Impact of cigarette‐smoking on sperm DNA methylation and its effect on sperm parameters

DNA methylation is an epigenetic modification of the genome. The purpose of this study was to determine the influence of cigarette‐smoking on sperm DNA methylation from a genomewide survey of sperm samples and to ascertain its effect on sperm parameters. Twenty‐eight sperm DNA samples (from 14 fertile smokers as a case study and 14 proven fertile nonsmokers as controls) were subjected to Infinium 450K BeadChip arrays to identify the changes in the DNA methylation level between the two groups. Then, deep bisulphite sequencing was used to validate five CpGs on 78 samples. The results from the Infinium 450K found that only 11 CpGs showed a significant difference in DNA methylation between the case and the control groups. Five CpGs of the eleven (cg00648582, cg0932376, cg19169023, cg23841288 and cg27391564) underwent deep bisulphite sequencing where cg00648582, related to the PGAM5 gene, and the cg23841288 CpGs, related to the PTPRN2 gene amplicons, showed a significant increase in their DNA methylation level in more than one CpG in the case group. In contrast, a significant decrease was found at cg19169023 and at its various neighbouring CpGs in the TYRO3 gene‐related amplicons. Furthermore, this study demonstrated a significant correlation between the variation in sperm DNA methylation level and standard sperm parameters in the case group.     

Association between alterations in DNA methylation level of spermatozoa at CpGs dinucleotide and male subfertility problems

The purpose of this study was to assess the relationship between alterations in sperm DNA methylation levels and sperm count and sperm motility. Five CpG sites underwent deep bisulphite sequencing to validate the observed methylation difference in 78 samples (28 proven fertile males “controls,” and 50 subfertile males “cases”). The results showed that variation in methylation levels was found in more than one CpG: the DNA methylation levels in CpG1, CpG2 and CpG3 of the PRRC2A gene‐related amplicon showed high significant differences in the case group compared to the control group (p ≤ .0001, p ≤ .003, and p ≤ .0001 respectively). Moreover, three CpGs of the four CpGs tested within the ANXA2 gene‐related amplicon (CpG1, CpG3 and CpG4) were significantly different (p ≤ .002, p ≤ .001, and p ≤ .0001, respectively) in the case group compared to the control group. In addition, a significant difference was found in seven CpGs of the twenty‐two CpGs tested within the MAPK8Ip3 gene‐related amplicon, besides six CpGs of the ten CpGs tested within the GAA gene‐related amplicon between case and control groups. In conclusion, this study identifies that CpGs have a significantly different in methylation levels of sperm DNA for subfertile males.     

Alterations in DNA methylation patterns and gene expression in spermatozoa of subfertile males

This study was designed to evaluate the DNA methylation patterns and gene expression in spermatozoa from subfertile males. Thirty samples were subjected to 450K arrays as a screening study to evaluate the variation in sperm DNA methylation levels between cases and controls groups, and then three CpG sites (cg07227024, cg05813498 and cg23081194) have the highest difference in methylation levels and located within ALS2CR12, GAA and UBE2G2 genes respectively; these were selected for further analysis using deep bisulphite sequencing and qPCR in 136 samples (64 proven fertile males “controls” and 72 subfertile males “cases”). A significant difference in the methylation level was found between cases and controls at two CpGs, six CpGs and three CpGs in ALS2CR12, GAA and UBE2G2 gene‐related amplicon respectively. Besides, the qPCR results showed a significant change in the expression levels of GAA, UBE2G2 and ALS2CR12 gene in cases compared to the controls (p ≤ .00001). In conclusion, the methylation levels at CpGs in GAA, UBE2G2 and ALS2CR12 gene amplicons were significantly different in subfertile compared to proven fertile males. In addition, a significantly different was showed in the expression levels of GAA, UBE2G2 and ALS2CR12 genes in subfertile males compared to proven fertile males.     

Aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity

The purpose of this study was to evaluate the aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity. A total of 108 males (65 males suffering from reduced fecundity as cases and 43 proven fertile males as a control) were included in the study. Thirty samples were subjected to 450K arrays as a screening phase, and then, three CpG sites located in the following genes: TYRO3, CGβ and FAM189A1 were selected to validate on 78 samples using deep bisulphite sequencing. A significant difference in the methylation level was found between cases and controls at all CpGs in TYRO3 gene‐related amplicon (CpG1, p ≤ .003, CpG2, p ≤ .0001, CpG3, p ≤ .003 and CpG4, p ≤ .030) and CpG1 in CGβ gene‐related amplicon (p ≤ .0001). Besides, a significant difference was found at two CpGs (CpG1, p ≤ .004 and CpG2, p ≤ .002) tested in the FAM189A1 gene‐related amplicon. A significant correlation was found between the methylation level at CpG1 in the FAM189A1 gene and the different types of sperm motility. In conclusion, an alteration in the methylation levels of sperm DNA from males with reduced fecundity was showed. In addition, a relationship between variations in the methylation level of these CpGs and sperm motility has been observed.     

Cigarette smoking induces only marginal changes in sperm DNA methylation levels of patients undergoing intracytoplasmic sperm injection treatment

DNA methylation plays important roles in genome stability and regulation of gene expression. This study was designed to determine the influence of cigarette smoking on sperm DNA methylation. From a genome‐wide survey on sperm samples, differentially methylated target CpGs should be selected and subjected to local deep bisulphite sequencing. Obtained methylation data are compared to sperm parameters and (ICSI) outcome. Similar to pilot study, samples were subjected to Infinium 450K BeadChip arrays to identify alterations in sperm DNA methylation between smokers and nonsmokers males. Routine testing on a significantly altered CpG site was performed on more samples using local deep bisulphite sequencing. Of approximately 485,000 CpG sites analysed, only seven CpGs were found to show a significant DNA methylation difference of >20% with the top six CpGs overlapping common SNP sites. The remaining CpG site (cg19455396) is located in intron 12 of the TAP2 gene. The results of deep bisulphite sequencing showed only a tendency towards hypomethylation in the smoking group. This study could not detect biologically relevant CpG positions that are altered in sperm DNA methylation on the influence of cigarette smoking beyond individual‐specific effects that may be caused by other environmental factors.     

Methylation levels of IGF2 and KCNQ1 in spermatozoa from infertile men are associated with sperm DNA damage

Abnormal imprinted genes methylation in spermatozoa has been shown to be associated with subfertility. However, the relationship between sperm DNA damage and specific imprinted genes methylation remains unclear. In this study, DNA methylation levels were determined at seven imprinted genes loci (H19, INS‐IGF2, KCNQ1, MEG3, MEST, PEG3 and SNRPN) in 66 semen samples using the MSRE‐qPCR method. The semen samples were divided into two groups according to the threshold value (25%) of DNA fragmentation index (DFI). We found that the mean methylation level at IGF2 (cg17037101) in the group with DFI ≥ 25% was lower than that in the group with DFI < 25% (13.7 ± 3% vs. 31.5 ± 5.3%, p = 0.0053). However, the methylation levels of other CpGs did not differ from the imprinted genes. Correlation analysis of DFI with the methylation levels of imprinted genes demonstrated that the IGF2 (cg17037101) methylation level was negatively correlated with sperm DFI (r = ?0.448, p = 0.0038), and the KCNQ1 (cg24932449) methylation level was positively correlated with sperm DFI (r = 0.354, p = 0.0273). Our results suggest that the aberrant methylation of IGF2 and KCNQ1 genes may be associated with sperm DNA damage.     

差异甲基化位点对肝细胞癌预后的相关性分析

目的 通过TCGA和GEO数据库关于肝细胞癌病例的数据挖掘,扫描全基因组范围内的肝细胞癌预后相关的甲基化位点。方法 本研究采用TCGA、GSE54503、GSE57956和GSE37988肝细胞癌病例的数据集分析共同的甲基化位点,比较肝细胞癌和癌旁组织甲基化水平差异,得出共同的差异甲基化位点。采用Cox比例风险模型分析各差异位点甲基化水平对肝细胞癌总生存率影响,评估其与对应基因mRNA 表达的相关性,进一步评估mRNA表达对肝细胞癌预后的影响。结果 肝细胞癌预后相关最显著的甲基化位点为位于基因TFCP2区域的cg20927059（P=0.003）,对应的HR值及95%CI为10.53（2.26～49.03）。筛选的与肝细胞癌预后有关联性的15个甲基化位点中,13个甲基化位点的甲基化水平与对应基因的mRNA表达相关,其中cg13984181对应的EPHX4基因和cg14541950对应的HIST3H2BB基因的mRNA表达与肝细胞癌的预后有关（HR：1.17,95%CI：1.06～1.29,P=0.002;HR：1.06,95%CI：1.00～1.13P=0.043）。结论?本研究首次发现EPHX4和HIST3H2BB基因甲基化位点的甲基化水平对肝细胞癌的预后有影响。     

