Methylation: An Ineluctable Biochemical and Physiological Process Essential to the Transmission of Life
甲基化：一种不可避免的生化和生理过程，对生命传递至关重要

1.1. A Reminder of the Methylation Process
1.1. 甲基化过程的提醒

S-adenosyl methionine (SAM) is the universal cofactor for methylation as the methyl group donor (Figure 1). Methionine adenosyltransferase (MAT) catalyzes SAM formation by linking methionine and ATP. Methylation is then carried out by DNA methyltransferase and histone methyltransferase enzymes. After the targets have been methylated, S-adenosyl homocysteine (SAH) is formed, and homocysteine (HCY) is then released. Hcy must be recycled: it is a toxic metabolite that inhibits the methylation process and can also inactivate some proteins via homocysteinilation, which leads to structural modifications. The one-carbon cycle is the major cellular pathway that recycles Hcy, together with methionine synthase associated with the folate cycle (see Figure 1). Methyl tetrahydrofolate (MTHF) is the active compound for folate cycle methionine synthase activity, and correct methylation depends upon a supply of MTHF. Some types of cells can also recycle Hcy via the cystine beta synthase (CBS) pathway, releasing cysteine. A third mechanism, available primarily in liver cells but only marginal in other cells, uses the betaine homocysteine pathway. Cysteine plays an important role in the redox homeostasis that is necessary for correct methylation, and errors in methylation are heavily linked to unbalanced oxidative stress. Abnormally low/inadequate concentrations of methionine and cystine may have an effect on epigenetic processes, and mechanisms for cellular transport and/or synthesis of methionine, cystine, glycine, and glutamic acid are crucial in supporting correct methylation. The availability of these compounds for direct (methionine) or indirect (via the synthesis of glutathione) participation in methylation as well as protection of the process are essential for correct establishment and maintenance of epigenetic and imprinting methyl tags.
S-腺苷甲硫氨酸（SAM）是甲基化的通用辅因子，作为甲基供体（图 1）。蛋氨酸腺苷转移酶（MAT）通过连接蛋氨酸和 ATP 催化 SAM 的形成。然后，DNA 甲基转移酶和组蛋白甲基转移酶酶催化甲基化。目标被甲基化后，形成 S-腺苷高半胱氨酸（SAH），然后释放出高半胱氨酸（HCY）。HCY 必须被回收：它是一种有毒代谢物，会抑制甲基化过程，并且还可以通过高半胱氨酸化作用使一些蛋白质失活，从而导致结构改变。一碳循环是主要的细胞途径，用于回收 HCY，与叶酸循环相关的蛋氨酸合酶一起（见图 1）。甲基四氢叶酸（MTHF）是叶酸循环蛋氨酸合酶活性的活性化合物，正确的甲基化依赖于 MTHF 的供应。一些类型的细胞还可以通过半胱氨酸β合酶（CBS）途径回收 HCY，释放半胱氨酸。第三种机制主要在肝细胞中可用，但在其他细胞中仅边缘可用，使用甜菜碱高半胱氨酸途径。 半胱氨酸在氧化还原平衡中起着重要作用，这对于正确的甲基化是必要的，而甲基化错误与不平衡的氧化应激密切相关。甲硫氨酸和半胱氨酸浓度异常低/不足可能会对表观遗传过程产生影响，细胞内甲硫氨酸、半胱氨酸、甘氨酸和谷氨酸的运输和/或合成机制对于支持正确的甲基化至关重要。这些化合物的可用性，无论是直接参与甲硫氨酸还是通过谷胱甘肽的合成间接参与甲基化，以及对过程的保护，对于正确建立和维持表观遗传和印记甲基标记至关重要。

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Figure 1 图 1

The one-carbon cycle (1-CC) and the folates cycle (MTHFR: methyltetrahydrofolate reductase, THF tetrahydrofolate). CBS: Cystathionine beta synthase, CoQ10: Coenzyme Q10, CH3: methyl group, DHFR: Dihydrofolate reductase, MS: methionine synthase, SAM S Adenosyl Methionine, SAH: S Adenosyl Homocysteine, Zn: Zinc.
单碳循环（1-CC）和叶酸循环（MTHFR：甲基四氢叶酸还原酶、THF：四氢叶酸）。CBS：半胱氨酸β合成酶、CoQ10：辅酶 Q10、CH3：甲基基团、DHFR：二氢叶酸还原酶、MS：甲硫氨酸合成酶、SAM：S 腺苷甲硫氨酸、SAH：S 腺苷同型半胱氨酸、Zn：锌。

