Minor DNA methylation changes are observed in spermatozoa prepared using different protocols

Abstract

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 with 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 = .335) and of spermatozoa with fragmented DNA of patients with RRBS analysis (22.9 ± 21.1% vs. 34.4 ± 21.2; P = .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.

Potential implications of sperm DNA methylation functional properties in aquaculture management

“…[11][12][13][14][15][16] The evident correlation of these phenomena opened new questions regarding the impact of assisted reproductive techniques (ART) with particular consideration to the possible negative impact of ARTs in human reproduction, and also on the potential incidence of developmental and phenotypic abnormalities in ART-conceived children. [17][18][19] Similar concerns have been also equally recognized in other model organisms such as reproduction of livestock, poultry or fish. 8,20,21 In fish with external fertilization, variations in the properties of the aquatic environment, where the sperm and oocyte meet upon fertilization, not only influence gamete activation and fertilization success, but also may cause another degree of variations to their epigenome that eventually alter the phenotype of the resultant offspring.…”

“…Seemingly non‐hereditary environmental factors and lifestyle of an individual are reflected in the DNA methylation pattern of the sperm genome that has shown to result in phenotypic changes in the next generations 11–16 . The evident correlation of these phenomena opened new questions regarding the impact of assisted reproductive techniques (ART) with particular consideration to the possible negative impact of ARTs in human reproduction, and also on the potential incidence of developmental and phenotypic abnormalities in ART‐conceived children 17–19 . Similar concerns have been also equally recognized in other model organisms such as reproduction of livestock, poultry or fish 8,20,21 …”

Association between Sperm Morphology and Altered Sperm microRNA Expression

“…Twenty-five women underwent a short antagonist protocol, while three women underwent a long agonist protocol for ovarian hyperstimulation. Both protocols were conducted as described previously [ 20 ]. Male patients were invited to participate in the study based on their diagnostic spermiograms.…”