Comparative analysis of chorionic methylation abnormalities in various pregnancy pathologies

The development of normal pregnancy at early stages is conditioned by epigenetic programs and is impossible without proper formation of the placenta and embryo. In the first trimester, epigenetic mechanisms are involved in these processes, one of which is DNA methylation. DNA methylation is involved in the regulation of various biological processes, such as cell differentiation and development, regulation of gene expression (as a rule, genes with reduced methylation levels in promoters are expressed), X-chromosome inactivation, genome imprinting, maintenance of chromosome stability, and others. Disruption of this process can lead to the formation of pathologic phenotypes such as spontaneous abortion (SA), pre-eclampsia (PE), and gestational diabetes mellitus (GDM). Currently, researchers have found many methylation abnormalities in these pathologies: for example, 54 differentially methylated regions (DMRs) associated with blood pressure, 1703 differentially methylated sites (DMSs) in early-onset pre-eclampsia, more than 200 DMRs in gestational diabetes mellitus, and 4 differentially expressed genes associated with spontaneous abortion have been identified in the trophoblast of chorionic villi. The emerging findings in such studies indicate that some epigenetic abnormalities and gene expression disorders may be involved in the development of gestational pathologies, affecting the cause of their formation -abnormal placentation. The methylation abnormalities observed in the considered pathologies of pregnancy may be particular cases of more general placental methylome disorders, the severity of phenotypic manifestation of which may vary from embryo death at early stages of development to term delivery. Given this and current theories of the pathogenesis of these diseases based on disorders of trophoblast development, it can be assumed that these pathologies may share common DNA methylation alterations. On the other hand, differences in the severity of clinical manifestations suggest the presence of unique methylation patterns, particularly in spontaneous abortion as an extreme manifestation of the phenotype. Detection of such abnormalities will have important diagnostic value for prognosis and preservation of pregnancy. In this study, we comparatively analysed differential DNA methylation data in chorionic villi from PE, GDM and SA. Methylation data in PE and GSD were obtained from the publicly available Genome Expression Omnibus (GEO) data repository - GSE200659, GSE100197, and GSE98224 datasets. Data for SA were obtained using reduced representation bisulfite sequencing (RRBS) on spontaneous abortus material with normal karyotype (n = 7) compared to medical abortus (n = 7). Methylation data were analysed in R 4.3.0., using function packages in R (dplyr,data.table,tibble,tidyr,stringr, tibble) and function packages in UNIX (bedtools) and BMIQ and NOOB normalisation methods (function packages in R - ChAMP, minifi). Thus, 7 differentially methylated genes (DMGs) common to SA, PE and GDM were obtained: 3 hypomethylated genes - WDR5, TMC4, MIR4533; 3 hypermethylated genes - ADARB2, INPPP5E, TUBB4A; and 1 gene with different methylation differences in different parts of gene - PRDMl 6. There were 114 DMGs unique to SA: 32 hypomethylated and 81 hypermethylated genes. Enrichment analyses were performed using the DAVID v2024q2 database (https://da-vid.ncifcrf.gov/home.jsp). Common DMGs were found to be involved in pathways related to transmembrane transport processes, regulation of transcription and differentiation, RNA processing, cell adhesion, lipid metabolism and mitotic cycle regulation. DMGs unique to SA have been implicated in processes related to the regulation of transcription, translation, mitotic cycle, histogenesis, migration, cell proliferation and differentiation. Some of the unique DMGs, according to the mammalian phenotype database (https://www.informatics.jax.org/batch), were found to be associated with the em-bryolethality phenotype - LRRC8A, HDLBP, HIC1 and ST14, while others were involved in the process of placenta development - BOK, BOK-AS1. The analysis showed that the pathologies under consideration have common and unique patterns of differential DNA methylation. We identified 7 common (3 hypo-methylated, 3 hypermethylated and 1 with different methylation patterns) and 114 differentially methylated genes unique to SA (33 hypomethylated and 81 hypermethylated). However, the effects of methylation disruption of the identified genes remain unclear, and further studies at the transcriptional level in larger samples are needed. The article contains 15 References. The Authors declare no conflict of interest.

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