Biology subjects

Aston, K. I.

Publications and source records attributed to Aston, K. I..

10 recordsLinked to original sources

bioRxiv2026

PRDM9-mediated meiotic hotspot specification is constrained in humans despite extensive sequence diversity

PRDM9 specifies meiotic recombination hotspots through a rapidly evolving C2H2 zinc-finger (ZNF) coding minisatellite that determines DNA-binding specificity. Although this minisatellite harbors extraordinary allelic diversity in humans, the functional consequences of most naturally occurring variants remain unknown. Here we functionally characterize 80 human PRDM9 alleles using genome-wide chromatin profiling. Despite extensive sequence diversity within the ZNF array, most alleles function indistinguishably from common A and C hotspot-specifying alleles, revealing that human PRDM9 function is more constrained than its sequence diversity predicts. In contrast, rare and infertility-associated variants occupy two functional extremes: either abundant and novel DNA binding specificity or minimal DNA binding, suggesting that both gain- and loss-of-function alleles may disrupt symmetric hotspot specification during meiosis, thus representing a plausible contributor to human infertility. Together, our findings define the functional landscape of human PRDM9 variation and provide a framework for interpreting the impact of newly discovered PRDM9 alleles.

genomics↗

bioRxiv2023

AXDND1 is required to balance spermatogonial commitment and for sperm tail formation in mice and humans

Dynein complexes are large, multi-unit assemblies involved in many biological processes including male fertility via their critical roles in protein transport and axoneme motility. Previously we identified a pathogenic variant in the dynein gene AXDND1 in an infertile man. Subsequently we identified an additional four potentially compound heterozygous variants of unknown significance in AXDND1 in two additional infertile men. We thus tested the role of AXDND1 in mammalian male fertility by generating a knockout mouse model. Axdnd1-/- males were sterile at all ages but could undergo one round of histologically complete spermatogenesis. Subsequently, a progressive imbalance of spermatogonial commitment to spermatogenesis over self-renewal occurred, ultimately leading to catastrophic germ cell loss, loss of blood-testis barrier patency and immune cell infiltration. Sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively, our data highlight the essential roles of AXDND1 as a regulator of spermatogonial commitment to spermatogenesis and during the processes of spermiogenesis where it is essential for sperm tail development, release and motility.

cell biology↗

bioRxiv2023

The Human Infertility Single-cell Testis Atlas (HISTA): An interactive molecular scRNA-Seq reference of the human testis.

BackgroundThe Human Infertility Single-cell Testis Atlas (HISTA) is an interactive web tool and a reference for navigating the transcriptome of the human testis. It was developed using joint analyses of scRNA-Seq datasets derived from a dozen donors, including healthy adult controls, juveniles, and several infertility cases. HISTA is very different than other websites of testis scRNA-seq data, providing visualization and hypothesis testing tools on a batch-removed and integrated dataset of 23429 genes measured across 26093 cells using. ObjectiveThe main goal of this manuscript is to describe HISTA in detail and highlight its unique and novel features. MethodsTherefore, we used HISTA as a guide for its application and demonstrated HISTAs translational capacity to follow up on two observations of biological relevance. ResultsOur first analytical vignette identifies novel groupings of tightly regulated long non-coding RNA (lncRNA) molecules throughout spermatogenesis, suggesting specific functional genomics of these groupings. This analysis also found highly controlled expression of pairs of sense and antisense transcripts, suggesting conjoined regulatory mechanisms. In the next investigative vignette, we examined gene patterns in undifferentiated spermatogonia (USgs). We found the NANOS family of genes function as key drivers of transcriptomic signatures involved in human spermatogonial self-renewal programming; for the first time, demonstrating the relationship of NANOS1/2/3 transcripts in humans with scRNA-seq. Discussion and ConclusionsUsing HISTA, we found new observations that contribute to unraveling the mechanisms behind transcriptional regulation and maintenance germ cells across spermatogenesis. Furthermore, our findings provide guidance on future validation studies and experimental direction. Overall, HISTA continues to be utilized in testis-related research, and thus is updated regularly with new analytical methods, visualizations, and data. We aim to have it serve as a research environment for a broad range of investigators looking to explore the testis tissue and male infertility. Availability and ImplementationHISTA is available as an interactive web tool: https://conradlab.shinyapps.io/HISTA Source code and documentation for HISTA are provided on GitHub: https://github.com/eisascience/HISTA

