Mapping of long interspersed element-1 (L1) insertions by TIPseq provides information about sub chromosomal genetic variation in human embryos.

PURPOSE: Retrotransposons play important roles during early development when they are transiently de-repressed during epigenetic reprogramming. Long interspersed element-1 (L1), the only autonomous retrotransposon in humans, comprises 17% of the human genome. We applied the Single Cell Transposon Insertion Profiling by Sequencing (scTIPseq) to characterize and map L1 insertions in human embryos. METHODS: Sixteen cryopreserved, genetically tested, human blastocysts, were accessed from consenting couples undergoing IVF at NYU Langone Fertility Center. Additionally, four trios (father, mother, and embryos) were also evaluated. scTIPseq was applied to map L1 insertions in all samples, using L1 locations reported in the 1000 Genomes as controls. RESULTS: Twenty-nine unknown and unique insertions were observed in the sixteen embryos. Most were intergenic; no insertions were located in exons or immediately upstream of genes. The location or number of unknown insertions did not differ between euploid and aneuploid embryos, suggesting they are not merely markers of aneuploidy. Rather, scTIPseq provides novel information about sub-chromosomal structural variation in human embryos. Trio analyses showed a parental origin of all L1 insertions in embryos. CONCLUSION: Several studies have measured L1 expression at different stages of development in mice, but this study for the first time reports unknown insertions in human embryos that were inherited from one parent, confirming no de novo L1 insertions occurred in parental germline or during embryogenesis. Since one-third of euploid embryo transfers fail, future studies would be useful for understanding whether these sub-chromosomal genetic variants or de novo L1 insertions affect embryo developmental potential.

Competition between two HUSH complexes orchestrates the immune response to retroelement invasion.

The human silencing hub (HUSH) preserves genome integrity through the epigenetic repression of invasive genetic elements. However, despite our understanding of HUSH as an obligate complex of three subunits, only loss of MPP8 or Periphilin, but not TASOR, triggers interferon signaling following derepression of endogenous retroelements. Here, we resolve this paradox by characterizing a second HUSH complex that shares MPP8 and Periphilin but assembles around TASOR2, an uncharacterized paralog of TASOR. Whereas HUSH represses LINE-1 retroelements marked by the repressive histone modification H3K9me3, HUSH2 is recruited by the transcription factor IRF2 to repress interferon-stimulated genes. Mechanistically, HUSH-mediated retroelement silencing sequesters the limited pool of the shared subunits MPP8 and Periphilin, preventing TASOR2 from forming HUSH2 complexes and hence relieving the HUSH2-mediated repression of interferon-stimulated genes. Thus, competition between two HUSH complexes intertwines retroelement silencing with the induction of an immune response, coupling epigenetic and immune aspects of genome defense.

An Evaluation of DNA Methylation Levels and Sleep in Relation to Hot Flashes: A Cross-Sectional Study.

Objectives: We aimed to evaluate the DNA methylation levels in perimenopausal and postmenopausal women, measured through Long Interspersed Element-1 (LINE-1) and Alu, and the sleep parameters in relation to the presence of hot flashes (HFs). Methods: This cross-sectional study included 30 peri- or postmenopausal women aged between 45 and 55. The menopausal status was determined according to STRAW + 10 criteria and all participants had a low cardiovascular disease (CVD) risk profile determined by Framingham risk score. The sample was divided into two groups based on the presence or absence of HFs documented in their medical history during their initial visit: Group 1 (n = 15) with HFs present and Group 2 (n = 15) with HFs absent. The patients had polysomnography test and HFs were recorded both by sternal skin conductance and self-report overnight. Genomic DNA was extracted from the women's blood and methylation status was analyzed by fluorescence-based real-time quantitative PCR. The quantified value of DNA methylation of a target gene was normalized by ß-actin. The primary outcome was the variation in methylation levels of LINE-1 and Alu and sleep parameters according to the presence of HFs. Results: LINE-1 and Alu methylation levels were higher in Group 1 (HFs present), although statistically non-significant. LINE-1 methylation levels were negatively correlated with age. Sleep efficiency was statistically significantly lower for women in Group 1 (HFs present) (74.66% ± 11.16% vs. 82.63% ± 7.31%; p = 0.03). The ratio of duration of awakening to total sleep time was statistically significantly higher in Group 1 (HFs present) (22.38% ± 9.99% vs. 15.07% ± 6.93, p = 0.03). Objectively recorded hot flashes were significantly higher in Group 1 (4.00 ± 3.21 vs. 1.47 ± 1.46, p = 0.03). None of the cases in Group 2 self-reported HF despite objectively recorded HFs during the polysomnography. The rate of hot flash associated with awakening was 41.4% in the whole sample. Conclusions: Women with a history of hot flashes exhibited lower sleep efficiency and higher awakening rates. Although a history of experiencing hot flashes was associated with higher LINE-1 and Alu methylation levels, no statistical significance was found. Further studies are needed to clarify this association. This study was funded by the Scientific Research Projects Coordination Unit of Istanbul University-Cerrahpasa. Project number: TTU-2021-35629.

