Comprehensive DNA methylation profiling of sperm in male partners of couples with unexplained recurrent pregnancy loss

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The male factors were associated with RPL in male partners, including chromosome abnormality and sperm DNA fragmentation. DNA methylation is one of the most extensively studied epigenetic factors that could help elucidate the mechanism underlying RPL in male partners. Results: We revealed DNA methylation alternations occurring in sperm of RPL patients compared with the controls by genome-wide DNA methylation beadchip, including a series of differentially methylated CpG positions and genes. Importantly, we validated that the CpG site cg17985533 and the region chr11:1997780-1997899 from the H19 imprinted maternally expressed transcript were significantly hypermethylated in sperm of RPL-related men with > 10% mean methylation difference by targeted bisulfite sequencing. Moreover, the receiver operating characteristic analysis showed that CpG site cg17985533 and region chr11:1997780-1997899 could distinguish RPL patients from controls, with an area under the curve of 0.7838 and 0.8125, sensitivity of 80% and 80%, and specificity of 80% and 75%, respectively. These results indicated that they could be potential biomarker for diagnosis of RPL in male partners. Conclusions: This study highlighted the importance of H19 gene methylation in differentiating RPL and control, and provided new insight for revealing potential epigenetic mechanisms for RPL in male partners. Recurrent pregnancy loss Sperm DNA methylation H19 Biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Recurrent pregnancy loss (RPL), defined as two or more spontaneous abortions before 20 weeks of gestation, affects fertility problems in approximately 5% of couples [ 1 ], and the cause of RPL remains unknown in up to 50% of RPL cases [ 2 , 3 ], which is called unexplained recurrent pregnancy loss (URPL) [ 4 ]. Initially, research on factors influencing RPL primarily focused on women, including endocrine abnormalities, thyroid disease, hyperprolactinemia, uncontrolled diabetes, uterine abnormality and some environmental factors [ 5 , 6 ]. However, further investigations have revealed some factors in men also play significant roles in the occurrence of RPL [ 7 – 9 ]. Currently, there is an increasing necessity on exploring the factors and potential mechanisms affecting RPL in male partners of couples experiencing it. The male factors that contribute to RPL mainly include chromosome abnormality (like aneuploidy, Y chromosome microdeletion, chromatin integrity) [ 10 – 12 ], sperm DNA fragmentation [ 7 ], virus infection [ 13 , 14 ], and other related diseases. Interestingly, studies have shown that epigenetic mechanisms were also associated with RPL in male partners, mainly focused on DNA methylation [ 15 – 17 ] and RNA methylation [ 18 ]. However, these studies about DNA methylation have some limitations for further uncovering the mechanisms of RPL in male partners. For example, Irani et al. just determined global methylation level in sperm of male partners of women experiencing idiopathic RPL, which did not find the methylation alterations of specific genes [ 16 ]. Some studies only explored the methylation effect of single gene or several genes on male-related RPL, which did not systematically reveal the genome-wide methylation signatures [ 15 , 17 ]. Therefore, we intended to comprehensively reveal the dysregulated methylation in sperm at the genome-wide level to identify possible causes of RPL in male partners. Although some studies analyzed the genome-wide alterations in sperm DNA methylation in male partners of idiopathic RPL, the corresponding validation about methylation levels were still absent [ 19 ]. In this study, we firstly profiled the differential methylation signatures of sperm from male patients with couples experiencing RPL (hereafter called RPL group or RPL patients) and healthy controls by genome-wide DNA methylation beadchip, some differentially methylated positions (DMPs) were then screened for further validation by targeted bisulfite sequencing. Importantly, multiple CpG sites showed significant hypermethylation in RPL group, especially in H19 imprinted maternally expressed transcript (H19), and the DMP cg17985533 and the region chr11:1997780–1997899 from H19 had consistently high methylation level in RPL, which might become a diagnosis biomarker for RPL in male partners. Methods Study subjects The 25 male patients with couples experiencing RPL and 25 healthy controls were recruited from Jinan Maternity and Child Care Hospital Affiliated to Shandong First Medical University from October 2021 to April 2024. The age of RPL group was 26-35y, the control group was 22-40y, both average age (p = 0.4378) and BMI (p = 0.0557) had no significant differences (t-test) between RPL group and the control group (Table S1 ). The RPL group needed to meet the following inclusion criteria, before 20 weeks of gestation, those female partners of male patients who had two or more consecutive pregnancy loss, and chromosomal, anatomical, endocrine, infection, immune abnormalities and other causes were excluded in these couples. In addition, all men had no other diseases or received related treatment. The healthy controls were confirmed to be fertile, and the inclusion criteria included the following information, a healthy child was confirmed to have been born, and the man had normal chromosomes, and there was no history of spontaneous abortion, ectopic pregnancy, premature delivery and stillbirth. In addition, both men and women are no more than the age of 40. All subjects signed informed consent forms for the following semen collection. The study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Ethics Review Committee of Jinan Maternity and Child Care Hospital Affiliated to Shandong First Medical University. Sample collection, DNA extraction and bisulfite conversion Sample collection, DNA extraction and bisulfite conversion Semen samples were collected from RPL patients and healthy controls after 2–7 days of abstinence, and semen analysis was performed according to WHO guidelines as previously described, including sperm volume, concentration, total motility, forward motility and morphology (Table S1 ) [ 20 ]. These clinical parameters had no significant differences (t-test) between RPL group and the control group, the p-values of sperm volume, concentration, total motility, forward motility and morphology were 0.1681, 0.3941, 0.7623, 0.8891, and 0.4493, respectively. The semen samples were washed and removed seminal plasma, somatic cells were further removed by somatic cell lysis buffer (0.1% sodium dodecyl sulphate, and 0.5% Triton X-100 in diethyl pyrocarbonate water) treatment as described previously [ 21 ]. The sperm samples were then washed twice with PBS and stored at − 80°C until genomic DNA extraction. Genomic DNA from sperm was isolated by using HiPurA Sperm Genomic DNA Purification Kit (HiMedia, India) as the manufacturer’s instructions, DNA purity and concentration were determined by Qubit3.0 fluorometer (Thermo Fisher Scientific, USA). For bisulfite conversion, 1 µg of sperm DNA was processed with the EZ DNA Methylation-Gold Kit (Zymo Research, USA) according to the manufacturer’s instructions. The bisulfite-converted DNA was then used for the detection of DNA methylation levels by microarray and sequencing. Infinium MethylationEPIC BeadChip analysis The converted DNA from 5 RPL patients and 5 healthy controls were subjected to the Infinium MethylationEPIC BeadChip v1.0 (Illumina, USA) analysis which contains > 850,000 CpG sites according to the manufacturer’s instructions, and raw IDAT files were obtained using the iScan SQ fluorescent scanner (Illumina, USA). The methylation data were analyzed by the ChAMP package (v 2.18.2) in R 4.3.3. The methylation level for each CpG was scored as a β-value [β = intensity of the methylated allele (M) / (intensity of the unmethylated allele (U) + intensity of the methylated allele (M) + 100)] according to the fluorescent intensity ratio, and it represented a continuous variable that ranged from 0 (no methylation) to 1 (full methylation). First, we filtered out the probes with detection p-value > 0.01, non-CpG probes, probes located on chromosome X/Y, and SNP-related probes via ChAMP [ 22 ]. Then, the β-values were normalized using BMIQ, and the singular value decomposition (SVD) analysis was used to evaluate the batch effect of normalized β-values [ 23 ]. Finally, a total of 742,000 CpG sites were used for differential analysis, and CpGs having |Δβ| ≥ 0.1 and p-value ≤ 0.05 were considered as DMPs between RPL patients and controls. Targeted bisulfite sequencing The methylation levels of candidate target regions were detected and analyzed by targeted bisulfite sequencing (Genesky Biotechnologies Inc., Shanghai, China), which was called MethylTarget, a multiplex-targeted CpG methylation analysis technology based on next-generation sequencing [ 24 , 25 ]. Bisulfite conversion of genomic DNA was subjected to sodium bisulfite treatment using the EZ DNA methylation kit (Zymo Research, USA) according to the manufacturer’s protocol. Multiplex PCR of 12 regions was performed using an optimized primer combination by Genesky, after PCR amplification and library construction, samples were sequenced on an Illumina HiSeq platform by pair-end 150 bp (Illumina, USA). The sequenced reads were mapped to the human reference genome GRCh38/hg38. The methylation level of each CpG site was calculated as the percentage of methylated cytosines in total cytosines, and DMP was considered by the difference of average methylation level between RPL patients and controls with p-value ≤ 0.05 (t-test). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis The differentially methylated genes (DMGs) from the Infinium MethylationEPIC BeadChip results were used for KEGG pathway enrichment analysis, and KEGG pathways with p-value < 0.05 were considered as significantly enriched terms. The KEGG pathway analysis was carried out by clusterProfiler package in R 4.3.3. Statistical analysis The methylation levels of candidate DMPs were subjected to multiple logistic regression to obtain a predicted probability score for each sample. The probability score was then subjected to the receiver operating characteristic (ROC) analysis to obtain a ROC curve, and the area under the curve (AUC) was calculated to assess the discrimination of candidate CpGs using “pROC” R package. All the statistical analyses were conducted in R 4.3.3, and p-value < 0.05 was considered significant. Results Genome-wide DNA methylation profiling identified the differential methylation signatures in sperm between RPL patients and controls The detailed clinical characteristics of the RPL patients (n = 25) and healthy controls (n = 25) were shown in Table S1 . To reveal DNA methylation alternations occurring in sperm of RPL patients, we performed a genome-wide DNA methylation profiling assay in 5 RPL patients and 5 healthy controls by Infinium MethylationEPIC BeadChip. After quality control and normalization, about 742,000 methylation positions were subject to subsequent differential analysis. These CpG positions showed obvious enrichment in high (β-value close to 1) and low methylation levels (β-value close to 0) in case and control group (Fig. 1 A). The PCA plot exhibited relatively little differences between RPL patients and normal control (Fig. 1 B). The CpG positions with |Δβ| ≥ 0.1 and p-value ≤ 0.05 were defined as the differentially methylated CpG positions (DMPs), a total of 960 DMPs were identified with 847 hypermethylated (88.2%) and 113 hypomethylated (11.8%) positions (Fig. 1 C, Table S2 ). The Manhattan plot exhibited the chromosomal positions of all methylation sites, including the above DMPs (Fig. 1 D), and the hypermethylated and hypomethylated positions showed significantly different distribution features between RPL and control, including in the island, N-Shelf/Shore, S-Shelf/Shore and the opensea regions (Figure S1 ). The genomic distribution of DMPs showed that more hyper-DMPs and hypo-DMPs were located in the intergenic region and gene body region, respectively (Figure S2 ). Next, we focused on the genes with the most DMPs, top genes included H19, TBC1D16, C7orf50, DLG2, JSRP1, and PFKP (Fig. 2 A). For example, the H19 imprinted maternally expressed transcript (H19) had 11 hyper-DMPs between RPL and control, including cg18362496, cg11735853, cg27300742, cg24605090, cg01539474, cg15886040, cg15922305, cg17985533, cg04975775, cg01977486, and cg11753499 (Fig. 2 B). The differentially methylated genes (DMGs) were then subjected to pathway analysis, the KEGG results displayed that drug metabolism-cytochrome P450, serotonergic synapse, arachidonic acid metabolism, retinol metabolism, and linoleic acid metabolism were significantly enriched (Fig. 2 C). Among them, cytochrome P450-related genes were reported to be associated with RPL or spermatogenesis [ 26 – 28 ]. Screening the differentially methylated genes related to RPL Studies found that a significant association between H19 gene methylation and male hypospermatogenesis [ 29 ] or male infertility [ 30 , 31 ]. Additionally, evidences showed that some DMGs found by our genome-wide screening were related spermatogenesis and male infertility, including cilia and flagella associated protein 61 (CFAP61) [ 32 , 33 ], major histocompatibility complex, class II, DQ beta 1 (HLA-DQB1) [ 34 ] and lysine demethylase 4D (KDM4D) [ 35 ]. However, most of these studies focused on the roles of genetic variants of the above genes. The epigenetic effects of them, especially DNA methylation, was not clear in RPL. Therefore, we firstly selected the DMPs in spermatogenesis-related gene H19, CFAP61, HLA-DQB1 and KDM4D (Fig. 3 ) for following methylation validation, among them, 11 hyper-DMPs were found in H19 (Fig. 2 B), 3 hyper-DMPs in CFAP61 (Fig. 3 A), both HLA-DQB1 and KDM4D had 2 hyper-DMPs (Fig. 3 B, C). Validation of DMPs by targeted bisulfite sequencing between RPL patients and controls In order to validate the candidate DMPs of the above 4 genes involved in RPL, we screened a total of 12 target regions (fragments) to compare the methylation differences of 89 CpGs by the targeted bisulfite sequencing method. The differential methylation analysis revealed that 3 CpG sites in H19 and 2 CpG sites in KDM4D had statistically significant differences (p-value ≤ 0.05) between 20 RPL patients and 20 healthy controls (Fig. 4 A, B), while only 3 CpG sites in H19 (chr11:1997806, cg13210239, and cg17985533) had relatively higher methylation levels with > 10% mean methylation difference in sperm of RPL patients compared with the controls (Fig. 4 A). Importantly, these 3 CpG sites were not in the imprinting control region (ICR) of H19 gene, which was previously reported to be subject to differential methylation [ 36 ]. Among them, the DMP cg17985533 was consistently hypermethylated in RPL group by both microarray and targeted sequencing methods (Fig. 4 A). We examined the performance of cg17985533, which exhibited AUC (0.7838), sensitivity (80%), and specificity (80%) by ROC analysis (Fig. 4 C, Table 1). It suggested that high methylation level of cg17985533 might be a potential biomarker for diagnosis of RPL. On the level of genomic fragment, only the region (chr11:1997780–1997899) in H19 was identified significantly hypermethylated with > 10% mean methylation difference (p = 0.032) in sperm of RPL patients compared with the controls (Fig. 5 A), this region included the above 3 differential CpG sites in H19, chr11:1997806, cg13210239, and cg17985533. All 6 CpG sites in region chr11:1997780–1997899 showed higher methylation levels in RPL group compared with the control group (Fig. 5 B). Interestingly, the abundance analysis of methylation haplotypes in this region showed that the CCCCCC and CCCTCC haplotypes (C represented methylated cytosine, T represented unmethylated cytosine) were higher in RPL group, and CTTTTT haplotype was higher in control group (Fig. 5 C). We also examined the performance of this region, which exhibited AUC (0.8125), sensitivity (80%), and specificity (75%) by ROC analysis (Fig. 5 D, Table 1). It also validated that this region was significantly hypermethylated in sperm of RPL patients. Therefore, the mean methylation level of this region in H19 could also be a diagnostic biomarker for RPL. Discussion The proper functioning of germ cells is crucial for both fertility and embryonic development. As we all know, the impact of DNA methylation on spermatogenesis and male infertility has got a lot of attention. Study has definitely confirmed that DNA methyltransferase 3A-dependent DNA methylation was required for spermatogonial stem cells to commit to spermatogenesis [ 37 ]. Moreover, genome-wide DNA methylation was dynamically changed during human spermatogenesis and germ cells exhibited considerable DNA methylation changes in disturbed spermatogenesis [ 38 ]. Therefore, a lot of evidences also characterized the links between DNA methylation and male infertility. Han et al. found that methylated inactivation of SOX30 uniquely impaired spermatogenesis, and further caused non-obstructive azoospermia disease which is the most severe form of male infertility [ 39 ]. Interestingly, DNA methylation could interplay with histone H3 lysine 4 tri-methylation (H3K4me3) throughout the genome of human sperm, and it might be related with fertility and development [ 40 ]. Currently, male factors are known to affect pregnancy loss, abortion, and infertility. Various factors such as smoking, obesity, advanced age, and other environmental factors could impact sperm quality and male infertility [ 41 ]. DNA methylation is one of the most extensively studied epigenetic factor that could help elucidate the mechanism underlying URPL. Therefore, this study aimed to reveal the relationships between DNA methylation modifications of sperm and URPL in male partners. Four genes (H19, CFAP61, HLA-DQB1, and KDM4D) were screened as differential methylation candidates between URPL and control groups in our study. These genes were reported to be associated with male infertility or RPL in male partners. Of these genes, KDM4D is a histone demethylase that has been shown to be involved in defects in elongated sperm production and changes in H3K9me3 distribution in round sperm upon deletion [ 35 ]. However, there was a report which found that KDM4D regulated methylation of histone H3 lysine 9 (H3K9) during spermatogenesis in the mouse but was dispensable for fertility [ 42 ]. Therefore, it was necessary to examine the genome methylation level of KDM4D to evaluate its potential effect on gene expression. The flagellum is an essential structure for sperm morphology and function, with CFAP61 locating centrally within the sperm and playing a role in flagellum formation in human and mouse [ 32 , 43 ]. Moreover, studies have demonstrated that biallelic variants in CFAP61 could cause multiple morphological abnormalities of the flagella and male infertility in human and mouse [ 33 , 44 ]. Further, the knockdown of CFAP61 aggravated male infertility by inhibiting testosterone secretion by Leydig cells via the MAPK/COX-2 pathway [ 45 ]. However, there were not relevant reports about the relation between KDM4D or CFAP61 and RPL. Interestingly, two studies from