Bisulfite Sequencing Research Articles

DNA methylation-regulated DDX27 promotes colorectal cancer progression through EZH2.

Early postnatal overnutrition as a contributor to metabolic dysregulation: Insights into hepatic epigenetic mechanisms.

Both absolute and presumably active rDNA (with a hypomethylated promoter region) copy number (CN) in the haploid human sperm genome are highly variable among individuals. Using a combination of droplet digital PCR and deep bisulfite sequencing, we have quantified absolute and presumably active rDNA CN in sperm samples (N = 190) with normal (NSPs) vs. abnormal semen parameters (ASPs), as well as in samples leading or not leading to a clinical pregnancy. ASP samples had a significantly lower presumably active CN (104 ± 31) than normozoospermic samples (115 ± 31). The loss of presumably active rDNA copies is explained by an increased promoter methylation (13.9% in ASP vs. 12.1% in NSP). When correcting for confounding factors, most importantly semen quality, samples not leading to a clinical pregnancy after IVF/ICSI displayed a significantly lower absolute (225 ± 51) and presumably active CN (103 ± 30) than samples with pregnancy (249 ± 62 and 115 ± 31, respectively). This between-group difference was most noticeable in normozoospermic males: absolute CN 220 ± 54; presumably active CN 107 ± 32 in samples without pregnancy and absolute CN 246 ± 63; presumably active CN 120 ± 28 in samples with pregnancy. We propose that absolute/active rDNA CN in sperm is a modulating factor contributing to idiopathic male infertility. In NSP samples, presumably active CN increases with absolute CN, which may have a positive impact on fertility and ART outcome. Our results suggest that approximately 60 active sperm rDNA copies are sufficient to establish a pregnancy.

Epigenetic transgenerational effects of prenatal exposure to 2,2',4,4'-tetrabromodiphenyl ether on sperm function and DNA methylation in rat offspring.

Developmental programming: Differing impact of prenatal testosterone and prenatal bisphenol-A -treatment on hepatic methylome in female sheep.

Functional characterization and epigenetic regulation of Tcf3a and Tcf3b during IgM+ B cell activation in large yellow croaker (Larimichthys crocea).

BackgroundOral squamous cell carcinoma (OSCC) remains a major global health burden, often diagnosed at advanced stages, with limited survival improvement. Epigenetic dysregulation, particularly promoter hypermethylation, plays a significant role in OSCC pathogenesis. Previous study by Demokan et al. identified Gamma-Aminobutyric Acid Receptor Beta-2 (GABRB2) as a candidate gene subject to methylation-dependent transcriptional silencing. This study aimed to evaluate the methylation and expression profiles of GABRB2 in OSCC and to explore its potential as a diagnostic biomarker.MethodsTissue and serum samples from 48 OSCC patients and 15 healthy controls were analyzed for GABRB2 promoter methylation via quantitative methylation-specific PCR and for gene expression levels via quantitative real-time PCR. Relationships between methylation, expression levels, clinicopathological parameters, and patient outcomes were statistically assessed.ResultsGABRB2 promoter hypermethylation was detected in 14.6% of tumor samples, predominantly in floor of the mouth and buccal tumors. Decreased GABRB2 expression was observed in 52.1% of tumor tissues compared to matched-normal tissues, while 22.9% of tumors exhibited increased expression. Methylation-dependent expression loss was confirmed in 71.4% of methylated tumors. Notably, decreased expression and hypermethylation of GABRB2 were correlated with poor prognosis parameters (p < 0.05). ROC analysis showed moderate discrimination for GABRB2 expression in distinguishing tumor from normal tissues (AUC = 0.635, p = 0.022), while serum-based analysis demonstrated poor diagnostic performance. Survival analysis revealed no statistically significant prognostic impact of GABRB2 expression levels (p > 0.05). However, alcohol consumption (p = 0.006) and recurrence developed (p = 0.030) as independent predictors of poor prognosis in multivariate analysis.ConclusionOur study suggests that there was association between methylation-based expression loss of GABRB2 with OSCC’ subgroups. To our knowledge, this is the first study to investigate GABRB2 gene methylation and expression profiling in both invasive/non-invasive samples from OSCC patients. Hypermethylation in the newly identified candidate GABRB2 gene may play a role in the development of tumors originating from the mouth floor and buccal anatomical regions of the oral cavity. However, since the loss of expression seen in the majority of our patients, we thought that only methylation may not the inhibition mechanism for GABRB2 gene decreased expression. The new identified candidate gene may be specific for the OSCC’s subgroups.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12903-025-06899-y.

