Comprehensive analysis of chromosomal breakpoints and candidate genes associated with male infertility: insights from cytogenetic studies and expression analyses.

The study aimed to investigate prevalent chromosomal breakpoints identified in balanced structural chromosomal anomalies and to pinpoint potential candidate genes linked with male infertility. This was acchieved through a comprehensive approach combining RNA-seq and microarray data analysis, enabling precise identification of candidate genes. The Cytogenetics data from 2,500 infertile males referred to Royan Research Institute between 2009 and 2022 were analyzed, with 391 cases meeting the inclusion criteria of balanced chromosomal rearrangement. Of these, 193 cases exhibited normal variations and were excluded from the analysis. By examining the breakpoints, potential candidate genes were suggested. Among the remaining 198 cases, reciprocal translocations were the most frequent anomaly (129 cases), followed by Robertsonian translocations (43 cases), inversions (34 cases), and insertions (3 cases).Some patients had more than one chromosomal abnormality. Chromosomal anomalies were most frequently observed in chromosomes 13 (21.1%), 14 (20.1%), and 1 (16.3%) with 13q12, 14q12, and 1p36.3 being the most prevalent breakpoints, respectively. Chromosome 1 contributed the most to reciprocal translocations (20.2%) and inversions (17.6%), while chromosome 14 was the most involved in the Robertsonian translocations (82.2%). The findings suggested that breakpoints at 1p36.3 and 14q12 might be associated with pregestational infertility, whereas breakpoints at 13q12 could be linked to both gestational and pregestational infertility. Several candidate genes located on common breakpoints were proposed as potentially involved in male infertility. Bioinformatics analyses utilizing three databases were conducted to examine the expression patterns of 78 candidate genes implicated in various causes of infertility. In azoospermic individuals, significant differential expression was observed in 19 genes: 15 were downregulated (TSSK2, SPINK2, TSSK4, CDY1, CFAP70, BPY2, BTG4, FKBP6, PPP2R1B, SPECC1L, CENPJ, SKA3, FGF9, NODAL, CLOCK), while four genes were upregulated ( HSPB1, MIF, PRF1, ENTPD6). In the case of Asthenozoospermia, seven genes showed significant upregulation (PRF1, DDX21, KIT, SRD5A3, MTCH1, DDX50, NODAL). Though RNA-seq data for Teratozoospermia were unavailable, microarray data revealed differential expression insix genes: three downregulated (BUB1, KLK4, PIWIL2) and three upregulated (AURKC, NPM2, RANBP2). These findings enhance our understanding of the molecular basis of male infertility and could provide valuable insights for future diagnostic and therapeutic strategies.

Comparative Epigenetic Analysis of Imprinting Genes Involved in Fertility, in Cryopreserved Human Sperms with Rapid Freezing versus Vitrification Methods.

OBJECTIVE: Choosing the optimal method for human sperm cryopreservation seems necessary to reduce cryoinjury. The aim of this study is to compare two cryopreservation methods including rapid-freezing and vitrification, in terms of cellular parameters, epigenetic patterns and expression of paternally imprinted genes (PAX8, PEG3 and RTL1) in human sperm which play a role in male fertility. MATERIALS AND METHODS: In this experimental study, semen samples were collected from 20 normozoospermic men. After washing the sperms, cellular parameters were investigated. DNA methylation and expression of genes were investigated using methylation-specific polymerase chain reaction (PCR) and real-time PCR methods, respectively. RESULTS: The results showed a significant decrease in sperm motility and viability, while a significant increase was observed in DNA fragmentation index of cryopreserved groups in comparison with the fresh group. Moreover, a significant reduction in sperm total motility (TM, P<0.01) and viability (P<0.01) was determined, whereas a significant increase was observed in DNA fragmentation index (P<0.05) of the vitrification group compared to the rapid-freezing group. Our results also showed a significant decrease in expression of PAX8, PEG3 and RTL1 genes in the cryopreserved groups compared to the fresh group. However, expression of PEG3 (P<0.01) and RTL1 (P<0.05) genes were reduced in the vitrification compared to the rapid-freezing group. Moreover, a significant increase in the percentage of PAX8, PEG3 and RTL1 methylation was detected in the rapid-freezing group (P<0.01, P<0.0001 and P<0.001, respectively) and vitrification group (P<0.01, P<0.0001 and P<0.0001, respectively) compared to the fresh group. Additionally, percentage of PEG3 and RTL1 methylation in the vitrification group was significantly increased (P<0.05 and P<0.05, respectively) compared to the rapid-freezing group. CONCLUSION: Our findings showed that rapid-freezing is more suitable method for maintaining sperm cell quality. In addition, due to the role of these genes in fertility, changes in their expression and epigenetic modification may affect fertility.

Upregulation of KAT2B and ESCO2 gene expression level in patients with rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune condition that causes progressive inflammation. It seems that alternations in epigenetic modifications contribute to RA development. The present study aimed to assess the expression pattern of K (lysine) acetyltransferase 1 (KAT1; HAT1) and lysine acetyltransferase 2B (KAT2B; PCAF), and the establishment of sister chromatid cohesion N-acetyltransferase 2 (ESCO2) in peripheral blood mononuclear cells (PBMCs) from RA patients. METHOD AND MATERIAL: In this case-control study, we studied 50 cases with RA in comparison to 50 age- and gender-matched healthy subjects. Separation of PBMCs samples from whole blood, extraction of RNA, and reverse transcription were performed. Gene transcript levels of KAT1, KAT2B, and ESCO2 were determined using SYBR green real-time quantitative PCR. RESULTS: Our results exhibited a significant upregulation in the expression levels of ESCO2 and KAT2B genes in patients with RA compared to normal individuals (P-value < 0.0001). Similarly, we observed higher expression of KAT1 in the patients' group when compared to the healthy controls, although the difference in expression level failed to show any significant changes (P-value = 0.485). Also, we found a positive correlation between ESCO2 and the level of erythrocyte sedimentation rate (ESR) in patients. CONCLUSION: Collectively, our results suggest that upregulated expression of KAT2B and ESCO2 genes may be correlated to RA development. Further studies with larger sample sizes are required for understanding the potential contribution of these enzymes in the pathology of RA. Key Points • Dysregulated expression level of epigenetics enzymes was observed in PBMCs from RA patients. • The expression of KAT2B was 2.44 times higher in the PBMCs of RA patients than in the healthy subjects. • The expression of ESCO2 was upregulated (2.75 times) in the PBMCs of RA patients compared to the control group. • There was a positive correlation between ESCO2 expression and the ESR level in patients.