DNA methylation levels of imprinted and nonimprinted genes DMRs associated with defective human spermatozoa

Asthenozoospermia (AS) is a common cause of human male infertility. Recent studies have shown significant associations of aberrant DNA methylation in spermatozoa with male infertility. The aims of the this investigation were to assess the changes in DNA methylation of known imprinted genes (MEST, GNAS and H19), novel imprinted gene (FAM50B) and nonimprinted genes (LINE‐1 and P16) DMRs in the spermatozoa of infertile men with single‐factor AS. Semen samples were obtained from 46 AS patients and 49 age‐matched normal controls. DNA methylation levels of detected genes DMR were determined by MassARRAY quantitative methylation analysis. The average methylation level at the P16 and MEST DMRs was significantly lower in AS patients than in controls (patients 6.51 ± 0.32%, controls 7.66 ± 0.40%, P < 0.01). The methylation level of 6 CpG sites of P16 DMR, and 1 CpG site of MEST, GNAS, FAM50B and LINE‐1 DMRs, was lower in AS group than in control group. For the first time, the data presented here suggest that increased methylation defects of P16 DMR may be associated with low sperm motility. This study provides the potential association between low sperm motility infertile men and the aberrant DNA methylation of MEST, LINE‐1, GNAS and FAM50B DMRs.     

Analysis of the methylation pattern of six gene promoters in sperm of men with abnormal protamination

It has recently been shown that alteration of the methylation pattern of imprinted genes is associated with different types of male infertility. The objective of our study was to investigate the methylation pattern of selected gene promoters in sperm of patients with abnormal protamine replacement. The promoters of OCT4, SOX2, NANOG, HOXC11, miR-17 and CREM were analyzed using bisulfite sequencing and the percentage of DNA methylation was compared between patients with an abnormal protamine 1/protamine 2 (P1/P2) ratio and normozoospermic controls. No significant quantitative differences were found between groups of patients with either an abnormally high or low P1/P2 ratio compared to normal controls. However, two individual samples from infertile subjects (2/20, 10%) showed an altered methylation pattern for the CREM gene promoter that was not found in control samples. These two samples had a significantly higher (P<0.05) promoter methylation (5.58 and 4.23%, respectively) compared to the control group (0.46%). In conclusion, in our pilot study, extreme methylations defects were not seen broadly in severely infertile men. However, two patients exhibited altered methylation of the CREM gene, which may be either causative or a result of abnormal protmaine replacement.     

父源印记基因H19印记控制区域DNA甲基化与少、弱精子症相关性分析

目的:研究印记基因H19印记控制区域的DNA甲基化程度与男性少、弱精子症的相关性。方法:通过染色体核型分析和Y染色体微缺失检测排除染色体因素干扰,进一步借助精液常规参数、伊红染色以及精子形态等指标,筛选出18例单一因素少精子症患者(浓度<15×106/ml,其余指标均正常)和20例单一因素弱精子症患者(前向运动精子百分率<32%,其余指标均正常)用于DNA甲基化检测;提取精子样本全基因组DNA,进行亚硫酸氢盐处理、PCR扩增目的基因片段并测序;通过BIQ Analyzer软件对测序结果进行质控和DNA甲基化程度分析。20例正常生育男性精液标本作为对照组。结果:与对照组甲基化丢失率[(0.30±0.06)%]相比,少精子症患者的H19印记控制区域的DNA甲基化丢失程度显著增高[(9.19±2.45)%],尤其当精子浓度<3×106/ml时,差异达到极显著水平(P<0.01)。在弱精子症患者中H19印记控制区域的DNA甲基化丢失程度[(0.30±0.07)%]和模式均与对照组无显著差异(P=0.62)。进一步重点分析了CTCF6位点的DNA甲基化状态,少精子症患者的DNA甲基化丢失程度[(2.67±0.75)%]显著高于对照组[(0.05±0.03)%]和弱精子症组[(0.03±0.02)%],而后两者之间无显著差异(P=0.35)。结论:H19印记控制区域的DNA甲基化程度的降低与少精子症密切相关,且降低程度与精子浓度呈显著负相关,而与精子活力无关。     

