Department of Human Reproduction, Division of Gynaecology, University Medical Center Ljubljana, Ljubljana, Slovenia.
Andrology. 2020 Sep;8(5):1312-1323. doi: 10.1111/andr.12832. Epub 2020 Jun 21.
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.
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.
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.
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.
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.
DNA 甲基化模式可显示跨代遗传，并受生活方式和环境因素的影响。有人提出，这些模式可以通过辅助生殖技术改变。
评估两种不同的精子制备方法（常规密度梯度离心（DGC）与非凋亡精子的 DGC 后磁激活细胞分选（MACS））对精子 DNA 甲基化谱的影响。
我们分析了纳入我们的体外受精治疗计划的患者的精液。每位患者的一半精液用 DGC 方法进行 ICIS 准备，另一半用 DGC 后 MACS 准备。其余样本用降低代表性亚硫酸氢盐测序（RRBS）进行 DNA 甲基化分析。除了 DNA 甲基化谱外，我们还评估了精子的形态和 DNA 碎片化。
RRBS 分析显示，两组（DGC 与 MACS 组）的平均全基因组甲基化水平相似，范围在 0.53 到 0.56 之间。此外，RRBS 分析鉴定出 99 个差异甲基化区域（DMR）和 800 个差异甲基化位置（DMP）。在 DGC 组中，与 MACS 组相比，有 43 个 DMR 和 392 个 DMP 呈高甲基化，而有 56 个 DMR 和 408 个 DMP 呈低甲基化。当 DMR 和 DMP 被注释到基因时，发现了 3 个与印迹相关的基因：IGF2、PRDM16 和 CLF4/BRUNOL4。RRBS 分析中形态正常精子的百分比（MACS 与 DGC；14.0±10.8 与 13.2±10.0；P=.335）和具有碎片化 DNA 的精子百分比（22.9±21.1%与 34.4±21.2%；P=.529）在两组之间也相似。
尽管两组样本的全基因组 DNA 甲基化平均水平相似，但 DMR 和 DMP 水平的甲基化变化数量明显不同。应该分析更多的样本，并测试其他精子制备方法来证实我们的发现。