India found that the HLA-DQB1*02:01:01 and DQB1*03:03:02 alleles were associated with an increase in risk of RPL, while DQB1*02:02:01 and DQB1*06:03 alleles appeared to be protective against RPL [ 46 , 47 ]. Another report about Lebanese women identified that DPB1-DQB1-DRB1 loci were linked with altered RPL susceptibility by case-control study [ 48 ]. A Danish study also validated that the frequencies of RPL women carrying three haplotypes with DQB1*0501 or one haplotype with DQB1*0201 were significantly increased compared with controls [ 49 ]. These data suggested the polymorphism of HLA-DQB1 was associated with the risk of RPL, however, the methylation level of HLA-DQB1 has not been reported to be related with RPL. Importantly, we observed significant hypermethylation of 11 CpG sites in H19 gene in RPL patients compared to the control. H19 is an imprinted gene that can undergo both methylation and demethylation processes. During spermatogenesis, DNA methylation patterns of the H19 gene are epigenetically inherited by somatic cells in embryos, particularly in relation to imprinted genes. The methylation status of the H19 gene has been extensively studied in male infertility, however, Cannarella et al. found that H19 methylation levels were significantly lower in the group of infertile patients than in fertile controls by meta-analysis [ 50 ]. In addition, this study suggested that the hypomethylation of H19 was also associated with patients with oligozoospermia and RPL [ 50 ]. Similar study also revealed the overall methylation rate of H19 DMR was significantly decreased in male infertile patients [ 51 ] or in oligospermic patients [ 52 ]. However, Nasri et al. showed that the median of methylation percentage for H19 was not statistically significant between male infertility group and control group [ 53 ]. Therefore, the methylation status of H19 at specific sites needs to be further validated in male infertile patients or RPL patients. Moreover, study also validated that the hypomethylation at specific CpG sites of insulin like growth factor-2 (IGF2)-H19 was observed in sperm DNA of male patients with couples experiencing RPL [ 15 ]. Aberrant methylation patterns of the H19/IGF2 genes have been associated with increased incidence of sperm DNA fragmentation (SDF), while decreased levels of H19 gene methylation have been linked to higher rates of recurrent miscarriage [ 54 ]. In fact, research has demonstrated a correlation between impaired H19/IGF2 methylation and elevated levels of reactive oxygen species (ROS), which can induce DNA fragmentation and thus play a significant role in increasing SDF rates [ 55 ]. Elevated SDF levels are also considered one of the major causes for RPL [ 56 ]. Khambata et al. validated that a combination of five imprinted genes comprising IGF2-H19 DMR, IG-DMR, ZAC, KvDMR, and PEG3 could be used as a diagnostic tool for spermatozoa samples of RPL patients [ 57 ]. In contrast, our study reveals that cg17985533, a highly methylated CpG site and a region chr11:1997780–1997899 derived from H19, holds promise as a diagnostic biomarker for RPL in male partners. Of course, our research also has some shortcomings. Firstly, we screened and validated the methylation-related genes and loci in a small number of sperm samples, and we will need more samples to confirm our current findings in the future. Secondly, we only verified the methylation levels of a few genes related to spermatogenesis or male infertility, and subsequently we need to further verify the methylation levels of more other genes between RPL and controls to discover new methylation biomarkers. Conclusions We have provided compelling evidence indicating altered methylation levels of H19, CFAP61, HLA-DQB1, and KDM4D genes in RPL patients compared with controls among male partners, notably highlighting that hypermethylation levels of CpG site cg17985533 and region chr11:1997780–1997899 derived from H19 represented a potential biomarker for RPL. Abbreviations RPL recurrent pregnancy loss URPL unexplained recurrent pregnancy loss DMPs differentially methylated positions H19 H19 imprinted maternally expressed transcript SVD singular value decomposition DMGs differentially methylated genes ROC receiver operating characteristic AUC area under the curve CFAP61 flagella associated protein 61 HLA-DQB1 major histocompatibility complex, class II, DQ beta 1 KDM4D lysine demethylase 4D ICR imprinting control region H3K9 histone H3 lysine 9 SDF sperm DNA fragmentation ROS reactive oxygen species. Declarations Ethics approval and consent to participate The study was approved by the Ethics Review Committee of Jinan Maternity and Child Care Hospital Affiliated to Shandong First Medical University. Consent for publication Not applicable. Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the High-level Talents in Medical and Health Industry of Jinan City, the Shandong Provincial Natural Science Foundation (No.ZR2020QH276) and the Jinan City Clinical Medicine Technology Innovation Program (No.202019173). Authors' contributions LW was a major contributor in writing the manuscript. LW and FL collected all sperm samples and recorded the individual information. LW, QL and RW analyzed and interpreted the DNA methylation data. 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Malcher A, Kamieniczna M, Rozwadowska N, Stokowy T, Berger A, Jedrzejczak P, Wolski JK, Kurpisz M: HLA-DQB1 as a potential prognostic biomarker of hormonal therapy in patients with non-obstructive azoospermia . Reprod Biol 2024, 24 (4):100949. Xu Z, Fujimoto Y, Sakamoto M, Ito D, Ikawa M, Ishiuchi T: Kdm4d mutant mice show impaired sperm motility and subfertility . J Reprod Dev 2024, 70 (5):320-326. Drewell RA, Goddard CJ, Thomas JO, Surani MA: Methylation-dependent silencing at the H19 imprinting control region by MeCP2 . Nucleic Acids Res 2002, 30 (5):1139-1144. Dura M, Teissandier A, Armand M, Barau J, Lapoujade C, Fouchet P, Bonneville L, Schulz M, Weber M, Baudrin LG et al : DNMT3A-dependent DNA methylation is required for spermatogonial stem cells to commit to spermatogenesis . Nat Genet 2022, 54 (4):469-480. Siebert-Kuss LM, Dietrich V, Di Persio S, Bhaskaran J, Stehling M, Cremers JF, Sandmann S, Varghese J, Kliesch S, Schlatt S et al : Genome-wide DNA methylation changes in human spermatogenesis . Am J Hum Genet 2024, 111 (6):1125-1139. Han F, Jiang X, Li ZM, Zhuang X, Zhang X, Ouyang WM, Liu WB, Mao CY, Chen Q, Huang CS et al : Epigenetic Inactivation of SOX30 Is Associated with Male Infertility and Offers a Therapy Target for Non-obstructive Azoospermia . Mol Ther Nucleic Acids 2020, 19 :72-83. Lambrot R, Chan D, Shao X, Aarabi M, Kwan T, Bourque G, Moskovtsev S, Librach C, Trasler J, Dumeaux V et al : Whole-genome sequencing of H3K4me3 and DNA methylation in human sperm reveals regions of overlap linked to fertility and development . Cell Rep 2021, 36 (3):109418. Eisenberg ML, Esteves SC, Lamb DJ, Hotaling JM, Giwercman A, Hwang K, Cheng YS: Male infertility . Nat Rev Dis Primers 2023, 9 (1):49. Iwamori N, Zhao M, Meistrich ML, Matzuk MM: The testis-enriched histone demethylase, KDM4D, regulates methylation of histone H3 lysine 9 during spermatogenesis in the mouse but is dispensable for fertility . Biol Reprod 2011, 84 (6):1225-1234. Huang T, Yin Y, Liu C, Li M, Yu X, Wang X, Zhang H, Muhammad T, Gao F, Li W et al : Absence of murine CFAP61 causes male infertility due to multiple morphological abnormalities of the flagella . Sci Bull (Beijing) 2020, 65 (10):854-864. Ma A, Zeb A, Ali I, Zhao D, Khan A, Zhang B, Zhou J, Khan R, Zhang H, Zhang Y et al : Biallelic Variants in CFAP61 Cause Multiple Morphological Abnormalities of the Flagella and Male Infertility . Front Cell Dev Biol 2021, 9 :803818. Zhu W, Mao J, Qin J, Chen X: CFAP61 knockdown aggravates male infertility by inhibiting testosterone secretion by Leydig cells via the MAPK/COX-2 pathway . Funct Integr Genomics 2023, 23 (4):340. Shetty S, Santhosh A, SP SP, Gunasheela D, Nayak R, Shetty S: HLA allele frequency of HLA-A, -B, -C, -DRB1 and -DQB1 in Indian recurrent implantation failure and recurrent pregnancy loss couples - A retrospective study . J Reprod Immunol 2024, 163 :104225. Aruna M, Nagaraja T, Andal Bhaskar S, Tarakeswari S, Reddy AG, Thangaraj K, Singh L, Reddy BM: Novel alleles of HLA-DQ and -DR loci show association with recurrent miscarriages among South Indian women . Hum Reprod 2011, 26 (4):765-774. Aimagambetova G, Hajjej A, Malalla ZH, Finan RR, Sarray S, Almawi WY: Maternal HLA-DR, HLA-DQ, and HLA-DP loci are linked with altered risk of recurrent pregnancy loss in Lebanese women: A case-control study . Am J Reprod Immunol 2019, 82 (4):e13173. Christiansen OB, Rasmussen KL, Jersild C, Grunnet N: HLA class II alleles confer susceptibility to recurrent fetal losses in Danish women . Tissue Antigens 1994, 44 (4):225-233. Cannarella R, Crafa A, Barbagallo F, Lundy SD, La Vignera S, Condorelli RA, Calogero AE: H19 Sperm Methylation in Male Infertility: A Systematic Review and Meta-Analysis . Int J Mol Sci 2023, 24 (8). Li XP, Hao CL, Wang Q, Yi XM, Jiang ZS: H19 gene methylation status is associated with male infertility . Exp Ther Med 2016, 12 (1):451-456. He W, Sun U, Zhang S, Feng X, Xu M, Dai J, Ni X, Wang X, Wu Q: Profiling the DNA methylation patterns of imprinted genes in abnormal semen samples by next-generation bisulfite sequencing . J Assist Reprod Genet 2020, 37 (9):2211-2221. Nasri F, Gharesi-Fard B, Namavar Jahromi B, Farazi-Fard MA, Banaei M, Davari M, Ebrahimi S, Anvar Z: Sperm DNA methylation of H19 imprinted gene and male infertility . Andrologia 2017, 49 (10). Cannarella R, Crafa A, Condorelli RA, Mongioi LM, La Vignera S, Calogero AE: Relevance of sperm