low in soluble fiber are associated with vaginal GBS persistence, and changes in vaginal microbiota structure and composition due to diet contribute to GBS clearance patterns in nonpregnant mice. These findings underscore a critical need for studies on diet as a key determinant of vaginal microbiota composition and its relevance to reproductive and perinatal outcomes.IMPORTANCEThis work sheds light on diet as a key determinant influencing the composition of vaginal microbiota and its involvement in group B
(GBS) colonization in a mouse model. This study shows that mice fed diets with different nutritional composition display differences in GBS density and timing of clearance in the female reproductive tract. These findings are particularly significant given clear links between GBS and adverse reproductive and neonatal outcomes, advancing our understanding by identifying critical connections between dietary components, factors originating from the intestinal tract, vaginal microbiota, and reproductive outcomes.
Circadian rhythms dynamically regulate sex differences in metabolism and immunity, and circadian disruption increases the risk of metabolic disorders. We investigated the role of sex-specific ...intestinal microbial circadian rhythms in host metabolism using germ-free and conventionalized mice and manipulation of dietary-derived fat, fiber, and microbiota-accessible carbohydrates. Our findings demonstrate that sex differences in circadian rhythms of genes involved in immunity and metabolism depend on oscillations in microbiota, microbial metabolic functions, and microbial metabolites. Further, we show that consuming an obesogenic, high-fat, low-fiber diet produced sex-specific changes in circadian rhythms in microbiota, metabolites, and host gene expression, which were linked to sex differences in the severity of metabolic dysfunction. Our results reveal that microbial circadian rhythms contribute to sex differences in immunity and metabolism and that dietary factors can entrain new circadian rhythms and modify the magnitude of sex differences in host-microbe circadian dynamics.
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•Intestinal microbial circadian rhythms differ by sex•Sex-specific rhythms in host transcriptional networks are microbiome-dependent•Diet-induced obesity entrains new sex-specific microbial and host gene circadian rhythms•Timing of data collection influences the magnitude of sex differences
Human metabolism; Microbiology; Microbiome
Rainwater harvesting from Roads For Indigenous Pasture production and improved rural livelihoods in Kitui, Kenya (ROFIP) is an applied research project. It assessed the potential of combining ...multiple sustainable land management practices, for example native grass reseeding, rainwater harvesting from roads and in situ microcatchments to enhance vegetation cover in a semi‐arid dryland in Kenya.
Rural earth roads were used as a catchment. Runoff generated from rainfall events was diverted into reseeded pastures with trenches established at intervals, across a slope. The ROFIP project also integrated microcatchments created using ox‐driven ploughs, a traditional practice for seedbed preparation and harnessing in situ rainwater harvesting in African drylands.
Combining the diversion of runoff from roads and harvesting rainwater in situ improves and prolongs soil moisture availability in reseeded pastures. Consequently, this translated to higher biomass yields (i.e. forage for livestock) and vegetation cover (land degradation mitigation and enhanced soil health). This project clearly showed that combining rainwater harvesting and native pasture reseeding improves water retention and soil health, thus improving sustainable pasture production.
However, for this to be achieved, it is prudent to involve practitioners to co‐design practical solutions that are socially, economically and environmentally sustainable. Multi‐stakeholder engagement, effective knowledge sharing, and community involvement can be major enablers in the pursuit of environmental and socioeconomic relevant benefits in applied research projects in Africa. This approach enhances a sense of shared purpose among practitioners and empowers them to become points of reference to their peers.
Multi‐stakeholder participation, effective knowledge sharing and community involvement are major enablers in the pursuit of environmental and socioeconomic relevant benefits in applied research projects in Africa.
Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. ...Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (
n
= 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (
GCNA
). Together with a larger follow-up study (
n
= 2049), 7 likely clinically relevant
GCNA
variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human
GCNA
expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA’s
i
ntrinsically
d
isordered
r
egion, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified
GCNA
variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.
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