The transcription factor PAX5 activates human LINE1 retrotransposons to induce cellular senescence.

As a hallmark of senescent cells, the derepression of Long Interspersed Elements 1 (LINE1) transcription results in accumulated LINE1 cDNA, which triggers the secretion of the senescence-associated secretory phenotype (SASP) and paracrine senescence in a cGAS-STING pathway-dependent manner. However, transcription factors that govern senescence-associated LINE1 reactivation remain ill-defined. Here, we predict several transcription factors that bind to human LINE1 elements to regulate their transcription by analyzing the conserved binding motifs in the 5'-untranslated regions (UTR) of the commonly upregulated LINE1 elements in different types of senescent cells. Further analysis reveals that PAX5 directly binds to LINE1 5'-UTR and the binding is enhanced in senescent cells. The enrichment of PAX5 at the 5'-UTR promotes cellular senescence and SASP by activating LINE1. We also demonstrate that the longevity gene SIRT6 suppresses PAX5 transcription by directly binding to the PAX5 promoter, and overexpressing PAX5 abrogates the suppressive effect of SIRT6 on stress-dependent cellular senescence. Our work suggests that PAX5 could serve as a potential target for drug development aiming to suppress LINE1 activation and treat senescence-associated diseases.

Diagnostic value of combined detection of plasma cfDNA concentration and integrity in NSCLC.

Aim: To evaluate the value of combined detection of plasma cfDNA concentration and integrity in the early diagnosis of NSCLC. Methods: Real-time fluorescence quantitative PCR was used to determine the concentration and integrity of plasma cfDNA in 71 NSCLC patients and 53 healthy people. Results: Combined detection of plasma cfDNA concentration and integrity had higher diagnostic power in differentiating NSCLC patients with stage I/II from healthy people than detection of plasma cfDNA concentration alone or integrity alone. The AUC, sensitivity and specificity of the combined detection of plasma cfDNA concentration and integrity were 0.781, 0.62 and 0.85. Conclusion: Combined detection of plasma cfDNA concentration and integrity could improve the diagnostic value in NSCLC detection.

The discovery of cfDNA has opened up a wide range of new possibilities for the diagnosis of cancer. CfDNA provides a noninvasive diagnostic approach for early screening, early detection and monitoring of patients with cancer. Currently, the application of cfDNA in clinical practice for NSCLC patients has been widely reported, which mainly focused on DNA methylation detection, oncogenic driver gene mutation detection. However, few studies have evaluated the diagnostic value of combined detection of plasma cfDNA concentration and integrity for NSCLC patients. Our study suggests that the combination of plasma cfDNA concentration and integrity has higher AUC value in differentiating NSCLC patients from healthy individuals than plasma cfDNA concentration alone or integrity alone.

Accurate detection of copy number aberrations in FFPE samples using the mFAST-SeqS approach.