imprinted gene methylation on assisted reproductive technique outcomes and pregnancy loss: a systematic review . Syst Biol Reprod Med 2021, 67 (4):251-259. Darbandi M, Darbandi S, Agarwal A, Baskaran S, Dutta S, Sengupta P, Khorram Khorshid HR, Esteves S, Gilany K, Hedayati M et al : Reactive oxygen species-induced alterations in H19-Igf2 methylation patterns, seminal plasma metabolites, and semen quality . J Assist Reprod Genet 2019, 36 (2):241-253. McQueen DB, Zhang J, Robins JC: Sperm DNA fragmentation and recurrent pregnancy loss: a systematic review and meta-analysis . Fertil Steril 2019, 112 (1):54-60 e53. Khambata K, Begum S, Raut S, Mohan S, Irani D, Singh D, Bansal V, Patil A, Balasinor NH: DNA methylation biomarkers to identify epigenetically abnormal spermatozoa in male partners from couples experiencing recurrent pregnancy loss . Epigenetics 2023, 18 (1):2252244. Table Table 1 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx SupplementaryFigureLegends.docx FigureS1.tif FigureS2.tif SupplementaryTables.xlsx Cite Share Download PDF Status: Published Journal Publication published 31 Dec, 2025 Read the published version in Clinical Epigenetics → Version 1 posted Editorial decision: Revision requested 11 Sep, 2025 Reviews received at journal 25 Jul, 2025 Reviews received at journal 16 Jul, 2025 Reviewers agreed at journal 25 Jun, 2025 Reviewers agreed at journal 25 Jun, 2025 Reviewers agreed at journal 25 Jun, 2025 Reviewers invited by journal 25 Jun, 2025 Editor assigned by journal 10 Jun, 2025 Submission checks completed at journal 10 Jun, 2025 First submitted to journal 06 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6835526","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476590402,"identity":"fda7d952-41df-4187-9b72-5c72708ba731","order_by":0,"name":"Linping Wei","email":"","orcid":"","institution":"Jinan Maternity and Child Care Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Linping","middleName":"","lastName":"Wei","suffix":""},{"id":476590403,"identity":"05c99f26-c956-4537-bed2-5f7537996cfb","order_by":1,"name":"Fang Luan","email":"","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Luan","suffix":""},{"id":476590404,"identity":"30613644-13b2-4393-b093-e0320de03c35","order_by":2,"name":"Qining Liu","email":"","orcid":"","institution":"Jinan Maternity and Child Care Hospital, Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qining","middleName":"","lastName":"Liu","suffix":""},{"id":476590405,"identity":"65c11561-2db6-4d27-b0c6-ddfa8b422361","order_by":3,"name":"Rui Wang","email":"","orcid":"","institution":"Jinan Maternity and Child Care Hospital, Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Wang","suffix":""},{"id":476590406,"identity":"63581c88-1187-4f0e-bf91-9f7c4016daf0","order_by":4,"name":"Yang Fu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBAC9gYwJcHDxt584MCHH0Ro4TkApmxk+HiOJR6c2UO8ljQbOYkc48McbMRokcgx/Fzw6zAPG8+ZD4cZeBjk+cUOENRiLD2zD6iFvXfD4QILBsOZsxPwa7GXyDGQ5u0B2XJ2w+EZPAwJBrcJaAHZ8husRSLnAZAkTouZNM+PNJAWBiK18Dwrs+ZtsAE67JgBMJAlCPuFhz15822ePxL28u3Njz98+GEjzy9NQAsDA4cBA2MbnCdBSDkIsD9gYPhDjMJRMApGwSgYsQAAiBNB/LopvaQAAAAASUVORK5CYII=","orcid":"","institution":"Jinan Maternity and Child Care Hospital, Shandong First Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yang","middleName":"","lastName":"Fu","suffix":""}],"badges":[],"createdAt":"2025-06-06 09:08:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6835526/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6835526/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13148-025-02043-3","type":"published","date":"2025-12-31T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85819736,"identity":"3dc66408-43bc-4c8c-8b5a-d8ad0ebe243c","added_by":"auto","created_at":"2025-07-02 06:22:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14531148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe genome-wide DNA methylation analysis of sperm from 5 RPL patients and 5 healthy controls by Infinium MethylationEPIC BeadChip.\u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) The distribution of 742,742 methylation probes with different β values in RPL patients (case) and healthy controls (control); (\u003cstrong\u003eB\u003c/strong\u003e) The principal component analysis plot of 5 cases and 5 controls with all methylation probes; (\u003cstrong\u003eC\u003c/strong\u003e) Unsupervised hierarchical heatmap clustering of 960 DMPs that were differential between RPL and control; (\u003cstrong\u003eD\u003c/strong\u003e) The Manhattan plot exhibited the chromosomal distribution of all methylation probes.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6835526/v1/bf235ec0e26450efb14a7e3f.png"},{"id":85819389,"identity":"c2682e48-5b61-4fc3-8c90-b80bf60bf559","added_by":"auto","created_at":"2025-07-02 06:14:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11797549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially methylated positions and genes between RPL and control.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Bar plot showed the top 70 differentially methylated genes with the most DMPs, including hypermethylated (green) and hypomethylated (blue) probes; (\u003cstrong\u003eB\u003c/strong\u003e) The box plot showed the β-value distribution of 11 hyper-DMPs from H19 gene in case and control; (\u003cstrong\u003eC\u003c/strong\u003e) The KEGG enrichment results of differentially methylated genes, the size of the dot represented the number of differential genes, the color of the dot represented p-value.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6835526/v1/4986f5aaa74c54cfdafcb088.png"},{"id":85818015,"identity":"b6c54a59-2ca7-47d4-bc19-869e324b213a","added_by":"auto","created_at":"2025-07-02 06:06:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":357370,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially methylated positions in candidate gene\u003c/strong\u003e \u003cstrong\u003eCFAP61, HLA DQB1 and KDM4D by beadChip. \u003c/strong\u003eThree hyper-DMPs in CFAP61 (\u003cstrong\u003eA\u003c/strong\u003e), 2 hyper-DMPs in HLA DQB1 (\u003cstrong\u003eB\u003c/strong\u003e) and KDM4D (\u003cstrong\u003eC\u003c/strong\u003e) between RPL and control were screened for subsequent validation.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6835526/v1/703d51bdd04962e9f6a283f8.png"},{"id":85819395,"identity":"0623f8a3-e4ac-4fca-8a18-0cec1ed65ba5","added_by":"auto","created_at":"2025-07-02 06:14:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":462550,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially methylated positions in candidate gene by targeted bisulfite sequencing.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Three hyper-DMPs from H19 in RPL compared with the control (Con); (\u003cstrong\u003eB\u003c/strong\u003e) Two hyper-DMPs from KDM4D in RPL; (\u003cstrong\u003eC\u003c/strong\u003e) ROC analysis of DMP cg17985533 in the diagnosis of RPL.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6835526/v1/2fa8c0183d49777cab51fbc0.png"},{"id":85818021,"identity":"895e05b5-002f-41eb-a9a9-d1926e7bc440","added_by":"auto","created_at":"2025-07-02 06:06:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":392529,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially methylated region chr11:1997780-1997899 in H19 by targeted bisulfite sequencing.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) The box plot showed the mean β-value of the region chr11:1997780-1997899 between RPL and control (Con); (\u003cstrong\u003eB\u003c/strong\u003e) The differential methylation status of 6 CpG sites in region chr11:1997780-1997899 between RPL and control; (\u003cstrong\u003eC\u003c/strong\u003e) The read percentages of methylation haplotypes in this region between RPL and control, including CCCCCC, CCCTCC and CTTTTT; (\u003cstrong\u003eD\u003c/strong\u003e) ROC analysis of region chr11:1997780-1997899 in the diagnosis of RPL.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6835526/v1/a93b693cfcdc3f121f73fc67.png"},{"id":99545395,"identity":"e1dbc091-af68-4718-a0f9-d7980323d48f","added_by":"auto","created_at":"2026-01-05 16:07:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21204934,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6835526/v1/0a2c63ba-c262-4191-99d9-d204a6ed59a5.pdf"},{"id":85818014,"identity":"dfed55a6-b74f-4ef0-a287-2e02c29e1082","added_by":"auto","created_at":"2025-07-02 06:06:44","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11140,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6835526/v1/f60ae7dc26d6fd6c9b3b41e4.xlsx"},{"id":85819388,"identity":"0daa8de2-3a7f-4f16-b650-83bb683110e3","added_by":"auto","created_at":"2025-07-02 06:14:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13301,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6835526/v1/c7aaf8b3a1c16a39057dd263.docx"},{"id":85818045,"identity":"5a192038-3961-4cdf-9f3f-fa00136e8293","added_by":"auto","created_at":"2025-07-02 06:06:45","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2014708,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6835526/v1/092d11df48c3be076cc83b32.tif"},{"id":85818024,"identity":"02583d7d-c3b9-46bb-afb0-5534c0a8f414","added_by":"auto","created_at":"2025-07-02 06:06:44","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":205468,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6835526/v1/79f2fe85f38bc37fd126ff32.tif"},{"id":85819737,"identity":"bb64b440-718a-449e-bc76-1a277a83772b","added_by":"auto","created_at":"2025-07-02 06:22:45","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":111882,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6835526/v1/4d875cc81277ec536150d0c0.