bioinformatics↗

bioRxiv2023

Global effects of identity and aging on the human sperm methylome

As the average age of fatherhood increases worldwide, so too does the need for understanding effects of aging in male germline cells. Molecular change, including epigenomic alterations, may impact off-spring. Age-associated change to DNA cytosine methylation in the cytosine-guanine (CpG) context is a hallmark of aging tissues, including sperm. Prior studies have led to accurate models that predict a mans age based on specific methylation features in the DNA of sperm, but the relationship between aging and global DNA methylation in sperm remains opaque. Further clarification requires a more complete survey of the methylome with assessment of variability within and between individuals. We collected sperm methylome data in a longitudinal study of ten healthy fertile men. We used whole-genome bisulfite sequencing of samples collected 10 to 18 years apart from each donor. We found that, overall, variability between donors far exceeds age-associated variation. After controlling for donor identity, we see significant age-dependent genome-wide change to the methylome. Notably, trends of change with age depend on genomic location or annotation, with contrasting signatures that correlate with gene density and proximity to centromeres and promoter regions. These molecular signatures reflect a stable process that begins in early adulthood, progressing steadily through most of the lifespan, and warrants consideration in any future study of the aging sperm epigenome.

bioinformatics↗

bioRxiv2022

A tissue specific atlas of gene promoter DNA methylation variability and the clinical value of its assessment

BackgroundComplex diseases have multifactorial etiologies making clinically actionable diagnostic markers difficult to identify. Novel tools with higher diagnostic yield and utility in driving personalized care are needed. MethodsWe utilized Illumina methylation array data from over 2400 samples to assess DNA methylation patterns in 20 distinct cell types ranging from sperm to brain as well as various disease states. We generated a simple analysis pipeline for DNA methylation data that focuses on intra-individual methylation variability within gene promoters. The analysis is designed, not to identify single causative gene alterations but instead focuses on any movement away from "healthy" methylation. This approach identifies altered regulation across multiple genes in related pathways thus enabling us to detect shifts in gene regulatory activity associated with distinct tissues and phenotypes. We explored three distinct questions in our assessment. 1) Are patterns of epigenetic variability tissue specific? 2) Do diseased tissues exhibit altered variability patterns compared to normal tissue? 3) Can epigenetic variability be detected in complex disease? ResultsUnsupervised clustering analyses established that patterns of epigenetic variability are tissue specific and that these patterns are at least as predictive of tissue type as differential methylation analysis. We demonstrated the ability to use these patterns to differentiate between healthy and diseased tissue with unsupervised clustering even in cases of complex multifactorial diseases. We applied this method to the clinical use case of male infertility and found that men undergoing intrauterine insemination (IUI) with the lowest number of epigenetically dysregulated promoters in their sperm were almost twice as likely to father a child than men participating in IUI with the highest number of dysregulated promoters (p=0.011). We saw no significant difference in birth rates between groups of men with high and low numbers of dysregulated promoters undergoing in vitro fertilization (IVF), indicating IVF as a better treatment than IUI to achieve live birth in the presence of multi-pathway dysregulation in sperm. ConclusionsThis study demonstrates that patterns of epigenetic variability can differentiate between tissue types. While intuitive, this finding has never been demonstrated previously and suggests that specific epigenetic variability patterns may be used to predict phenotypic changes in disease states as these are, by definition, functional changes to cellular phenotypes. We demonstrate that the variability of gene regulatory marks are distinct between healthy and diseased tissue. This is particularly apparent at genes known to be important to cell function of the tissue of interest. While in some cases these regional alterations can be seen across the entire genome, more often the regulatory alterations that define a pathological phenotype are restricted to genes of known importance to a particular tissue. Importantly, in the case of sperm, we found that these patterns of variability did have utility in predicting infertile patients who would conceive through intrauterine insemination (IUI). We would propose that this discriminatory ability is due to the fact that the signature can be assessed in an n-of-1 context and that the patterns of variability identify any shift away from regulatory normalcy in pathways known to be impactful in the tissue of interest, and not only assessing the presence or absence of rare genetic variants. While the data presented here are encouraging, more work needs to be performed in other tissues to determine when, and in what context, these findings could be clinically actionable.

genomics↗

bioRxiv2021

Single-Cell Analysis of Human Testis Aging, and Impact of Elevated Body Mass Index

Aging human males display reduced reproductive health, however testis aging is poorly understood at the molecular and genomic level. Here, we utilized single-cell RNA-seq to profile over 44,000 cells from both young and older men (>60 years old) - and examined age-related changes in germline development and in the somatic niche. Interestingly, age-related changes in spermatogonial stem cells appeared modest, whereas age-related dysregulation of spermatogenesis and the somatic niche 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 younger men. Taken together, we reveal candidate molecular mechanisms underlying the complex testicular changes conferred by aging, and their exacerbation by concurrent chronic conditions such as obesity.

