Intrauterine adhesion (IUA) represents a prevalent cause of persistent infertility and continues to be a significant unresolved challenge in reproductive medicine. However, its full pathogenic mechanisms remain incompletely elucidated. We retrieved endometrial RNA-sequencing data from IUA patients and normal controls in the Gene Expression Omnibus (GEO) database, and screened 586 differentially expressed genes (DEGs) in the endometrium of IUA patients using the R package limma. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) revealed activation of immune-related pathways, including chronic inflammatory response, complement activation, and complement and coagulation cascades in IUA. Further single-sample gene set enrichment analysis (ssGSEA)-based relative immune cell enrichment analysis revealed decreased enrichment scores of activated CD4 T cells and type 2 T helper cells, alongside increased enrichment scores of effector memory CD8 T cells, macrophages, mast cells, natural killer cells, and T follicular helper cells in IUA patients, indicating their potential involvement in IUA pathogenesis. A protein-protein interaction (PPI) network was constructed, identifying 10 hub genes associated with IUA: CDK1, CCNB1, CCNA2, CENPA, BUB1, AURKB, KIF11, BUB1B, CDC20, and TOP2A. The expression patterns of these genes were further validated in two independent external IUA cohorts, supporting a potential correlative link between these genes and IUA pathogenesis. Our findings provide exploratory insights and potential candidate molecules for subsequent mechanistic studies of IUA onset and progression.
Oxidative stress (OS) in ejaculates is a key indicator of semen quality. The aim was to estimate global OS using chemiluminescence and malondialdehyde (MDA) based methods in fresh (0h) and extended preserved (48h and 96h) semen of boars with different fertility indices. Based on the boar fertility index (BFI >10, ∼7-8 and <6), boars (n = 6) with three fertility (high, medium and low) levels were used to collect semen (n = 72). Routine semen evaluation tests along with OS estimation by MDA and chemiluminescence methods were done in fresh (0h) and extended semen at 48, 96 h of preservation. In results, routine assessment tests were not significantly (p > 0.05) reliable in determining fertility according to the corresponding BFI. Only low (BFI < 6) fertile boar's extended ejaculates at only 96 h of preservation could be distinguished using MDA-based OS estimation (p < 0.05). There was a significant (p < 0.05) difference between the OS levels measured by chemiluminescence in fresh (5.81 ± 0.16 vs 7.36 ± 0.15 vs 9.05 ± 0.24 RLU/s) and extended ejaculates (48h, 96h) of high (BFI > 10), medium (BFI∼7-8) and low (BFI < 6) fertile group boars. A weak (r = -0.12) to moderately (r = -0.29) negative correlation was observed between routine semen quality assessment and OS estimated by chemiluminescence. However, the initial capital cost of the chemiluminometer was the major limitation apart from the study over larger population and samples. In conclusion, the chemiluminescence method was a more accurate method than MDA to assess global OS level in the semen and can be used to predict the boar fertility grades.
Male fertility supplements reduce oxidative stress, but whether they improve fertility is controversial. Since sperm epigenetic patterns correlate with fertility potential, we investigated whether a male fertility supplement (AlphaSperm (R)) impacts semen parameters or sperm epigenetics. In a prospective, multi-institutional, within-subject study, 3 groups were evaluated: young healthy men < 25 years old (C, n = 11), young infertile men < 40 years old (YI, n = 10), and older infertile men > 40 years old (OI, n = 10). While on daily AlphaSperm (R) supplementation, subjects provided serial semen samples. Conventional WHO semen parameters, sperm DNA fragmentation, and sperm DNA methylation were assessed at baseline and after 9 months of treatment. Beta values were generated from methylation array data, and differences in regional sperm DNA methylation with treatment were reported as differentially methylated regions (DMRs) by paired sliding-window analyses. The Stanford GREAT gene ontology tool revealed biological pathways associated with DMRs. SpermQT, a clinical diagnostic, was performed on all subjects. AlphaSperm (R) supplementation did not significantly alter semen parameters or sperm DNA fragmentation within groups. Regional sperm DNA hypermethylation was observed in all groups (eFDR < 0.0001), and mean methylation changes at DMRs ranged from 0.1% to 4.3%. Gene ontological analysis revealed 29 biological pathways significantly altered with treatment in the OI group (adjusted p < 0.05), though their functional significance needs validation. Supplement-induced DMRs included 5 genes whose methylation statuses are correlated with male infertility that were hypermethylated in the OI group; 1 was hypermethylated in the C group. Thus, although semen parameters and DNA fragmentation were largely unaltered, male fertility supplementation induced novel regional sperm DNA hypermethylation, most obvious in older infertile men. This study provides novel insights into the potentially large epigenetic effects of nutritional supplements occurring independent of semen analysis changes. Limitations include small sample size and lack of placebo controls.
