Epimutations in human sperm from patients with impaired spermatogenesis,Clinical Epigenetics

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Epimutations in human sperm from patients with impaired spermatogenesis
Clinical Epigenetics ( IF 4.8 ) Pub Date : 2020-11-17 , DOI: 10.1186/s13148-020-00919-0
Joana Marques , Filipa Carvalho , Alberto Barros , Mário Sousa

In this Letter to the Editor, we comment on the recent publication by Leitão et al. The sperm epigenome does not display recurrent epimutations in patients with severely impaired spermatogenesis (Clinical Epigenetics 2020, DOI: 10.1186/s13148-020-00854-0), where concerns about the validity of our studies reporting imprinting errors in human sperm from infertile patients have been raised.

We read with great interest this recent publication describing genome-wide methylation in human sperm from oligozoospermic patients, which could be an important resource to understand the extent of methylation defects and whether these are restricted to imprinted genes.

However, we were surprised to find that the Authors attributed imprinting methylation errors to contamination of somatic cells and even stated that they “suspect that other studies also suffer from DNA contamination issues”.

Thereby, we would like to clarify that, in our studies, in order to eliminate somatic cell contamination, we have prepared sperm by density gradient centrifugation (DGC) (using density gradients) prior to swim-up separation, as we previously described [1, 2]. The sequential method of DGC swim-up is the elective method for sperm preparation to be used in assisted reproductive treatments (ART) [3]. Additionally, we visually inspected, by optical inverted microscopy with Hoffman modulation contrast, a 20-μl droplet of all the samples that were included in our studies, before using sperm samples in experiments.

We noticed that in the study by Leitão and collaborators [4], density gradient separation was not performed and direct swim-up was employed, thereby increasing the likelihood of having somatic cells contamination in their sperm samples, as is suggestive from the author´s WGBS data, including for H19 methylation values (ranging from 75 to 82% methylation in normal controls in their study vs 95% in our study [2]. For density gradient centrifugation, we used Puresperm gradients (Nidacon, Gothenburg, Sweden) which contain silane-coated silica particles that enable motile sperm to be separated from non-germinal cells and seminal plasma. The advantages of the density gradient method is the attainment of an excellent yield of highly motile normal spermatozoa, whereas leukocytes, bacteria, epithelial cells, cell debris, and most abnormal sperm are eliminated, while sperm DNA fragmentation and reactive oxygen species are significantly reduced [3, 5]. In fact, our team has more than 20 years of experience in preparing semen samples for ICSI and to date sperm samples obtained after gradient centrifugation followed by swim-up were never found to be contaminated by leukocytes and, in ICSI, purified sperm are selected individually.

Moreover, in our studies conducted on testicular sperm, that were individually isolated by micromanipulation from testicular biopsies [6, 7], and therefore not contaminated with somatic cells, we have also observed imprinting errors, namely H19 DMR hypomethylation and MEST DMR hypermethylation.

Nevertheless, we agree with the Authors that it is necessary to reassure patients undergoing IVF treatments and our works contributed to this purpose by showing a very low frequency of sperm with completely altered methylation patterns, in infertile patients.

Furthermore, in most of the cases presented in our results, the patient producing sperm with imprinting errors also carried sperm with correct methylation patterns. In the future, we hope to understand how these imprinting errors occur, in order to contribute to improve the safety and efficacy of assisted reproduction techniques.

Elsa Leitão,
Sara Di Persio,
Sandra Laurentino,
Marius Wöste,
Martin Dugas,
Sabine Kliesch,
Nina Neuhaus &
Bernhard Horsthemke

Correspondence: Bernhard.horsthemke@uni-due.de

We appreciate the comments by Marques et al. and their efforts to purify spermatozoa, which go far beyond what most researchers in this field do. Most of the previous studies were performed on swim-up samples, and we have found that samples prepared in this way can be contaminated with somatic DNA. Of course, we could not reanalyze the samples that were used by others, but our lack of finding any evidence for an epimutation in 93 sperm samples, which make our study one of the largest in its field, justifies our conclusion that “the prevalence of aberrant methylation in swim-up purified sperm of infertile men has likely been overestimated” [4]. Irrespective of the purification procedure used, the DNA should always be prescreened for somatic DNA contamination by bisulfite sequencing of at least the four genes suggested in our paper (H19, MEST, DDX4, and XIST) or the four genomic loci suggested by Jenkins et al. [8].