1.2. The Beginning of Life: Fertilization and Immediately Postfertilization
1.2. 生命的开始：受精和受精后立即阶段

During oogenesis, the oocyte accumulates epigenetic marks both on histones and on DNA, and this process continues until just before ovulation. At the time of fertilization, major intermediate metabolic changes are immediately initiated, which are necessary to support correct epigenetic/methylation status. All of the regulatory processes are strictly dependent upon maternal reserves of proteins and messenger RNAs (mRNAs) stored during oocyte growth; no transcription takes place in the newly fertilized egg until the zygote genome is activated (maternal to zygotic transition, MZT) at the 4- to 8-cell stages in human embryos. Interference RNAs (iRNAs) play an important role in regulating the translation of stored polyadenylated mRNAs.
在卵母细胞发生过程中，卵母细胞在组蛋白和 DNA 上积累表观遗传标记，这个过程一直持续到排卵前。在受精时，立即开始重要的中间代谢变化，这些变化对于支持正确的表观遗传/甲基化状态是必要的。所有的调控过程严格依赖于卵母细胞生长期间储存的蛋白质和信使 RNA（mRNA）的母源储备；在人类胚胎的 4-8 细胞阶段，新受精卵中没有转录发生，直到启动合子基因组（母源到合子转变，MZT）。干扰 RNA（iRNA）在调控储存的多腺苷酸化 mRNA 的翻译中起着重要作用。

Upregulation of the pentose phosphate pathway permits the formation of NADPH, essential for glutathione (GSH) synthesis: γ-L-Glutamyl-L-cysteinylglycine is a universal antioxidant molecule. GSH is necessary for sperm head swelling by opening the protamines that padlock the DNA and in order to protect the methylation process and to maintain redox homeostasis in order to prevent errors in methylation linked to unbalanced oxidative stress. As mentioned previously, abnormally low/inadequate concentrations of methionine and cystine may affect epigenetic processes [2]. Glutaredoxins are red-ox enzymes, classified as “light proteins”, that use glutathione as a cofactor. These proteins are thiol-disulfide oxidoreductases with a glutathione-binding site and one or two active cysteines in their active site. They can reduce methionine sulfone (oxidized methionine) to active methionine. Glutaredoxins are oxidized by oxidized substrates and are nonenzymatically reduced by reduced glutathione, which in turn is oxidized but can be regenerated by glutathione reductase (GRX). In terms of physio-biochemistry, glutaredoxins are present and highly expressed in early embryos: they actively protect redox homeostasis and thus have an impact on imprinting processes. Hypotaurine (Htau) is also found in the in vivo embryonic environment as an efficient antioxidant synthesized by oviductal cells and released into the tubal lumen [3,4]
提高戊糖磷酸途径的活性可以产生 NADPH，这对谷胱甘肽（GSH）的合成至关重要：γ-L-谷氨酰-L-半胱氨酸甘氨酸是一种普遍的抗氧化分子。GSH 对于通过打开锁住 DNA 的精蛋白来使精子头部肿胀是必要的，同时也为了保护甲基化过程并维持氧化还原平衡，以防止与不平衡的氧化应激相关的甲基化错误。正如之前提到的，甲硫氨酸和半胱氨酸浓度异常低/不足可能会影响表观遗传过程[2]。谷胱氧还蛋白是一类被归类为“轻质蛋白”的氧化还原酶，它们使用谷胱甘肽作为辅因子。这些蛋白质是硫醇-二硫键氧化还原酶，在其活性位点上具有一个谷胱甘肽结合位点和一个或两个活性半胱氨酸。它们可以将甲硫氨酸磺酸盐（氧化甲硫氨酸）还原为活性甲硫氨酸。谷胱氧还蛋白被氧化底物氧化，而被还原的谷胱甘肽则通过谷胱甘肽还原酶（GRX）再生。 从生理生化角度来看，谷胱氧还蛋白在早期胚胎中存在并高度表达：它们积极保护氧化还原平衡，从而对印记过程产生影响。盐酸甜菜碱（Htau）也存在于体内胚胎环境中，是由输卵管细胞合成并释放到输卵管腔中的高效抗氧化剂[3, 4]

