Study question: How does magnetic-activated cell sorting (MACS) of non-apoptotic spermatozoa change the DNA-methylation profile of spermatozoa compared to spermatozoa prepared only by density gradient centrifugation (DGC)? Summary answer: Reduced Representation Bisulfite Sequencing revealed 99 differentially methylated regions (DMRs) and 800 differentially methylated positions (DMPs) between MACS selected and DGC-only prepared spermatozoa. What is known already: MACS procedure enables the selection of non-apoptotic spermatozoa with improved quality, especially in term of DNA fragmentation. Studies have shown that using such spermatozoa in ICSI/IVF procedures can improve fertilization rate, embryo/blastocyst quality (especially in women aged over 30 years) and pregnancy rate. Despite known positive effect for selection of spermatozoa with lower DNA fragmentation other influences of MACS on sperm genetic/epigenetic status are not known. Epigenetic status (especially methylation) becomes recently an important subject of interest, because it was shown that also epigenetic information can be transmitted transgenerational without changing the information on DNA level. Study design, size, duration: Our prospective sibling-oocyte study included 19 couples who were treated in 2018 with ICSI due to male infertility, more precisely due to teratozoospermia (defined by strict Kruger criteria). The women in the couple included in the study were not older than 36 years and were required to have at least 6 mature MII oocytes after controlled ovarian hyperstimulation. Half of MII oocytes were fertilized using spermatozoa prepared only with DGC and half with MACS. Participants/materials, setting, methods: MACS was performed after DGC using MACS® ART Annexin V System (Miltenyi). The remaining spermatozoa that were not used for ICSI, were evaluated for morphology, DNA fragmentation (HaloSperm) and stored in liquid nitrogen for epigenetic status in term of methylation using Reduced Representation Bisulfite Sequencing (RRBS). To detect differentially methylated regions (DMRs) and positions (DMPs) between DGC and MACS samples a two-dimensional Kolmogorov-Smirnov test (for DMRs) and Mann-Whitney U test (for DMPs) were used. Main results and the role of chance: Of all stored samples, samples from 7 patients were used for RRBS analysis, of which 5 were successfully analysed. The comparison of MACS-selected and DGC-only prepared spermatozoa of these 5 patients showed that the percentage of morphologically normal spermatozoa (14.0±10.8 vs. 13.2±10.0;P=0.335) and of spermatozoa with fragmented DNA (39.4±14.8 vs. 47.3±22.8;P=0.183) were similar between the groups, although both of DNA-fragmentation indexes were abnormal. Fertilization rate and the quality of embryos was not significantly different, although there was a trend towards higher blastocyst rate in MACS-group (25.0% vs. 52.6%;P=0.097). The RRBS analysis identified 99 DMRs and 800 DMPs. 43 DMRs and 392 DMPs were hypermethylated in DGC-group, while 56 DMRs and 408 DMPs were hypomethylated. When DMRs were annotated to genes, 8.8% of them were annotated to promoters, 11.8% to exons, 33.0% to introns and 46.4 to intergenic regions. In case of DMPs, 5.3% were annotated to promoters, 5.4% to exons, 39.3% to introns and the rest to intergenic regions. When DMRs were annotated to CpGs, 16.1% were annotated to CpG islands, 6.5% to CpG shores and 5.0% to CpG shelves, while in case of DMPs, 5.3% were annotated to CpG islands, 18.0% to CpG shores and 7.1% to CpG shelves. Limitations, reasons for caution: Due to protamine the DNA in spermatozoa is highly condensed, which makes RRBS analysis difficult and can negatively influence the quality of obtained sequencing data. Higher number of samples should be analysed to confirm obtained results. Wider implications of the findings: The sperm DNA methylation profile could be used as additional test to assess the quality of spermatozoa. It could be al
COBISS.SI-ID: 6618540