Unfortunately, Marques et al. have not considered genetic variation as another confounder in this type of studies. As shown by us, a common genetic variant at the H19 locus is associated with DNA hypomethylation, which should not be confused with an infertility-associated epimutation [4].

Not applicable.

DGC:: Density gradient centrifugation
ART:: Assisted reproductive treatments
WGBS:: Whole-genome bisulfite sequencing
ICSI:: Intracytoplasmic sperm injection
DMR:: Differentially methylated region
IVF:: In vitro fertilization

1.
Marques CJ, Carvalho F, Sousa M, Barros A. Genomic imprinting in disruptive spermatogenesis. Lancet. 2004;363(9422):1700–2.
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2.
Marques CJ, Costa P, Vaz B, Carvalho F, Fernandes S, Barros A, et al. Abnormal methylation of imprinted genes in human sperm is associated with oligozoospermia. Mol Hum Reprod. 2008;14(2):67–74.
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3.
Bucar S, Goncalves A, Rocha E, Barros A, Sousa M, Sa R. DNA fragmentation in human sperm after magnetic-activated cell sorting. J Assist Reprod Genet. 2015;32(1):147–54.
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4.
Leitao E, Di Persio S, Laurentino S, Woste M, Dugas M, Kliesch S, et al. The sperm epigenome does not display recurrent epimutations in patients with severely impaired spermatogenesis. Clin Epigenetics. 2020;12(1):61.
CAS Article Google Scholar
5.
Henkel RR, Schill WB. Sperm preparation for ART. Reprod Biol Endocrinol. 2003;1:108.
Article Google Scholar
6.
Marques CJ, Francisco T, Sousa S, Carvalho F, Barros A, Sousa M. Methylation defects of imprinted genes in human testicular spermatozoa. Fertil Steril. 2010;94(2):585–94.
CAS Article Google Scholar
7.
Marques PI, Fernandes S, Carvalho F, Barros A, Sousa M, Marques CJ. DNA methylation imprinting errors in spermatogenic cells from maturation arrest azoospermic patients. Andrology. 2017;5(3):451–9.
CAS Article Google Scholar
8.
Jenkins TG, Liu L, Aston KI, Carrell DT. Pre-screening method for somatic cell contamination in human sperm epigenetic studies. Syst Biol Reprod Med. 2018;64:146–55.
CAS Article Google Scholar

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CJM is funded by the Portuguese Foundation for Science and Technology (FCT) with a salary contract (IF/00047/2012; CEECIND/00371/2017).

Affiliations

Department of Genetics, Faculty of Medicine, University of Porto (FMUP), 4200-319, Porto, Portugal
Joana Marques, Filipa Carvalho & Alberto Barros
i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135, Porto, Portugal
Joana Marques, Filipa Carvalho & Alberto Barros
Centre for Reproductive Genetics A Barros, 4100-009, Porto, Portugal
Alberto Barros & Mário Sousa
Department of Microscopy, Laboratory of Cell Biology, Multidisciplinary Unit for Biomedical Research-UMIB, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Rua Jorge Viterbo Ferreira, 228, 4099-003, Porto, Portugal
Mário Sousa

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Joana MarquesView author publications
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Filipa CarvalhoView author publications
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Alberto BarrosView author publications
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Mário SousaView author publications
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Contributions

CJM, FC, AB, and MS wrote, read, and approved the final manuscript.