developmental biology↗

bioRxiv2021

A de novo paradigm for male infertility

IntroductionDe novo mutations (DNMs) are known to play a prominent role in sporadic disorders with reduced fitness1. We hypothesize that DNMs play an important role in male infertility and explain a significant fraction of the genetic causes of this understudied disorder. To test this hypothesis, we performed trio-based exome-sequencing in a unique cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare protein altering DNMs were classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of Loss-of-Function (LoF) DNMs in LoF-intolerant genes (p-value=1.00x10-5) as well as predicted pathogenic missense DNMs in missense-intolerant genes (p-value=5.01x10-4). One DNM gene identified, RBM5, is an essential regulator of male germ cell pre-mRNA splicing2. In a follow-up study, 5 rare pathogenic missense mutations affecting this gene were observed in a cohort of 2,279 infertile patients, with no such mutations found in a cohort of 5,784 fertile men (p-value=0.009). Our results provide the first evidence for the role of DNMs in severe male infertility and point to many new candidate genes affecting fertility.

genetics↗

bioRxiv2019

Biallelic mutations in M1AP are a frequent cause of meiotic arrest leading to male infertility

Male infertility affects [~]7% of men in Western societies, but its causes remain poorly understood. The most clinically severe form of male infertility is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis, but so far only few genes have been reported to cause germ cell arrest in males. To address this gap, whole exome sequencing was performed in 60 German men with complete meiotic arrest, and we identified in three unrelated men the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 arresting protein. Then, with collaborators from the International Male Infertility Genomics Consortium (IMIGC), we screened a Dutch cohort comprising 99 infertile men and detected the same homozygous variant c.676dup in a man with hypospermatogenesis predominantly displaying meiotic arrest. We also identified two Portuguese men with NOA carrying likely biallelic loss-of-function (LoF) and missense variants in M1AP among men screened by the Genetics of Male Infertility Initiative (GEMINI). Moreover, we discovered a homozygous missense variant p.(Pro389Leu) in M1AP in a consanguineous Turkish family comprising five infertile men. M1AP is predominantly expressed in human and mouse spermatogonia up to secondary spermatocytes and previous studies have shown that knockout male mice are infertile due to meiotic arrest. Collectively, these findings demonstrate that both LoF and missense M1AP variants that impair its protein cause autosomal-recessive meiotic arrest, non-obstructive azoospermia and male infertility. In view of the evidence from several independent groups and populations, M1AP should be included in the growing list of validated NOA genes.

genetics↗

bioRxiv2019

Paternal cigarette smoke alters DNA methylation in sperm and gene expression in offspring brain

Paternal cigarette smoke (CS) exposure is associated with increased risk of behavioral disorders and cancer in offspring, but the mechanism has not been identified. This study used mouse models to evaluate: 1) what impact paternal CS exposure has on sperm DNA methylation (DNAme), 2) whether sperm DNAme changes persist after CS exposure ends, 3) the degree to which DNAme and gene expression changes occur in offspring and 4) the mechanism underlying impacts of CS exposure. We demonstrate that CS exposure induces sperm DNAme changes that are partially corrected within 28 days of removal from CS exposure. Additionally, paternal smoking causes changes in neural DNAme and gene expression in offspring. Remarkably, the effects of CS exposure are largely recapitulated in oxidative stress-compromised Nrf2-/- mice and their offspring, independent of paternal smoking. These results demonstrate that paternal CS exposure impacts offspring phenotype and that oxidative stress underlies CS induced heritable epigenetic changes.

genetics↗

bioRxiv2017

Germ line aging and regional epigenetic instability: age prediction using human sperm DNA methylation signatures

BackgroundThe relationship between aging and epigenetic profiles has been highlighted in many recent studies. Models using somatic cell methylomes to predict age have been successfully constructed. However, gamete aging is quite distinct and as such age prediction using sperm methylomes is ineffective with current techniques.\n\nResultsWe have produced a model that utilizes human sperm DNA methylation signatures to predict chronological age by utilizing methylation array data from a total of 329 samples. The dataset used for model construction includes infertile patients, sperm donors, and individuals from the general population. Our model is capable of accurately predicting age with an R2 of 0.928 in our test data set. We additionally investigated the repeatability of prediction by processing the same sample on 6 different arrays and found very robust age prediction with an average standard deviation of only 0.877 years. Additionally, we found that smokers have approximately 5% increased age profiles compared to never smokers.\n\nConclusionsThe predictive model described herein was built to offer researchers the ability to assess \"germ line age\" by accessing sperm DNA methylation signatures at genomic regions affected by age. Our data suggest that this model can predict an individuals chronological age with a high degree of accuracy regardless of fertility status and with a high degree of repeatability. Additionally, our data appear to show age acceleration patterns as a result of smoking suggesting that the aging process in sperm may be impacted by environmental factors, though this effect appears to be quite subtle.

genetics↗