BACKGROUND: Shallow whole genome sequencing (Shallow-seq) is used to determine the copy number aberrations (CNA) in tissue samples and circulating tumor DNA. However, costs of NGS and challenges of small biopsies ask for an alternative to the untargeted NGS approaches. The mFAST-SeqS approach, relying on LINE-1 repeat amplification, showed a good correlation with Shallow-seq to detect CNA in blood samples. In the present study, we evaluated whether mFAST-SeqS is suitable to assess CNA in small formalin-fixed paraffin-embedded (FFPE) tissue specimens, using vulva and anal HPV-related lesions. METHODS: Seventy-two FFPE samples, including 36 control samples (19 vulva;17 anal) for threshold setting and 36 samples (24 vulva; 12 anal) for clinical evaluation, were analyzed by mFAST-SeqS. CNA in vulva and anal lesions were determined by calculating genome-wide and chromosome arm-specific z-scores in comparison with the respective control samples. Sixteen samples were also analyzed with the conventional Shallow-seq approach. RESULTS: Genome-wide z-scores increased with the severity of disease, with highest values being found in cancers. In vulva samples median and inter quartile ranges [IQR] were 1[0-2] in normal tissues (n = 4), 3[1-7] in premalignant lesions (n = 9) and 21[13-48] in cancers (n = 10). In anal samples, median [IQR] were 0[0-1] in normal tissues (n = 4), 14[6-38] in premalignant lesions (n = 4) and 18[9-31] in cancers (n = 4). At threshold 4, all controls were CNA negative, while 8/13 premalignant lesions and 12/14 cancers were CNA positive. CNA captured by mFAST-SeqS were mostly also found by Shallow-seq. CONCLUSION: mFAST-SeqS is easy to perform, requires less DNA and less sequencing reads reducing costs, thereby providing a good alternative for Shallow-seq to determine CNA in small FFPE samples.

Retrotransposons in embryogenesis and neurodevelopment.

Retrotransposable elements (RTEs) are genetic elements that can replicate and insert new copies into different genomic locations. RTEs have long been identified as 'parasitic genes', as their mobilization can cause mutations, DNA damage, and inflammation. Interestingly, high levels of retrotransposon activation are observed in early embryogenesis and neurodevelopment, suggesting that RTEs may possess functional roles during these stages of development. Recent studies demonstrate that RTEs can function as transcriptional regulatory elements through mechanisms such as chromatin organization and noncoding RNAs. It is clear, however, that RTE expression and activity must be restrained at some level during development, since overactivation of RTEs during neurodevelopment is associated with several developmental disorders. Further investigation is needed to understand the importance of RTE expression and activity during neurodevelopment and the balance between RTE-regulated development and RTE-mediated pathogenesis.

Insertion of short L1 sequences generates inter-strain histone acetylation differences in the mouse.

BACKGROUND: Gene expression divergence between populations and between individuals can emerge from genetic variations within the genes and/or in the cis regulatory elements. Since epigenetic modifications regulate gene expression, it is conceivable that epigenetic variations in cis regulatory elements can also be a source of gene expression divergence. RESULTS: In this study, we compared histone acetylation (namely, H3K9ac) profiles in two mouse strains of different subspecies origin, C57BL/6 J (B6) and MSM/Ms (MSM), as well as their F1 hybrids. This identified 319 regions of strain-specific acetylation, about half of which were observed between the alleles of F1 hybrids. While the allele-specific presence of the interferon regulatory factor 3 (IRF3) binding sequence was associated with allele-specific histone acetylation, we also revealed that B6-specific insertions of a short 3' fragment of LINE-1 (L1) retrotransposon occur within or proximal to MSM-specific acetylated regions. Furthermore, even in hyperacetylated domains, flanking regions of non-polymorphic 3' L1 fragments were hypoacetylated, suggesting a general activity of the 3' L1 fragment to induce hypoacetylation. Indeed, we confirmed the binding of the 3' region of L1 by three Krüppel-associated box domain-containing zinc finger proteins (KZFPs), which interact with histone deacetylases. These results suggest that even a short insertion of L1 would be excluded from gene- and acetylation-rich regions by natural selection. Finally, mRNA-seq analysis for F1 hybrids was carried out, which disclosed a link between allele-specific promoter/enhancer acetylation and gene expression. CONCLUSIONS: This study disclosed a number of genetic changes that have changed the histone acetylation levels during the evolution of mouse subspecies, a part of which is associated with gene expression changes. Insertions of even a very short L1 fragment can decrease the acetylation level in their neighboring regions and thereby have been counter-selected in gene-rich regions, which may explain a long-standing mystery of discrete genomic distribution of LINEs and SINEs.

SAFB restricts contact domain boundaries associated with L1 chimeric transcription.

Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.

Maternal non-compliance with recommended folic acid supplement use alters global DNA methylation in cord blood of newborns: A cohort study.

