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. 2025 Dec 31;18:20. doi: 10.1186/s13148-025-02043-3

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

Linping Wei 1, Fang Luan 2, Qining Liu 3, Rui Wang 3, Yang Fu 3,✉
PMCID: PMC12866396  PMID: 41476224

Abstract

Background

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.

Results

We revealed DNA methylation alternations occurring in sperm of RPL partners 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 chr11:1997780–1,997,899 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 chr11:1997780–1,997,899 could distinguish RPL-related men 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-related men and control, and provided new insight for revealing potential epigenetic mechanisms for RPL in male partners.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13148-025-02043-3.

Keywords: Recurrent pregnancy loss, Sperm, DNA methylation, H19, Biomarker

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 chr11:1997780–1,997,899 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

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 more than 0.05. The semen samples were washed and removed seminal plasma. Sperm were then purified to remove somatic contaminants by two sequential centrifugations through 40:80 discontinuous gradients of Percoll (Merck, Germany) as described previously [21]. 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 [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 (average conversion efficiency > 98.9%) 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 mapped to the human reference genome GRCh38/hg38 and 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, the methylation levels and read percentages of haplotypes were analyzed by Student’s t test in two groups, and p-value < 0.05 was considered statistically 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. 1A). The PCA plot exhibited relatively little differences between RPL patients and normal control (Fig. 1B). The inter-group similarity and intra-group heterogeneity were because the PCA analysis utilized all methylation sites in which a lot of CpG positions did not have significant methylation differences. 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 in the RPL patients compared to the control (Fig. 1C, Table S2). The Manhattan plot exhibited the chromosomal positions of all methylation sites, including the above DMPs (Fig. 1D), 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).

Fig. 1.

Fig. 1

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The genome-wide DNA methylation analysis of sperm from 5 RPL patients and 5 healthy controls by Infinium MethylationEPIC BeadChip.A The distribution of 742,742 methylation probes with different β values (x-axis) in RPL patients (case) and healthy controls (control), the y-axis represented the distribution density of all probes; B The principal component analysis plot of 5 cases and 5 controls which were analyzed by all methylation probes; C Unsupervised hierarchical heatmap showed that the methylation levels of 960 positions were differential between 5 RPL patients and 5 healthy controls; D The Manhattan plot exhibited the chromosomal distribution of all methylation probes, the top two CpGs cg18379824 (Chr1) and cg05453434 (Chr16) were shown with -log10(p) values > 5

Next, we focused on the genes with the most DMPs, top genes included H19, TBC1D16, C7orf50, DLG2, JSRP1, and PFKP (Fig. 2A). 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. 2B, C).

Fig. 2.

Fig. 2

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Differentially methylated positions and genes between RPL patients and healthy control revealed by Infinium MethylationEPIC BeadChip. A Bar plot showed the top 70 DMGs with the most DMPs, including hypermethylated (green) and hypomethylated (blue) probes, The y-axis represented the number of DMPs in each gene; B The schematic diagram illustrated the exact genomic locations of 11 DMPs within H19 gene; C The box plot showed the β-value distribution and relative methylation levels of 11 hyper-DMPs from H19 gene in case and control, all p-values were shown below the name of each DMP; D The KEGG enrichment results of DMGs, the x-axis represented the gene ratio of DMGs relative to all genes in corresponding pathways, the size of the dot represented the number of differential genes associated with each KEGG pathway, and the color of the dot represented p-value of each enriched pathway

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. 2D). Among them, cytochrome P450-related genes were widely 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. 2C), 3 hyper-DMPs in CFAP61 (Fig. 3A), both HLA-DQB1 and KDM4D had 2 hyper-DMPs (Fig. 3B, C).

Fig. 3.

Fig. 3

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Differentially methylated positions in candidate gene CFAP61, HLA DQB1 and KDM4D by Infinium MethylationEPIC BeadChip. The box plot showed relative methylation levels of three hyper-DMPs in CFAP61 (A), 2 hyper-DMPs in HLA DQB1 (B) and 2 hyper-DMPs in KDM4D (C) between RPL case and the control, all p-values were shown below the name of each DMP. These DMPs were screened for subsequent methylation validation by targeted bisulfite sequencing

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 (Table S3). 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. 4A, B), while only 3 CpG sites in H19 (chr11:1997806, cg13210239 (chr11:1997860), and cg17985533 (chr11:1997886)) had relatively higher methylation levels with > 10% mean methylation difference in sperm of RPL patients compared with the controls (Fig. 4A). Importantly, these 3 CpG sites were not in the imprinting control region (ICR) of H19 gene (a 2 kb CpG-rich differentially methylated domain upstream of H19), 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. 4A). We examined the performance of cg17985533, which exhibited AUC (0.7838), sensitivity (80%), and specificity (80%) by ROC analysis (Fig. 4C; Table 1). It suggested that high methylation level of cg17985533 might be a potential biomarker for diagnosis of RPL.

Fig. 4.