A large scale detection of MLH1 methylation is lacking in esophageal cancer. MLH1 is a well-known mismatch repair gene. The mechanism of MLH1 in DNA double strand break (DSB) repair remains unclear. Esophageal cancer cell lines and 1018 cases of primary cancer samples were employed. Methylation specific PCR, Western Blot, and CRISPR/Cas9 knockout technique were utilized. Methylation of MLH1 was detected in 3.93%. MLH1 methylation was significantly associated with tumor differentiation, male gender, smoking, and tumor size (all p < 0.05). The median overall survival (OS) was 24.7 months (95% CI 13.4-36.0) and 51.5 months (95% CI 40.4-62.5) in MLH1 methylated and unmethylated groups, respectively. OS was shorter in MLH1 methylated compared to unmethylated group patients (p < 0.01). Multivariate factor analysis indicated that MLH1 methylation is an independent poor prognosis marker (p < 0.05). MLH1 promotes ataxia telangiectasia mutated (ATM), ataxia telangiectasia and RAD3-related (ATR), and non-homologous end-joining repair (NHEJ), while inhibiting microhomology-mediated end joining (MMEJ) repair signaling pathways. Deletion of MLH1 sensitized esophageal cancer cells to novobiocin. MLH1 plays important roles in DSB repair and deletion of MLH1 sensitizes ESCC cells to Polθ inhibitor.

Background/ObjectivesEpstein-Barr Virus (EBV) is a ubiquitous virus associated with a variety of diseases including cancers. Evidence has emerged that the C promoter is methylated in many EBV-associated malignancies, whereas in free virion DNA it is unmethylated. We have developed and evaluated a methylation-specific PCR assay for the EBV C Promoter (MSPCP) that can be applied to human biological specimens to quantify EBV methylation.MethodsTwo sets of methylation-specific primers were designed to anneal to bisulfite-converted DNA sequences with 3 CpGs in the forward primer binding site, and 2 CpGs in the reverse primer binding site. We evaluated this method in synthetic oligonucleotides, DNA extracted from cell lines, virion supernatants, and a variety of clinical specimens. EBV methylation of Cp, as measured by MSPCP, was validated with two orthogonal methods in select samples.ResultsIn contrived samples, this method had a linear range between 0–100% methylation. Application of this assay to DNA extracted from 11 formalin-fixed paraffin-embedded biopsy specimens showed high-level C promoter methylation in EBV-associated tumors (94–100%) but not in EBV-associated lymphoid hyperplasia. High-level EBV methylation was also detected in cell-free DNA extracted from the plasma of 13 patients with EBV-associated Hodgkin lymphoma. In contrast, EBV methylation was either not-detected, or detected at very low levels, in saliva from 25 adults in a general university population consistent with the presence of virion DNA.ConclusionsMSPCP is a simple, rapid and accurate method that characterizes the methylation status of the EBV C promoter, which may be useful in a variety of research and clinical settings.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13027-025-00702-x.

Cholangiocarcinoma (CC) is a highly lethal malignancy that urgently requires reliable prognostic biomarkers. Although MUC1 expression and promoter methylation have been implicated in CC, the clinical significance of promoter methylation pattern composition, beyond average methylation levels, remains unclear. Here, we investigated the relationship between MUC1 promoter methylation heterogeneity, MUC1 mRNA expression, and prognosis in CC. We analyzed bisulfite amplicon sequencing data and mRNA expression of MUC1, DNA methylation-related enzymes (TET1, TET2, TET3, Dnmt1, and Dnmt3a), and tumor microenvironment stress markers in 131 CC tissues. In the neoplastic region, high MUC1 mRNA expression was associated with poor overall survival (HR = 0.131, 95% CI: 0.02to0.95, p = 0.042) and correlated with the abundance of completely unmethylated promoter patterns (r = 0.386, p < 0.001). Among the enzymes analyzed, only TET3 expression significantly correlated with the abundance of completely unmethylated patterns in the neoplastic region (Cohen's f2 = 0.108, p = 0.009), suggesting a potential region-specific regulatory association. We visualized beta-diversity in methylation pattern composition using t-SNE and classified samples into two groups based on a linear decision boundary in the t-SNE space. This classification stratified prognosis independently of clinical factors (HR = 0.291, 95% CI: 0.06to0.94, p = 0.037; multivariate p = 0.021). These findings propose a novel, composition-based epigenetic stratification framework in CC, revealing that MUC1 promoter methylation pattern structure-rather than average methylation level-has prognostic relevance. Our results highlight the potential of pattern-resolved methylation profiling in the development of clinically applicable epigenetic biomarkers.