Oocytes and early embryos have active mechanisms for cellular transport and/or synthesis of methionine, cystine, glycine, and glutamic acid. SAM is actively synthesized, and all of the enzymes involved in the methylation process are present, including the methionine synthase pathway that regenerates methionine from Hcy. The expression of CBS pathway enzymes is weak or absent, which means that homocysteine cannot be recycled to cystine, reinforcing the requirement for cystine and methionine. The betaine homocysteine pathway (BHMT) is only marginally represented. Human oocytes express high levels of folate receptor 1 and folate transporter1 (SLC19A1), indicating that these molecules play an important role during the first three to four days of development prior to the onset of genomic activation, MZT. Folates are central to a system that involves high molecular trafficking [5], and all of the enzymes involved in the folate and 1-carbon cycles are highly expressed in the oocyte. The embryo also finds important metabolites in its tubal fluid environment.
卵母细胞和早期胚胎具有细胞运输和/或甲硫氨酸、半胱氨酸、甘氨酸和谷氨酸的合成活性机制。 SAM 被积极合成，并且涉及甲基化过程的所有酶都存在，包括能够从 Hcy 再生甲硫氨酸的甲硫氨酸合成途径酶。CBS 途径酶的表达很弱或不存在，这意味着同型半胱氨酸不能被回收为半胱氨酸，进一步强化了对半胱氨酸和甲硫氨酸的需求。甜菜碱同型半胱氨酸途径（BHMT）仅略有代表。人卵母细胞高表达叶酸受体 1 和叶酸转运蛋白 1（SLC19A1），表明这些分子在基因组激活、MZT 之前的前三至四天的发育过程中起重要作用。叶酸是涉及高分子运输的系统的核心[5]，卵母细胞中涉及叶酸和 1 个碳循环的所有酶都高表达。胚胎也在其输卵管液环境中寻找重要的代谢产物。

A major upheaval occurs during and immediately after the fertilization period: the DNA of the zygote genome is thought to be rapidly de-methylated immediately postfertilization, and human in vitro fertilized IVF embryos apparently follow this scheme. High sperm methylation and retained methylation of the paternal genome represent major markers of fertility [6,7,8]. The observation that isoforms affecting the one-carbon and folate cycles have a negative impact on fertility confirms the importance of a high methylation status in sperm. Paternal effects on early embryonic development is a feature that is well documented in bovine embryos. Suboptimal sperm methylation in terms of “quality and quantity” (CpG island regions restricted to retained histones) has a negative impact on human embryo blastocyst development [9] Approximately 10–15% of histones are retained, but these are not randomly located: they bind to the “active part/coding area” of DNA and so play an important role in embryonic development [6,9].
受精期间和受精后立即发生了一次重大动荡：认为合子基因组的 DNA 在受精后立即迅速去甲基化，人类体外受精的试管婴儿显然也遵循这个模式。高度的精子甲基化和父源基因组的保留甲基化是生育力的主要标志[6, 7, 8]。影响一碳和叶酸循环的异构体对生育力的负面影响证实了精子高甲基化状态的重要性。父源对早期胚胎发育的影响是牛胚胎中有充分证据的特征。精子甲基化的“质量和数量”不佳（CpG 岛区域仅限于保留的组蛋白）对人类胚胎囊胚发育有负面影响[9]。大约有 10-15%的组蛋白被保留下来，但它们并不是随机分布的：它们结合到 DNA 的“活跃部分/编码区域”，在胚胎发育中起重要作用[6, 9]。

