RESUMO
Infertility, affecting â¼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p = 4.4 × 10-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.
Assuntos
Infertilidade Masculina , Humanos , Masculino , Infertilidade Masculina/genética , Adulto , Sequenciamento do Exoma , Fator Esteroidogênico 1/genética , Azoospermia/genética , Oligospermia/genética , Mutação , Espermatogênese/genética , Estudos de CoortesRESUMO
Klinefelter syndrome (KS), also known as 47, XXY, is characterized by a distinct set of physiological abnormalities, commonly including infertility. The molecular basis for Klinefelter-related infertility is still unclear, largely because of the cellular complexity of the testis and the intricate endocrine and paracrine signaling that regulates spermatogenesis. Here, we demonstrate an analysis framework for dissecting human testis pathology that uses comparative analysis of single-cell RNA-sequencing data from the biopsies of 12 human donors. By comparing donors from a range of ages and forms of infertility, we generate gene expression signatures that characterize normal testicular function and distinguish clinically distinct forms of male infertility. Unexpectedly, we identified a subpopulation of Sertoli cells within multiple individuals with KS that lack transcription from the XIST locus, and the consequence of this is increased X-linked gene expression compared to all other KS cell populations. By systematic assessment of known cell signaling pathways, we identify 72 pathways potentially active in testis, dozens of which appear upregulated in KS. Altogether our data support a model of pathogenic changes in interstitial cells cascading from loss of X inactivation in pubertal Sertoli cells and nominate dosage-sensitive factors secreted by Sertoli cells that may contribute to the process. Our findings demonstrate the value of comparative patient analysis in mapping genetic mechanisms of disease and identify an epigenetic phenomenon in KS Sertoli cells that may prove important for understanding causes of infertility and sex chromosome evolution.
Assuntos
Infertilidade Masculina/patologia , Síndrome de Klinefelter/complicações , Células Intersticiais do Testículo/patologia , Células de Sertoli/patologia , Análise de Célula Única/métodos , Testículo/patologia , Transcriptoma , Humanos , Infertilidade Masculina/etiologia , Infertilidade Masculina/metabolismo , Síndrome de Klinefelter/cirurgia , Células Intersticiais do Testículo/metabolismo , Masculino , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células de Sertoli/metabolismo , Espermatogênese , Testículo/metabolismo , Inativação do Cromossomo XRESUMO
MOTIVATION: Single-cell sequencing methods provide previously impossible resolution into the transcriptome of individual cells. Cell hashing reduces single-cell sequencing costs by increasing capacity on droplet-based platforms. Cell hashing methods rely on demultiplexing algorithms to accurately classify droplets; however, assumptions underlying these algorithms limit accuracy of demultiplexing, ultimately impacting the quality of single-cell sequencing analyses. RESULTS: We present Bimodal Flexible Fitting (BFF) demultiplexing algorithms BFFcluster and BFFraw, a novel class of algorithms that rely on the single inviolable assumption that barcode count distributions are bimodal. We integrated these and other algorithms into cellhashR, a new R package that provides integrated QC and a single command to execute and compare multiple demultiplexing algorithms. We demonstrate that BFFcluster demultiplexing is both tunable and insensitive to issues with poorly behaved data that can confound other algorithms. Using two well-characterized reference datasets, we demonstrate that demultiplexing with BFF algorithms is accurate and consistent for both well-behaved and poorly behaved input data. AVAILABILITY AND IMPLEMENTATION: cellhashR is available as an R package at https://github.com/BimberLab/cellhashR. cellhashR version 1.0.3 was used for the analyses in this manuscript and is archived on Zenodo at https://www.doi.org/10.5281/zenodo.6402477. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Software , Processamento Eletrônico de Dados , Análise de Sequência , Análise de Célula ÚnicaRESUMO
Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/genética , Genoma Viral/genética , Viremia , Animais , Linhagem Celular , Cromossomos Artificiais Bacterianos , Citomegalovirus/patogenicidade , DNA Recombinante , Modelos Animais de Doenças , Feminino , Fibroblastos/virologia , Humanos , Macaca mulatta , Masculino , Mutação , Fases de Leitura Aberta/genética , Filogenia , Especificidade da EspécieRESUMO
Allogeneic hematopoietic stem cell transplantation (HSCT) and xenotransplantation are accompanied by viral reactivations and virus-associated complications resulting from immune deficiency. Here, in a Mauritian cynomolgus macaque model of fully MHC-matched allogeneic HSCT, we report reactivations of cynomolgus polyomavirus, lymphocryptovirus, and cytomegalovirus, macaque viruses analogous to HSCT-associated human counterparts BK virus, Epstein-Barr virus, and human cytomegalovirus. Viral replication in recipient macaques resulted in characteristic disease manifestations observed in HSCT patients, such as polyomavirus-associated hemorrhagic cystitis and tubulointerstitial nephritis or lymphocryptovirus-associated post-transplant lymphoproliferative disorder. However, in most cases, the reconstituted immune system, alone or in combination with short-term pharmacological intervention, exerted control over viral replication, suggesting engraftment of functional donor-derived immunity. Indeed, the donor-derived reconstituted immune systems of two long-term engrafted HSCT recipient macaques responded to live attenuated yellow fever 17D vaccine (YFV 17D) indistinguishably from untransplanted controls, mounting 17D-targeted neutralizing antibody responses and clearing YFV 17D within 14 days. Together, these data demonstrate that this macaque model of allogeneic HSCT recapitulates clinical situations of opportunistic viral infections in transplant patients and provides a pre-clinical model to test novel prophylactic and therapeutic modalities.
Assuntos
Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas , Infecções Oportunistas , Viroses , Aloenxertos , Animais , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Macaca fascicularis , Infecções Oportunistas/virologiaRESUMO
BACKGROUND: Single-cell RNA-seq (scRNA-Seq) has been widely adopted to study gene expression of the human testis. Several datasets of scRNA-Seq from human testis have been generated from different groups processed with different informatics pipelines. An integrated atlas of scRNA-Seq expression constructed from multiple donors, developmental ages, and fertility states would be widely useful for the testis research community. OBJECTIVE: To describe the generation and use of the human infertility single-cell testis atlas (HISTA), an interactive web tool for understanding human spermatogenesis through scRNA-Seq analysis. METHODS: We obtained scRNA-Seq datasets derived from 12 donors, including healthy adult controls, juveniles, and several infertility cases, and reprocessed these data using methods to remove batch effects. Using Shiny, an open-source environment for data visualization, we created numerous interactive tools for exploring the data, some of which support simple statistical hypothesis testing. We used the resulting HISTA browser and its underlying data to demonstrate HISTA's value for testis researchers. RESULTS: A primary application of HISTA is to search by a single gene or a set of genes; thus, we present various analyses that quantify and visualize gene expression across the testis cells and pathology. HISTA also contains machine-learning-derived gene modules ("components") that capture the entire transcriptional landscape of the testis tissue. We show how the use of these components can simplify the highly complex data in HISTA and assist with the interpretation of genes with unknown functions. Finally, we demonstrate the diverse ways HISTA can be used for new data analysis, including hypothesis testing. DISCUSSION AND CONCLUSIONS: HISTA is a research environment that can help scientists organize and understand the high-dimensional transcriptional landscape of the human testis. HISTA has already contributed to published testis research and can be updated as needed with input from the research community or downloaded and modified for individual needs.