Introduction Nasopharyngeal carcinoma (NPC) is a serious cancer with a poor prognosis and a significant risk of metastasis. Although its epigenetic control mechanisms are still unknown, epigallocatechin-3-gallate (EGCG) exhibits strong anticancer properties. Promoter methylation in malignancies often silences PACRG, a putative tumor suppressor gene. This study aimed to determine whether EGCG suppresses cancer by altering PACRG gene expression in NPC cells. Material and methods NPC cells were subjected to EGCG or 5-aza-dC. Cellular functions were assessed by CCK-8, Transwell, and flow cytometry assays. Methylation-specific PCR (MSP) was used to identify PACRG promoter methylation, and gene expression was measured using quantitative real-time PCR (qRT-PCR) and Western blot (WB). The expression of DNA methyltransferase-related genes was evaluated. siRNA targeting PACRG was used to assess its functional involvement in the effects mediated by EGCG. Results EGCG exerted dose- and time-dependent effects on NPC cells by reducing proliferation and migration while inducing apoptosis and G2 phase cell cycle arrest. Mechanistically, EGCG significantly reduced the mRNA expression and enzymatic activity of DNMT1, DNMT3A, and DNMT3B, resulting in decreased PACRG promoter methylation and restored PACRG expression. Functional assays revealed that knockdown of PACRG diminished the inhibitory effect of EGCG on NPC cells, indicating that the epigenetic reactivation of PACRG partially mediates the tumor-suppressive function of EGCG. Conclusions Our results revealed that EGCG inhibits NPC progression by reducing PACRG promoter methylation, highlighting that PACRG demethylation and reactivation are a promising therapeutic strategy.

DNA methylation is a key epigenetic mechanism regulating gene expression during spermatogenesis. This study investigated the effects of experimental amebiasis induced by Entamoeba histolytica and its treatment with metronidazole (MTZ) on the methylation status of the Spermatogenesis Associated 6 (SPATA6) gene and male reproductive function. Twenty-four adult male rats were assigned to control, infected, and MTZ-treated groups. Following treatment, testicular tissues were analyzed for SPATA6 promoter methylation via bisulfite sequencing, infection confirmation by PCR, histopathological changes by hematoxylin and eosin (H&E) staining, Inhibin B and Androgen-Binding Protein (ABP) expression by immunohistochemistry, and sperm quality indices. The infected group exhibited distinct non-CpG methylation at a SPATA6 locus, confirmed reproductive tract infection, severe testicular damage, increased expression of Inhibin B and ABP, and significantly impaired sperm parameters. MTZ treatment successfully cleared the parasite and partially restored testicular architecture and sperm count; however, residual abnormalities in sperm motility, histology, and SPATA6 methylation persisted. While these findings suggest that parasitic infection and its treatment may induce epigenetic dysregulation in the testis, the direct functional link between the observed methylation change and reproductive outcomes remains inconclusive due to the limited scope of analysis. These results underscore the need for genome-wide methylation and transcriptomic profiling to better characterize the molecular basis of infection- and treatment-related reproductive effects. The study provides initial insights into infection-associated epigenetic modulation in male reproduction, with potential implications for fertility and reproductive health.