Fig. 4

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Differentially methylated positions in candidate gene by targeted bisulfite sequencing. A The box plot showed relative methylation levels of three hyper-DMPs from H19 in RPL case compared with the control; B The box plot showed relative methylation levels of two hyper-DMPs from KDM4D between RPL case and the control, all p-values were shown below the name of each DMP; C ROC analysis of DMP cg17985533 in the diagnosis of RPL, the AUC of cg17985533 was 0.7838

Table 1.

The ROC analysis of cg17985533 and region chr11:1997780-1997899

AUC AUC_CI 95 Threshold Specificity Sensitivity Accuracy
cg17985533 0.7838 0.6342 - 0.9333 0.5035 0.8 0.8 0.8
chr11:1997780-1997899 0.8125 0.6763 - 0.9487 0.4582 0.75 0.38 0.775
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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. 5A), this region included the above 3 differential CpG sites in H19, chr11:1997806, cg13210239 (chr11:1997860), and cg17985533 (chr11:1997886). All 6 CpG sites in region chr11:1997780–1,997,899 showed higher methylation levels in RPL group compared with the control group (Fig. 5B). 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. 5C). We also examined the performance of this region, which exhibited AUC (0.8125), sensitivity (80%), and specificity (75%) by ROC analysis (Fig. 5D; 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.

Fig. 5.

Fig. 5

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Differentially methylated region chr11:1997780–1,997,899 inH19by targeted bisulfite sequencing. A The box plot showed the mean methylation β-value of the region chr11:1997780–1,997,899 between RPL case and the control; B The differential methylation status of 6 CpG sites in region chr11:1997780–1,997,899 between RPL case and the control; C The read percentages of three methylation haplotypes in this region between RPL case and control, including CCCCCC, CCCTCC and CTTTTT, all p-values were shown below the name of the region or haplotype; D ROC analysis of region chr11:1997780–1,997,899 in the diagnosis of RPL, the AUC of chr11:1997780–1,997,899 was 0.8125

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 3 A-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 factors 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. The DMGs identified by Infinium BeadChip were significantly associated with several metabolism pathways. Polymorphisms of cytochrome P450-related genes were reported associated with RPL, including CYP1A1 [27, 42, 43], CYP1A2 [44], CYP17 [45], etc. Arachidonic acid metabolism could regulate spermatogenesis [46] or affect germ cells [47–49]. Retinol or vitamin A metabolism was widely validated to function in spermatogenesis [50]. For example, its active metabolite, retinoic acid is also critical for various spermatogenesis stages, including the differentiation of spermatogonia, meiosis in spermatogenic cells, and the production of mature spermatozoa [51]. The linoleic acid metabolism was found associated with pathological pregnancies and human reproduction process [52]. However, these metabolism pathways are rarely reported associated with RPL, especially RPL 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 [53]. 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, 54]. 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, 55]. Further, the knockdown of CFAP61 aggravated male infertility by inhibiting testosterone secretion by Leydig cells via the MAPK/COX-2 pathway [56]. 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 [57, 58]. Another report about Lebanese women identified that DPB1-DQB1-DRB1 loci were linked with altered RPL susceptibility by case-control study [59]. 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 [60]. 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 [61]. In addition, this study suggested that the hypomethylation of H19 was also associated with patients with oligozoospermia and RPL [61]. Similar study also revealed the overall methylation rate of H19 DMR was significantly decreased in male infertile patients [62] or in oligospermic patients [63]. However, Nasri et al. showed that the median of methylation percentage for H19 was not statistically significant between male infertility group and control group [64]. 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 [65]. 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 [66]. Elevated SDF levels are also considered one of the major causes for RPL [67]. Khambata et al. found 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 [68]. However, this study only validated the five known imprinted genes by pyrosequencing in the male partners of couples undergoing RPL. Our study screened more genes and sites by microarray, and validated the methylation levels of related genes (including H19) by sequencing between RPL and controls. In contrast, our study reveals that cg17985533 (chr11:1997886), a highly methylated CpG site and a region chr11:1997780–1,997,899 derived from H19, holds promise as a diagnostic biomarker for RPL in male partners. The site and region are located within 200 bp from transcriptional start site (TSS200), they may affect the transcriptional process of H19 gene and inhibit its expression.

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 need to quantify the sperm that are affected by the hypermethylation of H19 gene by other methods, like single-cell methylation analysis, because the current study obtained the pooled DNA of all sperm from single sample. Thirdly, 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 chr11:1997780–1,997,899 derived from H19 represented a potential biomarker for RPL.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (13KB, docx)
Supplementary Material 2 (1.9MB, tif)
Supplementary Material 3 (200.7KB, tif)
Supplementary Material 4 (116.9KB, xlsx)

Acknowledgements

Not applicable.

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

Author 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. All authors read and approved the final manuscript.

Funding

This study was supported by the Ji Nan Health High-Caliber Talent Project (202312), the Shandong Provincial Natural Science Foundation (No.ZR2020QH276) and the Jinan City Clinical Medicine Technology Innovation Program (No.202019173).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Supplementary Materials

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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