RESUMO
YAP plays a vital role in controlling growth and differentiation in various cell lineages. Although the expression of YAP in mice testicular and spermatogenic cells suggests its role in mammalian spermatogenesis, the role of YAP in the development of human male germ cells has not yet been determined. Using an in vitro model and a gene editing approach, we generated human spermatogonia stem cell-like cells (hSSLCs) from human embryonic stem cells (hESCs) and investigated the role of YAP in human spermatogenesis. The results showed that reducing YAP expression during the early stage of spermatogenic differentiation increased the number of PLZF+ hSSLCs and haploid spermatid-like cells. We also demonstrated that the up-regulation of YAP is essential for maintaining spermatogenic cell survival during the later stages of spermatogenic differentiation. The expression of YAP that deviates from this pattern results in a lower number of hSSLCs and an increased level of spermatogenic cell death. Taken together, our result demonstrates that the dynamic expression pattern of YAP is essential for human spermatogenesis. Modulating the level of YAP during human spermatogenesis could improve the production yield of male germ cells derived from hESCs, which could provide the optimization method for in vitro gametogenesis and gain insight into the application in the treatment of male infertility.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Diferenciação Celular , Células-Tronco Embrionárias Humanas , Espermatogênese , Fatores de Transcrição , Proteínas de Sinalização YAP , Masculino , Humanos , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Proteínas de Sinalização YAP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Espermatogônias/metabolismo , Espermatogônias/citologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genéticaRESUMO
RASopathies are syndromes caused by congenital defects in the Ras/mitogen-activated protein kinase (MAPK) pathway genes, with a population prevalence of 1 in 1,000. Patients are typically identified in childhood based on diverse characteristic features, including cryptorchidism (CR) in >50% of affected men. As CR predisposes to spermatogenic failure (SPGF; total sperm count per ejaculate 0-39 million), we hypothesized that men seeking infertility management include cases with undiagnosed RASopathies. Likely pathogenic or pathogenic (LP/P) variants in 22 RASopathy-linked genes were screened in 521 idiopathic SPGF patients (including 155 CR cases) and 323 normozoospermic controls using exome sequencing. All 844 men were recruited to the ESTonian ANDrology (ESTAND) cohort and underwent identical andrological phenotyping. RASopathy-specific variant interpretation guidelines were used for pathogenicity assessment. LP/P variants were identified in PTPN11 (two), SOS1 (three), SOS2 (one), LZTR1 (one), SPRED1 (one), NF1 (one), and MAP2K1 (one). The findings affected six of 155 cases with CR and SPGF, three of 366 men with SPGF only, and one (of 323) normozoospermic subfertile man. The subgroup "CR and SPGF" had over 13-fold enrichment of findings compared to controls (3.9% vs. 0.3%; Fisher's exact test, p = 5.5 × 10-3). All ESTAND subjects with LP/P variants in the Ras/MAPK pathway genes presented congenital genitourinary anomalies, skeletal and joint conditions, and other RASopathy-linked health concerns. Rare forms of malignancies (schwannomatosis and pancreatic and testicular cancer) were reported on four occasions. The Genetics of Male Infertility Initiative (GEMINI) cohort (1,416 SPGF cases and 317 fertile men) was used to validate the outcome. LP/P variants in PTPN11 (three), LZTR1 (three), and MRAS (one) were identified in six SPGF cases (including 4/31 GEMINI cases with CR) and one normozoospermic man. Undiagnosed RASopathies were detected in total for 17 ESTAND and GEMINI subjects, 15 SPGF patients (10 with CR), and two fertile men. Affected RASopathy genes showed high expression in spermatogenic and testicular somatic cells. In conclusion, congenital defects in the Ras/MAPK pathway genes represent a new congenital etiology of syndromic male infertility. Undiagnosed RASopathies were especially enriched among patients with a history of cryptorchidism. Given the relationship between RASopathies and other conditions, infertile men found to have this molecular diagnosis should be evaluated for known RASopathy-linked health concerns, including specific rare malignancies.
Assuntos
Infertilidade Masculina , Humanos , Masculino , Infertilidade Masculina/genética , Infertilidade Masculina/diagnóstico , Adulto , Proteínas ras/genética , Criptorquidismo/genética , Criptorquidismo/complicações , Sequenciamento do Exoma , MutaçãoRESUMO
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.