BACKGROUND & AIMS: Prenatal folate exposure may alter epigenetic marks in the offspring. We aimed to evaluate associations between prenatal exposure to folic acid (FA) in preconception and in utero with cord blood DNA methylation in long interspersed nuclear element 1 (LINE-1) and Alu short interspersed nuclear elements (SINEs) as markers of global DNA methylation levels. METHODS: Data come from 325 mother-child pairs participating in the Nutrition in Early Life and Asthma (NELA) birth cohort (2015-2018). Pregnant women were asked about supplement use, including brand name and dose, one month before pregnancy (preconception) and through the trimesters of pregnancy. Maternal dietary folate intake was assessed using a validated food frequency questionnaire with additional questions for FA supplement use. Folate serum levels were measured in mothers at 24 weeks of gestation and in cord blood of newborns. DNA methylation was quantitatively assessed by bisulfite pyrosequencing on 5 LINE-1 and 3 Alu different elements. Associations were estimated using multivariable linear regression models. RESULTS: A reduction in methylation levels of LINE-1 in newborns was associated with the use of FA supplements below the recommended doses (<400 ug/day) during preconception (-0.50; 95% CI: -0.91, -0.09; P = 0.016), and from preconception up to 12 weeks of gestation (-0.48; 95% CI: -0.88, -0.08; P = 0.018). Maternal use of FA supplements above the tolerable upper intake level of 1000 ug/day from preconception until 12 weeks of gestation was also related to lower methylation in LINE-1 at birth (-0.77; 95% CI: -1.52, -0.02; P = 0.044). Neither FA supplement use after 12 weeks of gestation nor maternal total folate intake (diet plus supplements) were associated with global DNA methylation levels at birth. CONCLUSIONS: Maternal non-compliance with the use of FA supplement recommendations from preconception up to 12 weeks of gestation reduces offspring global DNA methylation levels at birth.

Improved LINE-1 Detection through Pattern Matching by Increasing Probe Length.

Long Interspersed Element-1 (LINE-1 or L1) is an autonomous transposable element that accounts for 17% of the human genome. Strong correlations between abnormal L1 expression and diseases, particularly cancer, have been documented by numerous studies. L1PD (LINE-1 Pattern Detection) had been previously created to detect L1s by using a fixed pre-determined set of 50-mer probes and a pattern-matching algorithm. L1PD uses a novel seed-and-pattern-match strategy as opposed to the well-known seed-and-extend strategy employed by other tools. This study discusses an improved version of L1PD that shows how increasing the size of the k-mer probes from 50 to 75 or to 100 yields better results, as evidenced by experiments showing higher precision and recall when compared to the 50-mers. The probe-generation process was updated and the corresponding software is now shared so that users may generate probes for other reference genomes (with certain limitations). Additionally, L1PD was applied to other non-human genomes, such as dogs, horses, and cows, to further validate the pattern-matching strategy. The improved version of L1PD proves to be an efficient and promising approach for L1 detection.

N6-adenosine methylation and the regulatory mechanism on LINE-1.

Long interspersed elements-1(LINE-1) is the only autonomous transposon in human genome，and its retrotransposition results in change of cellular genome structure and function, leading occurrence of various severe diseases. As a central key intermediated component during life cycle of LINE-1 retrotransposition, the host modification of LINE-1 mRNA affects the LINE-1 transposition directly. N6-adenosine methylation(m6A), the most abundant epigenetic modification on eukaryotic RNA, is dynamically reversible. m6A modification is also found on LINE-1 mRNA, and it participants regulation of the whole LINE-1 replication cycle, with affecting LINE-1 retrotransposition as well as its adjacent genes expression, followed by influencing genomic stability, cellular self-renewal, and differentiation potential, which plays important roles in human development and diseases. In this review, we summarize the research progress in LINE-1 m6A modification, including its modification positions, patterns and related mechanisms, hoping to provide a new sight on the mechanism research and treatment of related diseases.

Navigating the brain and aging: exploring the impact of transposable elements from health to disease.

Transposable elements (TEs) are mobile genetic elements that constitute on average 45% of mammalian genomes. Their presence and activity in genomes represent a major source of genetic variability. While this is an important driver of genome evolution, TEs can also have deleterious effects on their hosts. A growing number of studies have focused on the role of TEs in the brain, both in physiological and pathological contexts. In the brain, their activity is believed to be important for neuronal plasticity. In neurological and age-related disorders, aberrant activity of TEs may contribute to disease etiology, although this remains unclear. After providing a comprehensive overview of transposable elements and their interactions with the host, this review summarizes the current understanding of TE activity within the brain, during the aging process, and in the context of neurological and age-related conditions.