Sperm DNA methylation of H19 imprinted gene and male infertility

Infertility affects up to 15% of reproductive‐aged couples worldwide, with male factor being detected in 40%–50% of the cases. Proper sperm production is associated with the establishment of appropriate epigenetic marks in developing germ cells. Several studies have demonstrated the association between abnormal spermatogenesis and epigenetic disturbances with the major focus on DNA methylation. Imprinted genes are expressed in a parent‐of‐origin‐specific manner, and the role of their DNA methylation in proper spermatogenesis has been documented recently. The existing evidence along with the absence of relevant data in south of Iran prompted us to study the methylation of H19 imprinted gene in spermatozoa of idiopathic infertile patients (males with abnormalities in sperm parameters) and healthy controls by Combined Bisulfite Restriction Analysis. According to our results, the lowest methylation percentage of H19 imprinted gene belongs to three cases with sperm characteristics under normal range (two cases Oligoasthenoteratozoospermia and one case Oligoteratozoospermia). However, our results show that the median of methylation percentage for H19 is not statistically significant between case and control groups. Our results and those of others introduce DNA methylation as a potential marker of fertility and should be investigated with more patients in future studies.     

原发性肝癌组织RASSF1A异常甲基化与DNMTs及HBV的关系

目的探讨原发性肝癌ras相关结构域家族1A（ras association domain family 1A,RASSF1A）异常甲基化与肿瘤组织中DNA甲基转移酶（DNA methyltransferases,DNMTs）转录水平变化及HBV感染的关系.方法采用甲基化特异性PCR（methylation specific PCR,MSP）技术检测61例原发性肝癌组织RASSF1A基因异常甲基化情况,RT-PCR法检测其中三种主要DNA甲基转移酶（DNMT1、DNMT3A、DNMT3B）水平的变化.结果61例肝癌标本中RASSF1A基因启动子区异常甲基化45例（73.8%）;HBV感染者47例（77.1%）,其中MSP阳性32例;无HBV感染者14例,其中MSP阳性13例,RASSF1A基因异常甲基化与HBV感染之间的关系无统计学意义（x2=2.260,P=0.133）.肝癌组、肝癌细胞系组及HBV感染组DNMTs表达水平与正常对照组相比均显著升高;组内比较显示肝癌组中DNMT3A、DNMT3B在MSP阳性患者中较阴性患者升高（分别为t=3.494,P＜0.01;t=4.258,P＜0.01）,而DNMT1下降（t=3.221,P＜0.05）;HBV感染组中,MSP阳性组织其DNMT3B较阴性者升高（t=12.171,P＜0.01）.结论肝癌组织DNMTs水平变化与RASSF1A异常甲基化有密切关系;HBV感染与DNMT3B转录水平变化相关.     

Aberrant methylation of the TDMR of the GTF2AIL promoter does not affect fertilisation rates via TESE in patients with hypospermatogenesis

Increasing evidence shows a relationship between epigenetic regulation and male infertility. The GTF2A1L gene promoter contains the DNA methylation site of a tissue-specific differentially methylated region （TDMR）. Eighty-six patients with non-obstructive azoospermia were assessed for the DNA methylation state of CpG islands in the GTF2AIL promoter using testicular genomic DNA. Based on histological criteria, 26 of the 86 patients had normal spermatogenesis （controls）, 17 had hypospermatogenesis and 26 had a Sertoli cell-only phenotype or tubular sclerosis. GTF2AIL TDMR methylation was significantly lower in testes DNA from control samples than from hypospermatogenic samples （P=0.029）. Patients with hypospermatogenesis were divided into two subgroups： high DNA methylation （HM, n=5） and low DNA methylation （LM, n= 12）. The GTF2AIL TDMR methylation rate differed significantly between the HM and LM groups （P=0.0019）, and GTF2A 1L expression was significantly higher among the LM than in the HM patients （P=0.023）. High TDMR methylation was correlated with low GTF2AIL gene expression levels. Both groups demonstrated relatively good outcomes with respect to sperm retrieval, fertilisation, pregnancy and childbirth rates. We observed that aberrant GTF2AIL gene expression was not correlated with fertilisation rates. The testicular sperm extraction （TESE） technique may be used to overcome male infertility due to aberrant TDMR methvlation.     