The prognosis and resistance to temozolomide in glioblastoma have been evaluated based on O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation using bulk tumor samples resected via surgery. However, it remains unclear whether MGMT promoter methylation differs between glioblastoma stem cells and the overall tumor within the patient's brain. Chemoresistance of glioblastoma stem cells is a critical factor in understanding the refractory nature of the disease. This study aimed to assess MGMT promoter methylation in glioblastoma stem cells and compare it with that in differentiated tumor cells. Glioblastoma stem-like cells (GSCs) and differentiated GSCs (dGSCs) were cultured from surgically resected tumors. Following DNA extraction and bisulfite conversion, real-time methylation-specific PCR was performed to quantify MGMT promoter methylation in three tumor cell types: GSCs, dGSCs, and surgically resected tumor samples. MGMT protein and CD133 expression levels were assessed via flow cytometry. Based on CD133 expression and microscopic evaluation, dGSCs exhibited a more differentiated phenotype than GSCs. No significant differences were observed in ΔCt values or MGMT promoter methylation ratios between GSCs and dGSCs. Similarly, ΔCt values and methylation ratios showed negligible differences between GSCs and surgically resected tumors. MGMT was highly expressed in both GSCs and dGSCs, consistent with an unmethylated MGMT promoter. MGMT promoter methylation status in glioblastoma stem cells remained unchanged during differentiation and was comparable to that in surgically resected tumors. As temozolomide is presumed to be ineffective against glioblastoma stem cells with unmethylated MGMT promoters, MGMT blockade may be a beneficial therapeutic strategy.

This study investigated the expression, methylation patterns, and clinicopathological implications of ARHGAP40 in renal cell carcinoma (RCC), the most common urinary malignancy. A total of 60 clear cell renal cell carcinomas (ccRCC), 30 papillary renal cell carcinomas (pRCC), 30 chromophobe renal cell carcinomas (chRCC), and 13 other RCC subtypes were enrolled. ARHGAP40 expression was analyzed in both RCC tissues and matched paracancerous normal tissues using immunohistochemistry (IHC). The methylation status of the ARHGAP40 promoter region was assessed in both normal and tumor samples by bisulfite sequencing PCR (BSP). Circulating tumor DNA (ctDNA) extracted from peripheral blood samples of RCC patients (20), patients with benign renal tumors (1), and healthy controls (14) was quantitatively analyzed for methylation using quantitative methylation-specific PCR (qMSP). ARHGAP40 expression was significantly downregulated in RCC compared to matched normal tissues (P < 0.001). This reduced expression correlated with tumor necrosis (P = 0.009) but showed no significant association with age, gender, tumor location, tumor diameter, TNM stage, or vascular invasion. In the ccRCC subtype, ARHGAP40 expression exhibited a progressive decrease with larger tumor diameter (P = 0.045), advancing histological grade (P = 0.032), and tumor necrosis (P = 0.011). The methylation status of ARHGAP40 was consistent with its expression level in both tumor and adjacent normal tissues. Methylated ARHGAP40 DNA was detectable only in RCC patient ctDNA samples. ARHGAP40 is epigenetically silenced in RCC through methylation-mediated downregulation, which correlates with tumor necrosis and grade. The detection of methylated ARHGAP40 in ctDNA holds promise as a potential biomarker for early RCC diagnosis.

Objective Paternal high-fat diet (HFD) has detrimental effects on offspring. However, the extent of comprehensive damage and the underlying mechanisms associated with sustained multigenerational HFD exposure remain unclear. This study aims to investigate intergenerational progressive accumulation of obesity and glycolipid metabolic disorders, as well as mechanisms mediated by DNA methylation. Methods We performed a novel paternally multi-generational HFD consumption model in male C57BL/6 J mice, while excluding maternal gestational effects and any confounding influences from females. The body weight and glycolipid metabolism indicators of each generation of male mice were determined. The intergenerational transmission of CpG methylation and gene expression variation was detected through mRNA microarray, methylated DNA immunoprecipitation (MeDIP)-chip, bisulfite sequencing, RT-qPCR, and Western blot etc. Analysis, to indicate genes involved in glycolipid metabolism related to the intergenerational reprogramming. Results The HFD caused intergenerational accumulation of body weight increase, disturbance of glycolipid metabolism, and insulin insensitivity in male offspring. MeDIP/gene-chip results indicated that paternal HFD significantly modified gene expression and DNA methylation profiles in the liver and sperm of offspring. The majority of differential genes exhibited hypermethylation in promoter regions and reduced expression in the liver, which were linked to the glucolipid metabolic signaling pathway. The elevated promoter methylation and expression states of Spns2 , Lonp1, and Hk1 , which are involved in glycolipid metabolism, were inherited by offspring. Conclusion This research shows that paternal sustained multi-generational HFD could induce intergenerational progressive accumulation of obesity and metabolic disorder through DNA methylation regulation, and identifies the target genes related to the intergenerational reprogramming, which provides new insights for the establishment of healthy diets and lifestyles.