Regulation and function of transposable elements in cancer genomes.

Over half of human genomic DNA is composed of repetitive sequences generated throughout evolution by prolific mobile genetic parasites called transposable elements (TEs). Long disregarded as "junk" or "selfish" DNA, TEs are increasingly recognized as formative elements in genome evolution, wired intimately into the structure and function of the human genome. Advances in sequencing technologies and computational methods have ushered in an era of unprecedented insight into how TE activity impacts human biology in health and disease. Here we discuss the current views on how TEs have shaped the regulatory landscape of the human genome, how TE activity is implicated in human cancers, and how recent findings motivate novel strategies to leverage TE activity for improved cancer therapy. Given the crucial role of methodological advances in TE biology, we pair our conceptual discussions with an in-depth review of the inherent technical challenges in studying repeats, specifically related to structural variation, expression analyses, and chromatin regulation. Lastly, we provide a catalog of existing and emerging assays and bioinformatic software that altogether are enabling the most sophisticated and comprehensive investigations yet into the regulation and function of interspersed repeats in cancer genomes.

Insights into LINE-1 reverse transcription guide therapy development.

Subsets of long interspersed nuclear element 1 (LINE-1) retrotransposons can 'retrotranspose' throughout the human genome at a cost to host cell fitness, as observed in some cancers. Pharmacological inhibition of LINE-1 retrotransposition requires a comprehensive understanding of the LINE-1 ORF2p reverse transcriptase. Two recent publications, by Thawani et al. and Baldwin et al., report structures of LINE-1 ORF2p and address long-standing mechanistic gaps regarding LINE-1 retrotransposition. Both studies will be critical to design new specific inhibitors of the LINE-1 ORF2p reverse transcriptase.

Pharmacological inhibition of neddylation impairs long interspersed element 1 retrotransposition.

Aberrant long interspersed element 1 (LINE-1 or L1) activity can cause insertional mutagenesis and chromosomal rearrangements and has been detected in several types of cancers. Here, we show that neddylation, a post-translational modification process, is essential for L1 transposition. The antineoplastic drug MLN4924 is an L1 inhibitor that suppresses NEDD8-activating enzyme activity. Neddylation inhibition by MLN4924 selectively impairs ORF2p-mediated L1 reverse transcription and blocks the generation of L1 cDNA. Consistent with these results, MLN4924 treatment suppresses the retrotransposition activity of the non-autonomous retrotransposons short interspersed nuclear element R/variable number of tandem repeat/Alu and Alu, which rely on the reverse transcription activity of L1 ORF2p. The E2 enzyme UBE2M in the neddylation pathway, rather than UBE2F, is required for L1 ORF2p and retrotransposition. Interference with the functions of certain neddylation-dependent Cullin-really interesting new gene E3 ligases disrupts L1 reverse transcription and transposition activity. Our findings provide insights into the regulation of L1 retrotransposition and the identification of therapeutic targets for L1 dysfunctions.

Locus-level L1 DNA methylation profiling reveals the epigenetic and transcriptional interplay between L1s and their integration sites.

Long interspersed element 1 (L1) retrotransposons are implicated in human disease and evolution. Their global activity is repressed by DNA methylation, but deciphering the regulation of individual copies has been challenging. Here, we combine short- and long-read sequencing to unveil L1 methylation heterogeneity across cell types, families, and individual loci and elucidate key principles involved. We find that the youngest primate L1 families are specifically hypomethylated in pluripotent stem cells and the placenta but not in most tumors. Locally, intronic L1 methylation is intimately associated with gene transcription. Conversely, the L1 methylation state can propagate to the proximal region up to 300 bp. This phenomenon is accompanied by the binding of specific transcription factors, which drive the expression of L1 and chimeric transcripts. Finally, L1 hypomethylation alone is typically insufficient to trigger L1 expression due to redundant silencing pathways. Our results illuminate the epigenetic and transcriptional interplay between retrotransposons and their host genome.

Long interspersed nuclear elements safeguard neural progenitors from precocious differentiation.