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comprehensive DNA methylation profiling of sperm in male partners of couples with unexplained recurrent pregnancy loss","fulltext":[{"header":"Background","content":"\u003cp\u003eRecurrent pregnancy loss (RPL), defined as two or more spontaneous abortions before 20 weeks of gestation, affects fertility problems in approximately 5% of couples [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], and the cause of RPL remains unknown in up to 50% of RPL cases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which is called unexplained recurrent pregnancy loss (URPL) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Initially, research on factors influencing RPL primarily focused on women, including endocrine abnormalities, thyroid disease, hyperprolactinemia, uncontrolled diabetes, uterine abnormality and some environmental factors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, further investigations have revealed some factors in men also play significant roles in the occurrence of RPL [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Currently, there is an increasing necessity on exploring the factors and potential mechanisms affecting RPL in male partners of couples experiencing it.\u003c/p\u003e \u003cp\u003eThe male factors that contribute to RPL mainly include chromosome abnormality (like aneuploidy, Y chromosome microdeletion, chromatin integrity) [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], sperm DNA fragmentation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], virus infection [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and other related diseases. Interestingly, studies have shown that epigenetic mechanisms were also associated with RPL in male partners, mainly focused on DNA methylation [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and RNA methylation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, these studies about DNA methylation have some limitations for further uncovering the mechanisms of RPL in male partners. For example, Irani et al. just determined global methylation level in sperm of male partners of women experiencing idiopathic RPL, which did not find the methylation alterations of specific genes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Some studies only explored the methylation effect of single gene or several genes on male-related RPL, which did not systematically reveal the genome-wide methylation signatures [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, we intended to comprehensively reveal the dysregulated methylation in sperm at the genome-wide level to identify possible causes of RPL in male partners. Although some studies analyzed the genome-wide alterations in sperm DNA methylation in male partners of idiopathic RPL, the corresponding validation about methylation levels were still absent [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we firstly profiled the differential methylation signatures of sperm from male patients with couples experiencing RPL (hereafter called RPL group or RPL patients) and healthy controls by genome-wide DNA methylation beadchip, some differentially methylated positions (DMPs) were then screened for further validation by targeted bisulfite sequencing. Importantly, multiple CpG sites showed significant hypermethylation in RPL group, especially in H19 imprinted maternally expressed transcript (H19), and the DMP cg17985533 and the region chr11:1997780\u0026ndash;1997899 from H19 had consistently high methylation level in RPL, which might become a diagnosis biomarker for RPL in male partners.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy subjects\u003c/h2\u003e \u003cp\u003e The 25 male patients with couples experiencing RPL and 25 healthy controls were recruited from Jinan Maternity and Child Care Hospital Affiliated to Shandong First Medical University from October 2021 to April 2024. The age of RPL group was 26-35y, the control group was 22-40y, both average age (p\u0026thinsp;=\u0026thinsp;0.4378) and BMI (p\u0026thinsp;=\u0026thinsp;0.0557) had no significant differences (t-test) between RPL group and the control group (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The RPL group needed to meet the following inclusion criteria, before 20 weeks of gestation, those female partners of male patients who had two or more consecutive pregnancy loss, and chromosomal, anatomical, endocrine, infection, immune abnormalities and other causes were excluded in these couples. In addition, all men had no other diseases or received related treatment. The healthy controls were confirmed to be fertile, and the inclusion criteria included the following information, a healthy child was confirmed to have been born, and the man had normal chromosomes, and there was no history of spontaneous abortion, ectopic pregnancy, premature delivery and stillbirth. In addition, both men and women are no more than the age of 40. All subjects signed informed consent forms for the following semen collection. The study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Ethics Review Committee of Jinan Maternity and Child Care Hospital Affiliated to Shandong First Medical University.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample collection, DNA extraction and bisulfite conversion\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eSample collection, DNA extraction and bisulfite conversion\u003c/div\u003e \u003cp\u003eSemen samples were collected from RPL patients and healthy controls after 2\u0026ndash;7 days of abstinence, and semen analysis was performed according to WHO guidelines as previously described, including sperm volume, concentration, total motility, forward motility and morphology (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These clinical parameters had no significant differences (t-test) between RPL group and the control group, the p-values of sperm volume, concentration, total motility, forward motility and morphology were 0.1681, 0.3941, 0.7623, 0.8891, and 0.4493, respectively. The semen samples were washed and removed seminal plasma, somatic cells were further removed by somatic cell lysis buffer (0.1% sodium dodecyl sulphate, and 0.5% Triton X-100 in diethyl pyrocarbonate water) treatment as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The sperm samples were then washed twice with PBS and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until genomic DNA extraction. Genomic DNA from sperm was isolated by using HiPurA Sperm Genomic DNA Purification Kit (HiMedia, India) as the manufacturer\u0026rsquo;s instructions, DNA purity and concentration were determined by Qubit3.0 fluorometer (Thermo Fisher Scientific, USA). For bisulfite conversion, 1 \u0026micro;g of sperm DNA was processed with the EZ DNA Methylation-Gold Kit (Zymo Research, USA) according to the manufacturer\u0026rsquo;s instructions. The bisulfite-converted DNA was then used for the detection of DNA methylation levels by microarray and sequencing.\u003c/p\u003e\n\u003ch3\u003eInfinium MethylationEPIC BeadChip analysis\u003c/h3\u003e\n\u003cp\u003eThe converted DNA from 5 RPL patients and 5 healthy controls were subjected to the Infinium MethylationEPIC BeadChip v1.0 (Illumina, USA) analysis which contains\u0026thinsp;\u0026gt;\u0026thinsp;850,000 CpG sites according to the manufacturer\u0026rsquo;s instructions, and raw IDAT files were obtained using the iScan SQ fluorescent scanner (Illumina, USA). The methylation data were analyzed by the ChAMP package (v 2.18.2) in R 4.3.3. The methylation level for each CpG was scored as a β-value [β\u0026thinsp;=\u0026thinsp;intensity of the methylated allele (M) / (intensity of the unmethylated allele (U)\u0026thinsp;+\u0026thinsp;intensity of the methylated allele (M)\u0026thinsp;+\u0026thinsp;100)] according to the fluorescent intensity ratio, and it represented a continuous variable that ranged from 0 (no methylation) to 1 (full methylation). First, we filtered out the probes with detection p-value\u0026thinsp;\u0026gt;\u0026thinsp;0.01, non-CpG probes, probes located on chromosome X/Y, and SNP-related probes via ChAMP [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Then, the β-values were normalized using BMIQ, and the singular value decomposition (SVD) analysis was used to evaluate the batch effect of normalized β-values [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Finally, a total of 742,000 CpG sites were used for differential analysis, and CpGs having |Δβ| \u0026ge; 0.1 and p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered as DMPs between RPL patients and controls.\u003c/p\u003e\n\u003ch3\u003eTargeted bisulfite sequencing\u003c/h3\u003e\n\u003cp\u003eThe methylation levels of candidate target regions were detected and analyzed by targeted bisulfite sequencing (Genesky Biotechnologies Inc., Shanghai, China), which was called MethylTarget, a multiplex-targeted CpG methylation analysis technology based on next-generation sequencing [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Bisulfite conversion of genomic DNA was subjected to sodium bisulfite treatment using the EZ DNA methylation kit (Zymo Research, USA) according to the manufacturer\u0026rsquo;s protocol. Multiplex PCR of 12 regions was performed using an optimized primer combination by Genesky, after PCR amplification and library construction, samples were sequenced on an Illumina HiSeq platform by pair-end 150 bp (Illumina, USA). The sequenced reads were mapped to the human reference genome GRCh38/hg38. The methylation level of each CpG site was calculated as the percentage of methylated cytosines in total cytosines, and DMP was considered by the difference of average methylation level between RPL patients and controls with p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 (t-test).