Quality of sperm obtained by penile vibratory stimulation and percutaneous vasal sperm aspiration in men with spinal cord injury

The aim of this study was to explore sperm chromosomal aneuploidy and DNA integrity in infertile patients with spinal cord injury (SCI). Semensamples were collected from 12 infertile menwith SCI by percutaneous vasal sperm aspiration (PVSA) and from 14 male SCI patients by penile vibratory stimulation (PVS). These semen samples as well as samples from 16 donors were analyzed using the hypo-osmotic swelling (HOS) test, the sperm chromatin dispersion test, terminal deoxynucleotidyl transferase-mediated terminal uridine nick-end labeling assay, and multicolor fluorescence in situ hybridization with probes specific for the chromosomes 13, 18, 21, X, and Y. There were significant differences in the percentages of motile sperm, normal morphologic sperm, normal HOS/eosin staining, and sperm DNA fragmentation between the infertile men with SCI and the control group (P < .05 and P < .01). The sperm forward motility was significantly greater in the PVSA group than in the PVS group (P < .01). The number of round cells per milliliter of semen obtained from the 14 SCI patients by PVS was between 1 million and 12 million. The rate of sperm DNA fragmentation, as identified by the sperm chromatin dispersion test, was higher in the PVS group than in the PVSA group (P < .05). The aneuploidy rates for the SCI patients were 1.5- to 1.6-fold higher for chromosomes 13, 18, and 21, and were 2.3- to 2.4-fold higher for chromosomes X and Y than for patients in the control group (P < .001). These results suggest that for men with SCI, the semen quality is poorer, the prevalence of abnormal HOS/eosin staining is greater, and sperm DNA fragmentation and sperm chromosomal aneuploidies are seen at a higher rate compared with healthy, fertile, and normospermic men.     

Sperm chromatin quality and DNA integrity in partial versus total globozoospermia

Globozoospermia is a severe form of teratozoospermia with low incidence in infertile patients, considered as one of the important causes of male infertility. The objective was to investigate the chromatin/DNA integrity as well as apoptosis in ejaculated spermatozoa of cases with partial or total globozoospermia. Fifty‐seven semen samples were divided into three groups of partial globozoospermia (n = 17), total globozoospermia (n = 10) and normozoospermia (control; n = 30). Sperm chromatin condensation, DNA integrity and apoptosis were assessed using cytochemical assays. The results showed significant differences in sperm parameters of count and motility between two case groups versus controls. The percentages of spermatozoa with abnormal chromatin packaging and protamine deficiency were significantly higher in total and partial globozoospermic men compared to normozoospermic samples. Also, the rates of TUNEL‐positive spermatozoa were significantly increased in both globozoospermic cases with respect to the control (18.3 ± 10.1 and 12.3 ± 9.2 versus 5.9 ± 3 respectively). However, no significant differences were noticed between two subgroups of patients with regard to sperm DNA denaturation, DNA fragmentation and apoptosis. Abnormal chromatin packaging, DNA damage and apoptosis were significantly higher in cases than controls. The sperm chromatin/DNA anomalies may be considered as one of the main aetiology of ART failure in globozoospermic patients.     

Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions

The aim of this study was to examine the possible relationship between sperm DNA integrity and chromatin packaging evaluated by cytochemical assays, traditional sperm parameters and recurrent spontaneous abortion (RSA) of unknown origin. In this cohort study, 40 couples with a history of RSA and 40 couples with proven fertility were considered as case and control groups respectively. The semen samples of all husbands were analysed for sperm parameters and also sperm chromatin and DNA integrity assessed using cytochemical tests including aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), acridine orange (AOT) and nuclear chromatin stability assay. Among different sperm parameters, only slow motility was significantly different between the two groups. In sperm chromatin evaluations, there were significant differences between the two groups in all of the tests. In addition, the majority of semen samples in RSA patients exhibited upper percentages of abnormal spermatozoa than the cut-off values regarding different cytochemical assays. Our study showed that in the cases of RSA, slow motility had a significant reduction in comparison with controls and also spermatozoa of men from RSA group had less chromatin condensation and poorer DNA integrity than spermatozoa that obtained from fertile men with no history of RSA.     