Long interspersed nuclear element-1 (L1 or LINE-1) is a highly abundant mobile genetic element in both humans and mice, comprising almost 20% of each genome. L1s are silenced by several mechanisms, as their uncontrolled expression has the potential to induce genomic instability. However, L1s are paradoxically expressed at high levels in differentiating neural progenitor cells. Using in vitro and in vivo techniques to modulate L1 expression, we report that L1s play a critical role in both human and mouse brain development by regulating the rate of neural differentiation in a reverse-transcription-independent manner.

Epigenetic role of LINE-1 methylation and key genes in pregnancy maintenance.

Spontaneous abortion is a pregnancy complication characterized by complex and multifactorial etiology. About 5% of childbearing women are globally affected by early pregnancy loss (EPL) and most of them experience recurrence (RPL). Epigenetic mechanisms and controlled inflammation are crucial for pregnancy maintenance and genetic predispositions may increase the risk affecting the maternal-fetal crosstalk. Combined analyses of global methylation, inflammation and inherited predispositions may contribute to define pregnancy loss etiopathogenesis. LINE-1 epigenetic regulation plays crucial roles during embryo implantation, and its hypomethylation has been associated with senescence and several complex diseases. By analysing a group of 230 women who have gone through pregnancy interruption and comparing those experiencing spontaneous EPL (n = 123; RPL, 54.5%) with a group of normal pregnant who underwent to voluntary interruption (VPI, n = 107), the single statistical analysis revealed significant lower (P < 0.00001) LINE-1 methylation and higher (P < 0.0001) mean cytokine levels (CKs: IL6, IL10, IL17A, IL23) in EPL. Genotyping of the following SNPs accounted for different EPL/RPL risk odds ratio: F13A1 rs5985 (OR = 0.24; 0.06-0.90); F13B rs6003 (OR = 0.23; 0.047-1.1); FGA rs6050 (OR = 0.58; 0.33-1.0); CRP rs2808635/rs876538 (OR = 0.15; 0.014-0.81); ABO rs657152 (OR = 0.48; 0.22-1.08); TP53 rs1042522 (OR = 0.54; 0.32-0.92); MTHFR rs1801133/rs1801131 (OR = 2.03; 1.2-3.47) and FGB rs1800790 (OR = 1.97; 1.01-3.87), although Bonferroni correction did not reach significant outputs. Principal Component Analysis (PCA) and logistic regression disclosed further SNPs positive/negative associations (e.g. APOE rs7412/rs429358; FGB rs1800790; CFH rs1061170) differently arranged and sorted in four significant PCs: PC1 (F13A, methylation, CKs); PC3 (CRP, MTHFR, age, methylation); PC4 (F13B, FGA, FGB, APOE, TP53, age, methylation); PC6 (F13A, CFH, ABO, MTHFR, TP53, age), yielding further statistical power to the association models. In detail, positive EPL risk association was with PC1 (OR = 1.81; 1.33-2.45; P < 0.0001) and negative associations with PC3 (OR = 0.489; 0.37-0.66; P < 0.0001); PC4 (OR = 0.72; 0.55-0.94; P = 0.018) and PC6 (OR = 0.61; 0.46-0.81; P = 0.001). Moreover, significant inverse associations were detected between methylation and CKs levels in the whole group (rIL10 = - 0.22; rIL17A = - 0.25; rIL23 = - 0.19; rIL6 = - 0.22), and methylation with age in the whole group, EPL and RPL subgroups (r2TOT = 0.147; r2EPL = 0.136; r2 RPL = 0.248), while VPI controls lost significance (r2VPI = 0.011). This study provides a valuable multilayer approach for investigating epigenetic abnormalities in pregnancy loss suggesting genetic-driven dysregulations and anomalous epigenetic mechanisms potentially mediated by LINE-1 hypomethylation. Women with unexplained EPL might benefit of such investigations, providing new insights for predicting the pregnancy outcome and for treating at risk women with novel targeted epidrugs.

RNA m6A modification regulates L1 retrotransposons in human spermatogonial stem cell differentiation in vitro and in vivo.

The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m6A) plays an essential role in regulating various biological processes. However, the function of m6A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m6A methyltransferase METTL3 or the m6A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m6A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m6A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m6A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m6A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m6A modification in restricting L1 retrotransposition.