Assuntos
Azoospermia , Infertilidade Masculina , Humanos , Masculino , Animais , Camundongos , Azoospermia/genética , Azoospermia/patologia , Testículo/patologia , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Espermatogênese/genéticaRESUMO
OBJECTIVE: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is now a global pandemic. Emerging results indicate a dysregulated immune response. Given the role of CCR5 in immune cell migration and inflammation, we investigated the impact of CCR5 blockade via the CCR5-specific antibody leronlimab on clinical, immunological, and virological parameters in severe COVID-19 patients. METHODS: In March 2020, 10 terminally ill, critical COVID-19 patients received two doses of leronlimab via individual emergency use indication. We analyzed changes in clinical presentation, immune cell populations, inflammation, as well as SARS-CoV-2 plasma viremia before and 14 days after treatment. RESULTS: Over the 14-day study period, six patients survived, two were extubated, and one discharged. We observed complete CCR5 receptor occupancy in all donors by day 7. Compared with the baseline, we observed a concomitant statistically significant reduction in plasma IL-6, restoration of the CD4/CD8 ratio, and resolution of SARS-CoV2 plasma viremia (pVL). Furthermore, the increase in the CD8 percentage was inversely correlated with the reduction in pVL (r = -0.77, p = 0.0013). CONCLUSIONS: Our study design precludes clinical efficacy inferences but the results implicate CCR5 as a therapeutic target for COVID-19 and they form the basis for ongoing randomized clinical trials.
Assuntos
Antagonistas dos Receptores CCR5/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Tratamento Farmacológico da COVID-19 , Citocinas/sangue , RNA Viral/sangue , SARS-CoV-2 , Adulto , Idoso , COVID-19/imunologia , COVID-19/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
Mucosal-associated invariant T (MAIT) cells typically express a TRAV1-2+ semi-invariant TCRα that enables recognition of bacterial, mycobacterial, and fungal riboflavin metabolites presented by MR1. MAIT cells are associated with immune control of bacterial and mycobacterial infections in murine models. Here, we report that a population of pro-inflammatory TRAV1-2+ CD8+ T cells are present in the airways and lungs of healthy individuals and are enriched in bronchoalveolar fluid of patients with active pulmonary tuberculosis (TB). High-throughput T cell receptor analysis reveals oligoclonal expansions of canonical and donor-unique TRAV1-2+ MAIT-consistent TCRα sequences within this population. Some of these cells demonstrate MR1-restricted mycobacterial reactivity and phenotypes suggestive of MAIT cell identity. These findings demonstrate enrichment of TRAV1-2+ CD8+ T cells with MAIT or MAIT-like features in the airways during active TB and suggest a role for these cells in the human pulmonary immune response to Mycobacterium tuberculosis.
Assuntos
Linfócitos T CD8-Positivos/citologia , Células T Invariantes Associadas à Mucosa/imunologia , Tuberculose Pulmonar/imunologia , Animais , Brônquios/microbiologia , Líquido da Lavagem Broncoalveolar , Broncoscopia , Linfócitos T CD8-Positivos/microbiologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Sistema Imunitário , Inflamação , Intestinos/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Antígenos de Histocompatibilidade Menor/imunologia , Células T Invariantes Associadas à Mucosa/microbiologia , Mycobacterium tuberculosis/imunologia , Oregon , Fenótipo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , África do Sul , Tuberculose Pulmonar/microbiologiaRESUMO
Live attenuated simian immunodeficiency virus (SIV) vaccines (LAVs) remain the most efficacious of all vaccines in nonhuman primate models of HIV and AIDS, yet the basis of their robust protection remains poorly understood. Here we show that the degree of LAV-mediated protection against intravenous wild-type SIVmac239 challenge strongly correlates with the magnitude and function of SIV-specific, effector-differentiated T cells in the lymph node but not with the responses of such T cells in the blood or with other cellular, humoral and innate immune parameters. We found that maintenance of protective T cell responses is associated with persistent LAV replication in the lymph node, which occurs almost exclusively in follicular helper T cells. Thus, effective LAVs maintain lymphoid tissue-based, effector-differentiated, SIV-specific T cells that intercept and suppress early wild-type SIV amplification and, if present in sufficient frequencies, can completely control and perhaps clear infection, an observation that provides a rationale for the development of safe, persistent vectors that can elicit and maintain such responses.