Corresponding author

Correspondence to Joana Marques.

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The authors declare that they have no competing interests.

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Marques, J., Carvalho, F., Barros, A. et al. Epimutations in human sperm from patients with impaired spermatogenesis. Clin Epigenet 12, 172 (2020). https://doi.org/10.1186/s13148-020-00919-0

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Received: 01 July 2020
Accepted: 17 August 2020
Published: 17 November 2020
DOI: https://doi.org/10.1186/s13148-020-00919-0

中文翻译：

精子发生受损的人的精子表位变异

在这封致编辑的信中，我们评论了Leitão等人的最新出版物。在精子发生严重受损的患者中，精子表观基因组未显示出反复的突变（Clinical Epigenetics 2020，DOI：10.1186 / s13148-020-00854-0），其中关于我们研究报告不育患者的精子印迹错误的有效性的担忧被提出。

我们非常感兴趣地阅读了这份最近的出版物，该出版物描述了少精症患者精子中全基因组甲基化的情况，这可能是了解甲基化缺陷程度以及这些缺陷是否仅限于印迹基因的重要资源。

但是，我们惊讶地发现作者将印迹甲基化错误归因于体细胞的污染，甚至说他们“怀疑其他研究也遭受DNA污染问题”。

因此，我们想澄清的是，在我们的研究中，为了消除体细胞污染，我们在游泳分离之前通过密度梯度离心（DGC）（使用密度梯度）制备了精子，如我们先前所述[1 ，2]。DGC游泳的顺序方法是用于辅助生殖治疗（ART）的精子制备的选择性方法[3]。此外，在实验中使用精子样本之前，我们先通过光学倒置显微镜和霍夫曼调制对比对20μl液滴进行了目视检查，这些液滴包括在我们的研究中。

我们注意到，在Leitão和合作者的研究中[4]，未进行密度梯度分离，而是采用直接游泳法，从而增加了精子样品中体细胞被污染的可能性，这是作者的暗示。 WGBS数据，包括针对H19的数据甲基化值（正常研究中甲基化为75％到82％，而我们的研究中为95％[2]。对于密度梯度离心，我们使用Puresperm梯度（Nidacon，哥德堡，瑞典），其中包含硅烷包覆的二氧化硅颗粒，使活动精子与非生殖细胞和精浆分离，密度梯度法的优势是获得了高活动性的正常精子，而白细胞，细菌，上皮细胞，细胞碎片和大多数异常精子的出众率消除，而精子DNA片段化和活性氧种类显着减少[3，5]。我们的团队在为ICSI制备精液样品方面拥有20多年的经验，迄今为止，从未发现白细胞会在梯度离心，随后游泳之后获得的精子样品被白细胞污染，并且在ICSI中，单独选择纯化的精子。

此外，在我们对睾丸精子进行的研究中，通过显微操作从睾丸活检组织中单独分离它们[6，7]，因此没有被体细胞污染，我们还观察到了印记错误，即H19 DMR低甲基化和MEST DMR高甲基化。

然而，我们同意作者的观点，有必要使接受IVF治疗的患者放心，我们的工作通过在不育患者中显示极低的精子频率和完全甲基化的模式，为实现这一目标做出了贡献。

此外，在我们结果中显示的大多数情况下，产生带有印迹错误精子的患者也带有正确甲基化模式的精子。将来，我们希望了解这些印记错误是如何发生的，从而有助于提高辅助复制技术的安全性和有效性。

ElsaLeitão，
萨拉·迪·佩尔西奥（Sara Di Persio），
Sandra Laurentino，
MariusWöste，
马丁·杜加斯
萨宾·凯里什（Sabine Kliesch），
妮娜·纽豪斯（Nina Neuhaus）
伯恩哈德·霍瑟姆克