\u003c/p\u003e\n\u003ch3\u003eKyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis\u003c/h3\u003e\n\u003cp\u003eThe differentially methylated genes (DMGs) from the Infinium MethylationEPIC BeadChip results were used for KEGG pathway enrichment analysis, and KEGG pathways with p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered as significantly enriched terms. The KEGG pathway analysis was carried out by clusterProfiler package in R 4.3.3.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe methylation levels of candidate DMPs were subjected to multiple logistic regression to obtain a predicted probability score for each sample. The probability score was then subjected to the receiver operating characteristic (ROC) analysis to obtain a ROC curve, and the area under the curve (AUC) was calculated to assess the discrimination of candidate CpGs using \u0026ldquo;pROC\u0026rdquo; R package. All the statistical analyses were conducted in R 4.3.3, and p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGenome-wide DNA methylation profiling identified the differential methylation signatures in sperm between RPL patients and controls\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe detailed clinical characteristics of the RPL patients (n\u0026thinsp;=\u0026thinsp;25) and healthy controls (n\u0026thinsp;=\u0026thinsp;25) were shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. To reveal DNA methylation alternations occurring in sperm of RPL patients, we performed a genome-wide DNA methylation profiling assay in 5 RPL patients and 5 healthy controls by Infinium MethylationEPIC BeadChip. After quality control and normalization, about 742,000 methylation positions were subject to subsequent differential analysis. These CpG positions showed obvious enrichment in high (β-value close to 1) and low methylation levels (β-value close to 0) in case and control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The PCA plot exhibited relatively little differences between RPL patients and normal control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The CpG positions with |Δβ| \u0026ge; 0.1 and p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 were defined as the differentially methylated CpG positions (DMPs), a total of 960 DMPs were identified with 847 hypermethylated (88.2%) and 113 hypomethylated (11.8%) positions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The Manhattan plot exhibited the chromosomal positions of all methylation sites, including the above DMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), and the hypermethylated and hypomethylated positions showed significantly different distribution features between RPL and control, including in the island, N-Shelf/Shore, S-Shelf/Shore and the opensea regions (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The genomic distribution of DMPs showed that more hyper-DMPs and hypo-DMPs were located in the intergenic region and gene body region, respectively (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we focused on the genes with the most DMPs, top genes included H19, TBC1D16, C7orf50, DLG2, JSRP1, and PFKP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). For example, the H19 imprinted maternally expressed transcript (H19) had 11 hyper-DMPs between RPL and control, including cg18362496, cg11735853, cg27300742, cg24605090, cg01539474, cg15886040, cg15922305, cg17985533, cg04975775, cg01977486, and cg11753499 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe differentially methylated genes (DMGs) were then subjected to pathway analysis, the KEGG results displayed that drug metabolism-cytochrome P450, serotonergic synapse, arachidonic acid metabolism, retinol metabolism, and linoleic acid metabolism were significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Among them, cytochrome P450-related genes were reported to be associated with RPL or spermatogenesis [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eScreening the differentially methylated genes related to RPL\u003c/h3\u003e\n\u003cp\u003eStudies found that a significant association between H19 gene methylation and male hypospermatogenesis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] or male infertility [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Additionally, evidences showed that some DMGs found by our genome-wide screening were related spermatogenesis and male infertility, including cilia and flagella associated protein 61 (CFAP61) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], major histocompatibility complex, class II, DQ beta 1 (HLA-DQB1) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and lysine demethylase 4D (KDM4D) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, most of these studies focused on the roles of genetic variants of the above genes. The epigenetic effects of them, especially DNA methylation, was not clear in RPL. Therefore, we firstly selected the DMPs in spermatogenesis-related gene H19, CFAP61, HLA-DQB1 and KDM4D (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) for following methylation validation, among them, 11 hyper-DMPs were found in H19 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), 3 hyper-DMPs in CFAP61 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), both HLA-DQB1 and KDM4D had 2 hyper-DMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eValidation of DMPs by targeted bisulfite sequencing between RPL patients and controls\u003c/h2\u003e \u003cp\u003eIn order to validate the candidate DMPs of the above 4 genes involved in RPL, we screened a total of 12 target regions (fragments) to compare the methylation differences of 89 CpGs by the targeted bisulfite sequencing method. The differential methylation analysis revealed that 3 CpG sites in H19 and 2 CpG sites in KDM4D had statistically significant differences (p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05) between 20 RPL patients and 20 healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B), while only 3 CpG sites in H19 (chr11:1997806, cg13210239, and cg17985533) had relatively higher methylation levels with \u0026gt;\u0026thinsp;10% mean methylation difference in sperm of RPL patients compared with the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Importantly, these 3 CpG sites were not in the imprinting control region (ICR) of H19 gene, which was previously reported to be subject to differential methylation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Among them, the DMP cg17985533 was consistently hypermethylated in RPL group by both microarray and targeted sequencing methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We examined the performance of cg17985533, which exhibited AUC (0.7838), sensitivity (80%), and specificity (80%) by ROC analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Table\u0026nbsp;1). It suggested that high methylation level of cg17985533 might be a potential biomarker for diagnosis of RPL.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the level of genomic fragment, only the region (chr11:1997780\u0026ndash;1997899) in H19 was identified significantly hypermethylated with \u0026gt;\u0026thinsp;10% mean methylation difference (p\u0026thinsp;=\u0026thinsp;0.032) in sperm of RPL patients compared with the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), this region included the above 3 differential CpG sites in H19, chr11:1997806, cg13210239, and cg17985533. All 6 CpG sites in region chr11:1997780\u0026ndash;1997899 showed higher methylation levels in RPL group compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Interestingly, the abundance analysis of methylation haplotypes in this region showed that the CCCCCC and CCCTCC haplotypes (C represented methylated cytosine, T represented unmethylated cytosine) were higher in RPL group, and CTTTTT haplotype was higher in control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). We also examined the performance of this region, which exhibited AUC (0.8125), sensitivity (80%), and specificity (75%) by ROC analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, Table\u0026nbsp;1). It also validated that this region was significantly hypermethylated in sperm of RPL patients. Therefore, the mean methylation level of this region in H19 could also be a diagnostic biomarker for RPL.