Rare earth elements (REEs) represent critical industrial resources, yet conventional extraction methods face substantial environmental and efficiency constraints. Fungal bioleaching emerges as an eco-friendly alternative, leveraging organic acid secretion to facilitate REEs dissolution and adsorption. However, progressive REEs accumulation inhibits microbial activity, with fungal resistance mechanisms remaining incompletely understood. Here, we report the discovery of Aspergillus niger FH1, a highly REEs-tolerant strain exhibiting remarkable Ce(III) tolerance (600 mg/L maximum) and achieving 74.05% adsorption efficiency under optimized conditions. Integrated physicochemical characterization (SEM, FTIR, XPS) revealed dual adsorption mechanisms: physical entrapment evidenced by Ce(III)-induced cellular invagination, and chemical monolayer binding via extracellular functional group coordination (amino, hydroxyl, carboxyl, carbonyl, phosphate), with specific moieties enabling Ce(III) capture through surface complexation. Transcriptomic analysis identified 3,733 differentially expressed genes under Ce(III) stress. Functional annotation (GO/KEGG) demonstrated: (1) Significant repression of oxidative phosphorylation genes; (2) Concomitant upregulation of glycolysis, pentose phosphate pathway, and amino acid metabolism genes indicating metabolic rerouting for energy maintenance; (3) Enhanced expression of antioxidative/chelating metabolite synthesis pathways. Whole-genome bisulfite sequencing revealed conserved global 5mC DNA methylation levels (0.32% vs. 0.36% in controls) with preferential CHH-context targeting. Collectively, these adaptation strategy combines extracellular sequestration, metabolic plasticity, and stress mitigation to confers exceptional resilience against rare earth metal toxicity. The demonstrated adsorption-tolerance synergy positions A. niger FH1 as an important bioagent for sustainable recovery of recalcitrant rare earth resources.

Background/PurposeSjögren's syndrome (SS) is a chronic systemic autoimmune disease characterized by lymphocytic infiltration and formation of lymphoepithelial lesions (LEL) in exocrine glands, leading to secretory dysfunction. DNA methylation, a dynamically regulated epigenetic mark, has been increasingly recognized as a key regulatory mechanism in the pathogenesis of autoimmune diseases including SS, and holds promise for identifying novel diagnostic and therapeutic strategies.MethodsReduced representation bisulfite sequencing (RRBS) was performed on 4 cases of SS and 3 controls to profile genome-wide DNA methylation patterns. Differentially methylated regions (DMRs) and associated differentially methylated genes (DMGs) were detected, followed by functional enrichment analysis. Integration with transcriptomic data (GSE40611) was performed to identify overlapping epigenetic and transcriptional changes.ResultsA total of 29,462 DMRs were detected, with 24,116 hypermethylated and 5,346 hypomethylated regions, indicating an overall increase in methylation levels of SS, and DMGs located in gene promoter regions were significantly enriched in pathways related to immune response, transcriptional regulation, and inflammation. Nine hub genes (LCP2, BTK, LAPTM5, ARHGAP9, IKZF1, WDFY4, CSF2RB, ARHGAP25, DOCK8) were identified, which displayed promoter hyper—or hypomethylation, indicating the complex epigenetic regulatory mechanisms.ConclusionThis study reveals extensive DNA methylation alterations in SS, providing new insights into the epigenetic mechanisms underlying pathogenesis. Moreover, these findings suggest potential biomarkers or therapeutic targets for further investigation to elucidate detailed molecular mechanisms of SS.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13072-025-00637-y.

Protein restriction reprograms the multi-organ proteomic landscape of mouse aging.

