Sperm DNA methylome abnormalities occur both pre- and post-treatment in men with Hodgkin disease and testicular cancer [methylation array]

Background: Combination chemotherapy has contributed to increased survival from Hodgkin disease (HD) and testicular cancer (TC). However, questions concerning the quality of spermatozoa after treatment have arisen. While studies have shown evidence of DNA damage and aneuploidy in spermatozoa years following anticancer treatment, the sperm epigenome has received little attention. Our objectives here were to determine the impact of HD and TC, as well as their treatments, on sperm DNA methylation. Semen samples were collected from community controls (CC) and from men undergoing treatment for HD or TC, both before initiation of chemotherapy and at multiple times post-treatment. Sperm DNA methylation was assessed using genome-wide and locus-specific approaches. Results: Imprinted gene methylation was not affected in the sperm of HD or TC men, before or after treatment. Prior to treatment, using Illumina HumanMethylation450 BeadChip (450K) arrays, a subset of 500 probes was able to distinguish sperm samples from TC, HD and CC subjects; differences between groups persisted post-treatment. Comparing altered sperm methylation between HD or TC patients versus CC men, twice as many sites were affected in TC versus HD men; for both groups, the most affected CpGs were hypomethylated. For TC patients, the promoter region of GDF2 contained the largest region of differential methylation. To assess alterations in DNA methylation over time/post-chemotherapy, serial samples from individual patients were compared. With restriction landmark genome scanning and 450K array analyses, some patients who underwent chemotherapy showed increased alterations in DNA methylation, up to two to three years post-treatment, when compared to the CC cohort. Similarly, a higher resolution human sperm-specific assay that includes assessment of environmentally-sensitive regions, or “dynamic sites”, also demonstrated persistently altered sperm DNA methylation in cancer patients post-treatment and suggested preferential susceptibility of “dynamic” CpG sites. Conclusions: Distinct sperm DNA methylation signatures were present pre-treatment in men with HD and TC and may help explain increases in birth defects reported in recent clinical studies. Epigenetic defects in spermatozoa of some cancer survivors were evident even up to two years post-treatment. Abnormalities in the sperm epigenome both pre- and post-chemotherapy may contribute to detrimental effects on future reproductive health. Sperm samples were collected from Community Control (CC. n=7), Hodgkin Disease (HD, n=7) and Testicular Cancer (TC, n=6) subjects. Baseline samples were taken prior to the initial of chemotherapy regimens (time 0). Post-treatment samples were collected at 6, 12, 18, 24, 36, 42 month after chemotherapy sessions concluded (times 1,2,3,4,5,6,7). Due to drop-out and/or lack of sperm production post-therapy, not all patients were able to provide post-treatment samples. DNA methylation of sperm DNA was measured using the Illumina HumanMethylation450 BeadChip (platform GPL13534) and a subset was further examined using a custom 5-methyl-cytosine capture sequencing (MCC-Seq)

背景：联合化疗已显著提升霍奇金病（Hodgkin Disease, HD）与睾丸癌（Testicular Cancer, TC）患者的生存率。然而，治疗后精子细胞的质量问题逐渐受到关注。既往研究已证实，抗癌治疗数年后的精子中存在DNA损伤与非整倍体现象，但精子表观组的相关研究仍较为匮乏。本研究旨在明确HD、TC本身及其治疗方案对精子DNA甲基化的影响。研究收集了社区对照（Community Controls, CC）人群以及接受HD或TC治疗的男性的精液样本，采样时间涵盖化疗开始前，以及化疗结束后的多个时间节点。采用全基因组及位点特异性分析方法，对精子DNA甲基化水平进行检测。
结果：HD或TC患者无论治疗前后，其精子中的印记基因甲基化水平均未受影响。化疗前，通过Illumina HumanMethylation450 BeadChip（450K芯片）阵列检测发现，500个探针组成的子集可有效区分TC、HD患者与CC人群的精子样本；且该组间差异在治疗后仍持续存在。对比HD、TC患者与CC人群的精子甲基化改变情况，TC患者受影响的甲基化位点数量约为HD患者的2倍；两组中受影响最显著的CpG位点均呈现低甲基化状态。在TC患者中，GDF2基因的启动子区域存在最大范围的差异甲基化。为评估化疗后不同时间点的DNA甲基化变化，研究对同一患者的系列样本进行了比对分析。通过限制性地标基因组扫描与450K芯片阵列分析，与CC队列相比，部分接受化疗的患者在治疗后2~3年内仍可检测到不断增加的DNA甲基化异常。同样，一种分辨率更高的人类精子特异性检测方法（可评估环境敏感区域即“动态位点”）也证实，癌症患者治疗后精子DNA甲基化持续存在异常，并提示“动态”CpG位点具有更高的易感性。
结论：HD与TC患者在治疗前即存在独特的精子DNA甲基化特征，该特征或可解释近期临床研究中报道的出生缺陷发生率升高现象。部分癌症幸存者在治疗后长达2年内，其精子中仍可检测到表观遗传缺陷。化疗前后精子表观组的异常，或可对未来生殖健康产生不良影响。
本研究共收集样本如下：社区对照（CC）7例、霍奇金病（HD）患者7例、睾丸癌（TC）患者6例的精液样本。基线样本采集于化疗方案开始前（时间点0）。治疗后样本采集于化疗结束后的6、12、18、24、36、42个月（对应时间点1至7）。由于部分患者失访或治疗后出现精子生成障碍，并非所有患者均能提供治疗后样本。精子DNA甲基化水平通过Illumina HumanMethylation450 BeadChip（平台编号GPL13534）进行检测，其中部分样本进一步采用定制化5-甲基胞嘧啶捕获测序（5-methyl-cytosine capture sequencing, MCC-Seq）进行分析。