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe proper functioning of germ cells is crucial for both fertility and embryonic development. As we all know, the impact of DNA methylation on spermatogenesis and male infertility has got a lot of attention. Study has definitely confirmed that DNA methyltransferase 3A-dependent DNA methylation was required for spermatogonial stem cells to commit to spermatogenesis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Moreover, genome-wide DNA methylation was dynamically changed during human spermatogenesis and germ cells exhibited considerable DNA methylation changes in disturbed spermatogenesis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, a lot of evidences also characterized the links between DNA methylation and male infertility. Han et al. found that methylated inactivation of SOX30 uniquely impaired spermatogenesis, and further caused non-obstructive azoospermia disease which is the most severe form of male infertility [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Interestingly, DNA methylation could interplay with histone H3 lysine 4 tri-methylation (H3K4me3) throughout the genome of human sperm, and it might be related with fertility and development [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, male factors are known to affect pregnancy loss, abortion, and infertility. Various factors such as smoking, obesity, advanced age, and other environmental factors could impact sperm quality and male infertility [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. DNA methylation is one of the most extensively studied epigenetic factor that could help elucidate the mechanism underlying URPL. Therefore, this study aimed to reveal the relationships between DNA methylation modifications of sperm and URPL in male partners. Four genes (H19, CFAP61, HLA-DQB1, and KDM4D) were screened as differential methylation candidates between URPL and control groups in our study. These genes were reported to be associated with male infertility or RPL in male partners. Of these genes, KDM4D is a histone demethylase that has been shown to be involved in defects in elongated sperm production and changes in H3K9me3 distribution in round sperm upon deletion [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, there was a report which found that KDM4D regulated methylation of histone H3 lysine 9 (H3K9) during spermatogenesis in the mouse but was dispensable for fertility [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Therefore, it was necessary to examine the genome methylation level of KDM4D to evaluate its potential effect on gene expression. The flagellum is an essential structure for sperm morphology and function, with CFAP61 locating centrally within the sperm and playing a role in flagellum formation in human and mouse [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Moreover, studies have demonstrated that biallelic variants in CFAP61 could cause multiple morphological abnormalities of the flagella and male infertility in human and mouse [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Further, the knockdown of CFAP61 aggravated male infertility by inhibiting testosterone secretion by Leydig cells via the MAPK/COX-2 pathway [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. However, there were not relevant reports about the relation between KDM4D or CFAP61 and RPL. Interestingly, two studies from India found that the HLA-DQB1*02:01:01 and DQB1*03:03:02 alleles were associated with an increase in risk of RPL, while DQB1*02:02:01 and DQB1*06:03 alleles appeared to be protective against RPL [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Another report about Lebanese women identified that DPB1-DQB1-DRB1 loci were linked with altered RPL susceptibility by case-control study [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. A Danish study also validated that the frequencies of RPL women carrying three haplotypes with DQB1*0501 or one haplotype with DQB1*0201 were significantly increased compared with controls [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. These data suggested the polymorphism of HLA-DQB1 was associated with the risk of RPL, however, the methylation level of HLA-DQB1 has not been reported to be related with RPL.\u003c/p\u003e \u003cp\u003eImportantly, we observed significant hypermethylation of 11 CpG sites in H19 gene in RPL patients compared to the control. H19 is an imprinted gene that can undergo both methylation and demethylation processes. During spermatogenesis, DNA methylation patterns of the H19 gene are epigenetically inherited by somatic cells in embryos, particularly in relation to imprinted genes. The methylation status of the H19 gene has been extensively studied in male infertility, however, Cannarella et al. found that H19 methylation levels were significantly lower in the group of infertile patients than in fertile controls by meta-analysis [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In addition, this study suggested that the hypomethylation of H19 was also associated with patients with oligozoospermia and RPL [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Similar study also revealed the overall methylation rate of H19 DMR was significantly decreased in male infertile patients [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] or in oligospermic patients [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. However, Nasri et al. showed that the median of methylation percentage for H19 was not statistically significant between male infertility group and control group [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, the methylation status of H19 at specific sites needs to be further validated in male infertile patients or RPL patients. Moreover, study also validated that the hypomethylation at specific CpG sites of insulin like growth factor-2 (IGF2)-H19 was observed in sperm DNA of male patients with couples experiencing RPL [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Aberrant methylation patterns of the H19/IGF2 genes have been associated with increased incidence of sperm DNA fragmentation (SDF), while decreased levels of H19 gene methylation have been linked to higher rates of recurrent miscarriage [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In fact, research has demonstrated a correlation between impaired H19/IGF2 methylation and elevated levels of reactive oxygen species (ROS), which can induce DNA fragmentation and thus play a significant role in increasing SDF rates [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Elevated SDF levels are also considered one of the major causes for RPL [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Khambata et al. validated that a combination of five imprinted genes comprising IGF2-H19 DMR, IG-DMR, ZAC, KvDMR, and PEG3 could be used as a diagnostic tool for spermatozoa samples of RPL patients [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In contrast, our study reveals that cg17985533, a highly methylated CpG site and a region chr11:1997780\u0026ndash;1997899 derived from H19, holds promise as a diagnostic biomarker for RPL in male partners.\u003c/p\u003e \u003cp\u003eOf course, our research also has some shortcomings. Firstly, we screened and validated the methylation-related genes and loci in a small number of sperm samples, and we will need more samples to confirm our current findings in the future. Secondly, we only verified the methylation levels of a few genes related to spermatogenesis or male infertility, and subsequently we need to further verify the methylation levels of more other genes between RPL and controls to discover new methylation biomarkers.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe have provided compelling evidence indicating altered methylation levels of H19, CFAP61, HLA-DQB1, and KDM4D genes in RPL patients compared with controls among male partners, notably highlighting that hypermethylation levels of CpG site cg17985533 and region chr11:1997780\u0026ndash;1997899 derived from H19 represented a potential biomarker for RPL.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRPL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003erecurrent pregnancy loss\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eURPL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eunexplained recurrent pregnancy loss\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edifferentially methylated positions\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH19\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eH19 imprinted maternally expressed transcript\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSVD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esingular value decomposition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edifferentially methylated genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereceiver operating characteristic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003earea under the curve\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCFAP61\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eflagella associated protein 61\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHLA-DQB1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emajor histocompatibility complex, class II, DQ beta 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKDM4D\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elysine demethylase 4D\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimprinting control region\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH3K9\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehistone H3 lysine 9\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esperm DNA fragmentation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Review Committee of Jinan Maternity and Child Care Hospital Affiliated to Shandong First Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the High-level Talents in Medical and Health Industry of Jinan City, the Shandong Provincial Natural Science Foundation (No.ZR2020QH276) and the Jinan City Clinical Medicine Technology Innovation Program (No.202019173).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLW was a major contributor in writing the manuscript. LW and FL collected all sperm samples and recorded the individual information. LW, QL and RW analyzed and interpreted the DNA methylation data. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRPL EGGo, Bender Atik R, Christiansen OB, Elson J, Kolte AM, Lewis S, Middeldorp S, Nelen W, Peramo B, Quenby S\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eESHRE guideline: recurrent pregnancy loss\u003c/strong\u003e. \u003cem\u003eHum Reprod Open \u003c/em\u003e2018, \u003cstrong\u003e2018\u003c/strong\u003e(2):hoy004.\u003c/li\u003e\n\u003cli\u003eChristiansen OB: \u003cstrong\u003eSpecial Issue Recurrent Pregnancy Loss: Etiology, Diagnosis, and Therapy\u003c/strong\u003e. \u003cem\u003eJ Clin Med \u003c/em\u003e2021, \u003cstrong\u003e10\u003c/strong\u003e(21).