Background-Aim: Epigenetic changes are normal and are needed in terms of enabling the cells with same genetic code to perform different tasks in tissues and organs. But recent advancements in epigenetic studies suggest that also epigenetic informations can be transmitted through the germline and persist in subsequent generations. Since epigenetic changes can be influenced by environment, life style (e.g. smoking), age or even medicins, this raises a question if medically assisted reproduction could also have similar impact. For this reason we decided to study one type of epigenetic changes in sperm and this is DNA methylation, which when present, directly blocks DNA transcription. It was already shown before, that sperm DNA methylation pattern can be correlated to sperm quality, but there is little known how sperm preparation methods can influence it. Therefore we checked what is the sperm DNA methylation pattern after widely used density gradient centrifugation (DGC) compared to DGC followed with magnetic-activated cell sorting (MACS). Material&Methods: We analyzed the semen of patients, which were included in our IVF treatment programm. Half of the semen was prepared for ICSI using DGC method and other half with DGC followed by MACS (MACS® ART Annexin V System, Miltenyi). After performing ICSI (using sibling oocyte approach) the remaining of the samples was processed for DNA methylation analysis. DNA methylation was determined using reduced representation bisulfite sequencing of a whole genome. To detect significantly methylated positions, a Mann-Whitney test was applied to obtain p-values. P-values under 0.05 were recognized as being significant. Results: The analysis revealed 800 differentially methlyated positions, of which 392 were hypermethylated and 408 hypomethylated in DGC samples when compared to DGC+MACS samples. Of hypermethylated positions, there were several which were previously connected to reproduction/infertility, for instance the most interesting genes annotated to these positions were IGF2, UBE2W, ISG15, HIF3A, NOTCH1, FOXD1, CD24. Similar stands also for hypomethylated positions where PRDM16, ILF2, IFNGR2, FOXL1, AGTPBP1, GPM6A, PDGFA were annotated to these positions. There were identified also several other differentially methlyated positions, which annotated genes are associated with health problems, such as GNB1, PTDSS2, CDH12, NEUROG1 and BCAP31. Conclusions:Our study revealed that sperm epigenetic status in term of methylation pattern can be influenced by the sperm preparation method. Concerns are raised because some of differentially methylated position were annotated to genes important in reproduction/infertility and even to genes associated with health problems. The limitation of the study is low number of included patients (5). More patients should be included to confirm the study findings.
COBISS.SI-ID: 6621612
Background DNA methylation patterns can show transgenerational inheritance and are influenced by lifestyle and environmental factors. It is suggested that these patterns can be changed by assisted reproductive technology. Objectives To evaluate the impact of two different sperm preparation methods, conventional density gradient centrifugation (DGC) vs. density gradient centrifugation followed by magnetic-activated cell sorting (MACS) of non-apoptotic spermatozoa, on sperm DNA methylation profile. Materials and methods We analyzed semen of patients included in our IVF treatment program. Half of the semen from each included patient was prepared for ICSI using the DGC method and the other half with DGC followed by MACS. The remaining samples were processed for DNA methylation analysis with reduced representation bisulfite sequencing (RRBS). In addition to the DNA methylation profile, we assessed the morphology and DNA fragmentation of spermatozoa. Results RRBS analysis revealed that the average genome-wide methylation level was similar between both groups (DGC vs. MACS group) and ranged from 0.53 to 0.56. Furthermore, RRBS analysis identified 99 differentially methylated regions (DMRs) and 800 differentially methylated positions (DMPs). In the DGC group, 43 DMRs and 392 DMPs were hypermethylated whereas 56 DMRs and 408 DMPs were hypomethylated compared to those in the MACS group. When DMRs and DMPs were annotated to genes, 3 genes associated with imprinting were found: IGF2, PRDM16, and CLF4/BRUNOL4. The percentage of morphologically normal spermatozoa (MACS vs. DGC; 14.0±10.8 vs. 13.2±10.0;P=0.335) and of spermatozoa with fragmented DNA of patients with RRBS analysis (22.9±21.1% vs. 34.4±21.2;P=0.529) were also similar between groups. Discussion and Conclusion Although the average genome-wide level of sperm DNA methylation was similar in both sample groups, a distinctive number of methylation changes were observed in DMR and DMP levels. A larger number of samples should be analyzed and additional sperm preparation methods should be tested to confirm our findings.