IntroductionHemophilia is a rare X-linked bleeding disorder leading to recurrent hemarthroses, hemophilic arthropathy, and impaired quality of life. A chronic lifelong disease, hemophilia might be associated with accelerated biological aging. Here, we investigated whether biological age derived from epigenetic age estimators differs in hemophilia.Patients/MethodsWe collected blood samples from men with severe (<1 IU/dL; PWSH,n = 20) or mild hemophilia (≥5 IU/dL; PWMH,n = 20), and age-matched healthy male controls (n = 20). DNA methylation of cytosine–phosphate–guanine (CpG) dinucleotides at five genes (ASPA,ITGA2B,PDE4C,FHL2,CCDC10SB) was measured by bisulfite pyrosequencing. Biological age was estimated using two epigenetic aging signatures, each including three CpGs. We investigated differences in biological age and the rate of biological aging between study groups using separate linear regressions on chronological age and study group without and with an interaction, respectively.ResultsDeviations of epigenetic from chronological age were high for both 3-CpG age estimators, with results suggesting systematic overprediction. In both linear regressions using the two 3-CpG estimates, respectively, evidence for a different rate of biological aging in severe hemophilia was weak. The rate of biological aging in PWSH was 0.24 (95% CI, 0.01–0.48) and 0.21 (0.04–0.37) higher compared with PWMH, and 0.05 (−0.19–0.29) and 0.17 (−0.00–0.34) higher compared with healthy controls, respectively. Hemophilic arthropathy was associated with an increased rate of biological aging.ConclusionEvidence for a difference in epigenetic aging as reflected by two 3-CpG estimators in severe compared with mild hemophilia or healthy controls was weak.

Disclosure: L. Zoff: None. F.M. Garcia: None. G. Veneruzzo: None. G. Aschettino: None. M.C. Mattone: None. E.B. Berensztein: None. C. Terada: None. N. Perez Garrido: None. R.M. Marino: None. C. Alonso: None. G. Guercio: None. A. Belgorosky: None. M.S. Baquedano: None.Being born SGA in 46, XY DSD patients without molecular diagnosis and with no specific disorders of undermasculinization have a higher prevalence than expected for the general population. Numerous links have been made between infant growth restriction and specific epigenetic alterations, including changes in DNA methylation, thereby implicating such regulation in appropriate growth and development. Moreover, several studies have suggested that SGA children present with permanent alterations in the epigenome due to the programming of fetal tissues. The Growth hormone (GH)/ insulin-like growth factor type 1 (IGF1) and Insulin axis, crucial in human growth and development, is involved in adrenogonadal differentiation in mice. We hypothesized that epigenetic changes in the GH/IGFs and Insulin axis, could mediate the association between being born SGA and with DSD in humans. Our aim was to evaluate both the levels and patterns of methylation across the regulatory regions of GHR, IGF1R, and INSR genes in foreskin (FT) from 46,XY DSD children born either AGA (AGADSD-FT, n=7; 0.8-7.37y) or SGA (SGADSD-FT, n=7; 1.3-7.3y) vs age-matched control children (C-FT; n=7, 0.9-7y) using targeted deep-amplicon bisulfite sequencing on a MiSeq system. Sequencing libraries of 4 promoter CpG-rich regions for GHR, 3 for IGF1R and 5 for INSR (104, 103 and 141 CpG sites, respectively) were analyzed for mean methylation values of all CpG sites using amplikyzer2 software. The average CpG methylation levels of the SP1-responsive IGF1R proximal promoter [GRCh38:15:98648387: 98648592:1 (-152 to +53)] and the 5´UTR regulatory region [GRCh38:15:98649214: 98649496:1 (+676 to +958)] were significantly higher in SGADSD-FT than in C-FT and AGADSD-FT (Kruskal Wallis test, p<0.05). In locus-by-locus comparative analyses, higher methylation levels of CpG-23, CpG-17, CpG-15, CpG-13, and CpG-11 within the unique Sp1-dependent initiator motif of the IGF1R gene were detected in SGADSD-FT compared with C-FT and AGADSD-FT, p<0.05. The mean methylation levels of INSR promoter regions [GRCh38:19:7294796:7295022:-1 (-608 to -381) and GRCh38:19:7294410:7294702:1 (-288 to +5)], covering the binding site of activating transcription factors, were higher in DSD 46, XY patients, both in SGADSD-FT and in AGADSD-FT than in C-FT (p<0.05). There were no significant differences in GHR promoter methylation among the three FT groups. Promoter DNA methylation is a fundamental epigenetic mark associated with transcriptional repression during development. According to our findings, it could be speculated that epigenetic repression, mainly of IGF1R, might contribute to mediate the association between being born SGA and 46XYDSD in humans. Nevertheless, whether or not the epigenetic changes observed in postnatal foreskin are present at the time of sexual differentiation during intrauterine life must be determined.Presentation: Saturday, July 12, 2025