As mentioned earlier, global methylation in the sperm nucleus (DNA and histones) provides the key for correct spatial and biochemical conformation that will allow rapid access to the paternal genome after nuclear swelling in the sperm head [7,8]. This feature is mandatory for rapid S-phase activation and the major methylation/epigenetic modifications that will modify the male genome. Minor epigenetic alterations in sperm will immediately affect the early stages of preimplantation embryo developmental capacity. The paternal genome is rapidly demethylated initially, whereas the maternal genome is passively demethylated slowly during the subsequent cell cycle. The majority of the epigenetic methyl tags should be erased during de-methylation, but those that are retained and transmitted to the offspring probably transmit important epigenetic and imprint information. In human embryos, just prior to and during the second half of the pronuclear stage and before entering the first mitotic division, demethylated paternal DNA is immediately re-methylated, together with de novo H3-K9 tri-methylation [10]. A similar feature is observed in mouse, rabbit, and pig embryos. Oocytes have a significant endogenous pool of polyadenylated mRNAs coding for DNMT1 (DNA methyltransferase1), which is responsible for maintenance of methylation [3,5,11,12].
如前所述，精子核（DNA 和组蛋白）中的全局甲基化为正确的空间和生化构象提供了关键，这将在精子头部核肿胀后允许对父本基因组的快速访问[7, 8]。这个特征对于快速的 S 期激活和将修改男性基因组的主要甲基化/表观遗传修饰是必需的。精子中的微小表观遗传改变将立即影响到早期胚胎发育能力的阶段。父本基因组最初会迅速去甲基化，而母本基因组则在随后的细胞周期中缓慢地被动去甲基化。在去甲基化过程中，大部分表观遗传甲基标记应该被抹去，但那些保留并传递给后代的标记可能传递了重要的表观遗传和印记信息。在人类胚胎中，在第一次有丝分裂之前和第二个卵核阶段的后半段以及进入第一次有丝分裂之前，去甲基化的父本 DNA 会立即重新甲基化，同时进行 de novo H3-K9 三甲基化[10]。在小鼠、兔子和猪胚胎中也观察到类似的特征。 卵母细胞具有大量内源性的编码 DNMT1（DNA 甲基转移酶 1）的多聚腺苷酸化 mRNA，负责维持甲基化。[3, 5, 11, 12]

Global demethylation during preimplantation embryo development excludes imprinted genes: the parental imprints must be protected by DNMT1. The expression of DNMT3, responsible for de novo methylation, is weaker.
全球去甲基化在植入前胚胎发育过程中排除了印记基因：父母的印记必须由 DNMT1 保护。负责新甲基化的 DNMT3 的表达较弱。

Two major issues must be highlighted: maternal age and the environment and the significant genetic impact of mutations in the one-carbon and folate cycles [13,14,15] The negative impact of abnormal methylation on bovine blastocyst formation has been documented [16]. Methylene tétrahydrofolate reductase (MTHFR) knockout decreases the number of blastocysts obtained, and those remaining have fewer cells in both the inner cell mass and the trophectoderm. Older females have a reduced abundance of DNA methyltransferases in MII oocytes, as the majority of important oocyte mRNAS are involved in regulation of homeostasis (redox, intermediate metabolism, etc.). Incorrect regulation of DNA methylation will further lead to inappropriate gene expression [17].
必须强调两个重要问题：母亲年龄和环境，以及一碳和叶酸循环中突变的显著遗传影响[13, 14, 15]。已经有文献记录了异常甲基化对牛胚胎囊胚形成的负面影响[16]。甲烷四氢叶酸还原酶（MTHFR）基因敲除会减少获得的囊胚数量，并且剩下的囊胚内细胞团和滋养层细胞数量较少。年龄较大的雌性在 MII 卵母细胞中 DNA 甲基转移酶的丰度较低，因为大部分重要的卵母细胞 mRNA 参与了稳态调节（氧化还原、中间代谢等）。DNA 甲基化的错误调节将进一步导致基因表达不当[17]。