\u003c/li\u003e\n\u003cli\u003eMuncey W, Scott M, Lathi RB, Eisenberg ML: \u003cstrong\u003eThe paternal role in pregnancy loss\u003c/strong\u003e. \u003cem\u003eAndrology \u003c/em\u003e2024.\u003c/li\u003e\n\u003cli\u003eYu N, Kwak-Kim J, Bao S: \u003cstrong\u003eUnexplained recurrent pregnancy loss: Novel causes and advanced treatment\u003c/strong\u003e. \u003cem\u003eJ Reprod Immunol \u003c/em\u003e2023, \u003cstrong\u003e155\u003c/strong\u003e:103785.\u003c/li\u003e\n\u003cli\u003eIbrahim Y, Johnstone E: \u003cstrong\u003eThe male contribution to recurrent pregnancy loss\u003c/strong\u003e. \u003cem\u003eTransl Androl Urol \u003c/em\u003e2018, \u003cstrong\u003e7\u003c/strong\u003e(Suppl 3):S317-S327.\u003c/li\u003e\n\u003cli\u003eCao C, Bai S, Zhang J, Sun X, Meng A, Chen H: \u003cstrong\u003eUnderstanding recurrent pregnancy loss: recent advances on its etiology, clinical diagnosis, and management\u003c/strong\u003e. \u003cem\u003eMed Rev (2021) \u003c/em\u003e2022, \u003cstrong\u003e2\u003c/strong\u003e(6):570-589.\u003c/li\u003e\n\u003cli\u003eInversetti A, Bossi A, Cristodoro M, Larcher A, Busnelli A, Grande G, Salonia A, Di Simone N: \u003cstrong\u003eRecurrent pregnancy loss: a male crucial factor-A systematic review and meta-analysis\u003c/strong\u003e. \u003cem\u003eAndrology \u003c/em\u003e2023.\u003c/li\u003e\n\u003cli\u003eHedegaard S, Landersoe SK, Olsen LR, Krog MC, Kolte AM, Nielsen HS: \u003cstrong\u003eStress and depression among women and men who have experienced recurrent pregnancy loss: focusing on both sexes\u003c/strong\u003e. \u003cem\u003eReprod Biomed Online \u003c/em\u003e2021, \u003cstrong\u003e42\u003c/strong\u003e(6):1172-1180.\u003c/li\u003e\n\u003cli\u003ePeuranpaa PL, Gissler M, Peltopuro P, Tiitinen A, Hautamaki H: \u003cstrong\u003eThe effect of paternal and maternal factors on the prognosis of live birth in couples with recurrent pregnancy loss\u003c/strong\u003e. \u003cem\u003eActa Obstet Gynecol Scand \u003c/em\u003e2022, \u003cstrong\u003e101\u003c/strong\u003e(12):1374-1385.\u003c/li\u003e\n\u003cli\u003eGolin AP, Yuen W, Flannigan R: \u003cstrong\u003eThe effects of Y chromosome microdeletions on in vitro fertilization outcomes, health abnormalities in offspring and recurrent pregnancy loss\u003c/strong\u003e. \u003cem\u003eTransl Androl Urol \u003c/em\u003e2021, \u003cstrong\u003e10\u003c/strong\u003e(3):1457-1466.\u003c/li\u003e\n\u003cli\u003ePourmasumi S, Sabeti P, Ghasemi N: \u003cstrong\u003eMale factor testing in recurrent pregnancy loss cases: A narrative review\u003c/strong\u003e. \u003cem\u003eInt J Reprod Biomed \u003c/em\u003e2022, \u003cstrong\u003e20\u003c/strong\u003e(6):447-460.\u003c/li\u003e\n\u003cli\u003eGkeka K, Symeonidis EN, Tsampoukas G, Moussa M, Issa H, Kontogianni E, Almusafer M, Katsouri A, Mykoniatis I, Dimitriadis F\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eRecurrent miscarriage and male factor infertility: diagnostic and therapeutic implications. 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Sun U, Zhang S, Feng X, Xu M, Dai J, Ni X, Wang X, Wu Q: \u003cstrong\u003eProfiling the DNA methylation patterns of imprinted genes in abnormal semen samples by next-generation bisulfite sequencing\u003c/strong\u003e. \u003cem\u003eJ Assist Reprod Genet \u003c/em\u003e2020, \u003cstrong\u003e37\u003c/strong\u003e(9):2211-2221.\u003c/li\u003e\n\u003cli\u003eNasri F, Gharesi-Fard B, Namavar Jahromi B, Farazi-Fard MA, Banaei M, Davari M, Ebrahimi S, Anvar Z: \u003cstrong\u003eSperm DNA methylation of H19 imprinted gene and male infertility\u003c/strong\u003e. \u003cem\u003eAndrologia \u003c/em\u003e2017, \u003cstrong\u003e49\u003c/strong\u003e(10).\u003c/li\u003e\n\u003cli\u003eCannarella R, Crafa A, Condorelli RA, Mongioi LM, La Vignera S, Calogero AE: \u003cstrong\u003eRelevance of sperm imprinted gene methylation on assisted reproductive technique outcomes and pregnancy loss: a systematic review\u003c/strong\u003e. \u003cem\u003eSyst Biol Reprod Med \u003c/em\u003e2021, \u003cstrong\u003e67\u003c/strong\u003e(4):251-259.\u003c/li\u003e\n\u003cli\u003eDarbandi M, Darbandi S, Agarwal A, Baskaran S, Dutta S, Sengupta P, Khorram Khorshid HR, Esteves S, Gilany K, Hedayati M\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eReactive oxygen species-induced alterations in H19-Igf2 methylation patterns, seminal plasma metabolites, and semen quality\u003c/strong\u003e. \u003cem\u003eJ Assist Reprod Genet \u003c/em\u003e2019, \u003cstrong\u003e36\u003c/strong\u003e(2):241-253.\u003c/li\u003e\n\u003cli\u003eMcQueen DB, Zhang J, Robins JC: \u003cstrong\u003eSperm DNA fragmentation and recurrent pregnancy loss: a systematic review and meta-analysis\u003c/strong\u003e. \u003cem\u003eFertil Steril \u003c/em\u003e2019, \u003cstrong\u003e112\u003c/strong\u003e(1):54-60 e53.\u003c/li\u003e\n\u003cli\u003eKhambata K, Begum S, Raut S, Mohan S, Irani D, Singh D, Bansal V, Patil A, Balasinor NH: \u003cstrong\u003eDNA methylation biomarkers to identify epigenetically abnormal spermatozoa in male partners from couples experiencing recurrent pregnancy loss\u003c/strong\u003e. \u003cem\u003eEpigenetics \u003c/em\u003e2023, \u003cstrong\u003e18\u003c/strong\u003e(1):2252244.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-epigenetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clep","sideBox":"Learn more about [Clinical Epigenetics](http://clinicalepigeneticsjournal.biomedcentral.com/)","snPcode":"13148","submissionUrl":"https://submission.nature.com/new-submission/13148/3","title":"Clinical Epigenetics","twitterHandle":"@OAgenetics","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Recurrent pregnancy loss, Sperm, DNA methylation, H19, Biomarker","lastPublishedDoi":"10.21203/rs.3.rs-6835526/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6835526/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Recurrent pregnancy loss (RPL) affects fertility problems in approximately 5% of couples, while the cause of RPL remains unknown in about half RPL cases, which is also called unexplained RPL. The male factors were associated with RPL in male partners, including chromosome abnormality and sperm DNA fragmentation. DNA methylation is one of the most extensively studied epigenetic factors that could help elucidate the mechanism underlying RPL in male partners.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e We revealed DNA methylation alternations occurring in sperm of RPL patients compared with the controls by genome-wide DNA methylation beadchip, including a series of differentially methylated CpG positions and genes. Importantly, we validated that the CpG site cg17985533 and the region chr11:1997780-1997899 from the H19 imprinted maternally expressed transcript were significantly hypermethylated in sperm of RPL-related men with \u0026gt; 10% mean methylation difference by targeted bisulfite sequencing. Moreover, the receiver operating characteristic analysis showed that CpG site cg17985533 and region chr11:1997780-1997899 could distinguish RPL patients from controls, with an area under the curve of 0.7838 and 0.8125, sensitivity of 80% and 80%, and specificity of 80% and 75%, respectively. These results indicated that they could be potential biomarker for diagnosis of RPL in male partners.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e This study highlighted the importance of H19 gene methylation in differentiating RPL and control, and provided new insight for revealing potential epigenetic mechanisms for RPL in male partners.\u003c/p\u003e","manuscriptTitle":"Comprehensive DNA methylation profiling of sperm in male partners of couples with unexplained recurrent pregnancy loss","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-02 06:06:39","doi":"10.21203/rs.3.rs-6835526/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-11T13:04:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-25T12:56:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-16T07:42:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260413179867643169410718003006719298757","date":"2025-06-26T02:52:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336358584782467003035450728824179509998","date":"2025-06-26T00:47:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266166369594357584137337476717442540279","date":"2025-06-25T12:46:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-25T11:19:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-11T03:22:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T23:15:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Epigenetics","date":"2025-06-06T09:04:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-epigenetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clep","sideBox":"Learn more about [Clinical Epigenetics](http://clinicalepigeneticsjournal.biomedcentral.com/)","snPcode":"13148","submissionUrl":"https://submission.nature.com/new-submission/13148/3","title":"Clinical Epigenetics","twitterHandle":"@OAgenetics","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0dc4c086-f827-41a8-a936-65907fd11aff","owner":[],"postedDate":"July 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T16:02:32+00:00","versionOfRecord":{"articleIdentity":"rs-6835526","link":"https://doi.org/10.1186/s13148-025-02043-3","journal":{"identity":"clinical-epigenetics","isVorOnly":false,"title":"Clinical Epigenetics"},"publishedOn":"2025-12-31 15:57:37","publishedOnDateReadable":"December 31st, 2025"},"versionCreatedAt":"2025-07-02 06:06:39","video":"","vorDoi":"10.1186/s13148-025-02043-3","vorDoiUrl":"https://doi.org/10.1186/s13148-025-02043-3","workflowStages":[]},"version":"v1","identity":"rs-6835526","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6835526","identity":"rs-6835526","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}