Evaluation of zeta and HA-binding methods for selection of spermatozoa with normal morphology, protamine content and DNA integrity

Sperm selection parameters based on morphology and motility for ICSI might not be relevant to chromatin integrity. Thus sperm selection based on sperm characteristics has been suggested. Therefore, the aim of this study was to evaluate the efficiency of the zeta and hyaluronic acid (HA) sperm selection procedures with neat semen, for recovering spermatozoa with normal morphology, protamine content and DNA integrity in infertile men. Semen samples from 77 infertile couples were assessed during this study. Semen analysis was carried out according to World Health Organization criteria. Protamine content, DNA integrity and sperm morphology were assessed by chromomycin A3, sperm chromatin dispersion and Papanicolaou staining respectively. The results show that both HA and zeta methods were efficient to recover spermatozoa with normal morphology and protamine content. In terms of the latter parameters, there was no superiority between the two procedures. However, in terms of DNA integrity, the zeta method was more efficient compared with the control and HA procedure and no significant difference was observed between HA and the controls. Therefore, the zeta method appears to be a suitable procedure to recover spermatozoa with normal DNA integrity.     

DNA integrity and methylation changes of mouse spermatozoa following prolonged incubation

Sperm quality can be affected by different factors including the length of incubation time between sperm preparation and intracytoplasmic sperm injection. Here, we have evaluated the level of DNA methylation and expressions of related genes in mice spermatozoa. The spermatozoa were divided into three groups: fresh, spermatozoa incubated at room temperature (RT) and 37°C for 24 hr. The sperm chromatin structure assay was used to determine the DNA fragmentation index (DFI), and DNA methylation was analysed by flow cytometry. The expression levels of DNA methylation‐related genes were determined by quantitative real‐time PCR (qRT‐PCR). According to the results, we observed significantly higher sperm progressive motility and viability in the group incubated at RT compared to the spermatozoa incubated at 37°C (p < 0.05). Spermatozoa incubated at 37°C had a higher DFI compared to the other groups (p < 0.05), but the DNA methylation level significantly decreased (p < 0.05). qRT‐PCR analysis showed increased Dnmt‐1 expression in spermatozoa after 24‐hr incubation at 37°C. However, there were significantly higher expression levels of Dnmt‐3l, Dnmt‐3a and Dnmt‐3b after incubation at both RT and 37°C compared to the fresh group (p < 0.05). The 24‐hr incubation period affected both sperm DNA methylation and integrity. This study indicated that incubation at RT resulted in better sperm quality.     

A nomogram based on adipogenesis-related methylation sites in intraoperative visceral fat to predict EWL% at 1 year following laparoscopic sleeve gastrectomy

BackgroundLaparoscopic sleeve gastrectomy (LSG) is a crucial surgical procedure for patients with obesity. However, epigenetic research in LSG is still in its infancy from the perspective of adipogenesis.ObjectivesThis work aims to develop a model to predict 1 year excess weight loss percentage (EWL)% following LSG in Chinese patients with obesity by examining the DNA methylation profiles of intraoperative visceral fat.SettingUniversity hospital, Beijing, China.MethodsFirstly, we classified patients with obesity as either the satisfied group or unsatisfied group depending on whether their EWL% was 50% or higher at 1 year following LSG. After that, we analyzed differentially methylated sites (DMSs) between the satisfied group and unsatisfied group. DMSs were mapped to the corresponding differentially methylated genes. Then, we took the intersection of adipogenesis-related genes and differentially methylated genes and obtained adipogenesis-related DMSs. Next, hub methylation sites were identified by least absolute shrinkage and selection operator analysis. Finally, a nomogram was developed to predict EWL% of Chinese patients with obesity at 1 -year following LSG.ResultsA total of 26 patients with obesity were enrolled in the study, including 13 in the satisfied group and 13 in the unsatisfied group. A total of 16 genes and 31 DMSs were involved in the adipogenesis signaling pathway. Finally, 4 hub methylation sites (cg06093355, cg00294552, cg00753924, and cg17092065) were identified and a predictive nomogram was established.ConclusionsThe predictive nomogram based on methylation sites including cg06093355, cg00294552, cg00753924, and cg17092065 can predict EWL% at 1 year following LSG in Chinese patients with obesity efficiently.