通讯：Bernhard.horsthemke@uni-due.de

我们感谢Marques等人的评论。以及他们纯化精子的努力，这远远超出了该领域大多数研究人员的能力。以前的大多数研究都是对游动样本进行的，我们发现以这种方式制备的样本可能会被体液DNA污染。当然，我们无法重新分析其他人使用的样本，但是我们没有找到93个精子样本发生突变的任何证据，这使我们的研究成为该领域最大的研究之一，证明了我们的结论：“不育男性精制后的精子中的异常甲基化可能被高估了[4]。无论使用哪种纯化方法，都应始终通过亚硫酸氢盐测序至少对我们论文中建议的四个基因进行DNA筛查，以检测是否被体液DNA污染（H19，MEST，DDX4和XIST）或Jenkins等人建议的四个基因组位点。[8]。

不幸的是，Marques等。尚未将遗传变异视为此类研究的另一个混杂因素。正如我们所显示的，H19基因座的常见遗传变异与DNA低甲基化有关，不应与不育相关的基因突变相混淆[4]。

不适用。

DGC：: 密度梯度离心
艺术：: 辅助生殖治疗
WGBS：: 全基因组亚硫酸氢盐测序
ICSI：: 胞浆内精子注射
DMR：: 差异甲基化区域
试管婴儿: 体外受精

1。
Marques CJ，Carvalho F，Sousa M，Barros A.破坏性精子发生中的基因组印迹柳叶刀。2004; 363（9422）：1700-2。
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2。
Marques CJ，Costa P，Vaz B，Carvalho F，Fernandes S，Barros A等。人类精子中印迹基因的异常甲基化与少精症有关。摩尔嗡嗡声。2008; 14（2）：67-74。
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Bucar S，Goncalves A，Rocha E，Barros A，Sousa M，Sa R.磁激活细胞分选后人类精子中的DNA片段化。J助教基因。2015; 32（1）：147-54。
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4。
Leitao E，Di Persio S，Laurentino S，Woste M，Dugas M，Kliesch S等。在精子发生严重受损的患者中，精子表观基因组不会再出现突变。临床表观遗传学。2020; 12（1）：61。
CAS文章Google学术搜索
5，
汉高RR，Schill WB。精子准备艺术。生殖生物学内分泌。2003; 1：108。
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6。
Marques CJ，Francisco T，Sousa S，Carvalho F，Barros A，Sousa M.人类睾丸精子印迹基因的甲基化缺陷。Fertil Steril。2010; 94（2）：585–94。
CAS文章Google学术搜索
7。
侯爵PI，费尔南德斯S，卡瓦略F，巴罗斯A，索萨M，侯爵CJ。来自成熟的精子细胞中的DNA甲基化印迹错误会阻止无精子症患者。男科学。2017; 5（3）：451–9。
CAS文章Google学术搜索
8。
詹金斯TG，刘L，阿斯顿KI，卡雷尔DT。人类精子表观遗传研究中体细胞污染的预筛选方法。Syst Biol Reprod Med。2018; 64：146–55。
CAS文章Google学术搜索

下载参考

不适用。

CJM由葡萄牙科学技术基金会（FCT）资助并签订了工资合同（IF / 00047/2012; CEECIND / 00371/2017）。

隶属关系

波尔图大学医学院遗传学系（FMUP），葡萄牙波尔图4200-319
Joana Marques，Filipa Carvalho和Alberto Barros
i3S-波尔图大学，葡萄牙波尔图大学和Inovação研究所，葡萄牙，波尔图，4200-135
Joana Marques，Filipa Carvalho和Alberto Barros
生殖遗传学中心A Barros，4100-009，葡萄牙波尔图
Alberto Barros和MárioSousa
波尔图大学显微镜生物学系，细胞生物学实验室，生物医学研究多学科单位-UMIB，生物医学科学研究所Abel Salazar（ICBAS），鲁阿·豪尔赫·维泰博·费雷拉（228），4099-003，葡萄牙波尔图
马里奥·索萨（MárioSousa）

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