1.3. Problems Associated with Assisted Reproductive Technology
1.3. 辅助生殖技术相关问题

Several publications have indicated that babies born as a result of IVF procedures have different methylation patterns to those conceived naturally [13,18,19,20,21] and this is not related to intrinsic characteristics of the gametes or to the etiology of male and/or female infertility [2]. Controlled ovarian hyperstimulation (COH) is known to have a borderline negative effect, but there may be effects on implantation, placentation, and ultimately perinatal outcome [13,17]. Suboptimal in vitro culture conditions may have a negative impact on regulatory processes. A recent paper suggested that a rapid drop in methylation can be observed in human embryos shortly after fertilization [22]. This is based on data obtained from IVF embryos and may be an artifact of culture conditions [2,11,23]. Under natural conditions, the methylation status is stabilized by maintenance of methylation. Two recent papers have highlighted a significant stumbling block, in that there is no in vivo control for human embryos. A high incidence of four major imprinting disorders was reported in Japanese babies born after assisted reproduction technologies ART [19], and a second publication describes the risk of ART-imprinted disorders that are linked to epimutations [20]. Both reports suggest that these disorders may originate in the period immediately following fertilization under culture conditions in current use [20]. The possible source of these problems has been clearly advocated [2,11,23,24]; firstly, controlled ovarian stimulation increases the level of homocysteine (Hcy) in follicular fluid [25]. Hcy competes with methionine for the same amino acid transporter [26], which leads to abnormal accumulation of Homocysteine in the oocyte. Secondly, current culture media do not support correct maintenance of methylation, even in mouse embryos that have passed the “standard” Mouse Embryo Assay (MEA) for toxicity [24]. The majority of culture media lack both methyl donors and protection against methylation anomalies induced by oxidative stress. Methionine and cysteine are absent in some media [2,27]. Adding oviduct fluid to the medium might overcome these problems, but inclusion of methionine and cystine to support the one-carbon cycle could also provide a solution. Moreover, current culture media generate free radicals spontaneously [28]; these data highlight the fact that brief perturbation of the DNA methylation maintenance process in early stage embryos can influence development. In fact, DNA methylation, measured as the quantity of methylated CpG per embryo, does not decrease significantly until the time of maternal to zygotic transition and immediately after [29] (Figure 2). Errors in methylation may further impact placental function [30]. Phospholipid formation, another biochemical parameter linked to methylation, is also impaired by IVF and could affect the health of offspring [31].
有几份出版物指出，通过体外受精（IVF）程序出生的婴儿与自然受孕的婴儿具有不同的甲基化模式[13, 18, 19, 20, 21]，这与配子的内在特性或男性和/或女性不育的病因无关[2]。已知控制性卵巢过度刺激（COH）具有边缘负面影响，但可能会对植入、胎盘形成和最终围产期结果产生影响[13, 17]。不理想的体外培养条件可能对调控过程产生负面影响。最近的一篇论文提出，在受精后不久，人类胚胎的甲基化水平会迅速下降[22]。这是基于从 IVF 胚胎获得的数据，可能是培养条件的人为因素[2, 11, 23]。在自然条件下，甲基化状态通过甲基化的维持得到稳定。最近的两篇论文强调了一个重要障碍，即对人类胚胎没有体内控制。 辅助生殖技术（ART）后出生的日本婴儿中报告了高发病率的四种主要印记障碍[19]，第二篇文章描述了与 ART 印记障碍相关的表观突变的风险[20]。这两份报告都表明，这些障碍可能源于目前使用的培养条件下受精后的即时时期[20]。这些问题的可能来源已经得到明确提出[2, 11, 23, 24]；首先，控制性卵巢刺激会增加卵泡液中同型半胱氨酸（Hcy）的水平[25]。Hcy 与蛋氨酸竞争同一种氨基酸转运体[26]，导致卵母细胞中同型半胱氨酸异常积累。其次，目前的培养基无法正确维持甲基化，即使是通过了“标准”小鼠胚胎毒性试验（MEA）的小鼠胚胎[24]。大多数培养基既缺乏甲基供体，也缺乏对由氧化应激引起的甲基化异常的保护。某些培养基中缺乏蛋氨酸和半胱氨酸[2, 27]。 将输卵管液添加到培养基中可能能够克服这些问题，但是添加蛋氨酸和半胱氨酸以支持一碳循环也可能提供解决方案。此外，目前的培养基会自发产生自由基[28]；这些数据突显了早期胚胎 DNA 甲基化维持过程的短暂干扰可能会影响发育。事实上，DNA 甲基化（以每个胚胎甲基化 CpG 的数量来衡量）直到母源到合子转变之后和紧接着的时期才显著减少[29]（图 2）。甲基化错误可能进一步影响胎盘功能[30]。磷脂形成，另一个与甲基化相关的生化参数，也受到体外受精的影响，可能会影响后代的健康[31]。

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Figure 2 图 2

DNA methyl transferase activity in the mouse preimplantation embryo [32]. Yellow: caryogames, Blue: Ethanol activated parthenogenotes, Pink: Calcium ionophore activated parthenogenots.
小鼠囊胚中的 DNA 甲基转移酶活性[32]。黄色：有核配子体，蓝色：乙醇激活的孤雌生殖体，粉色：钙离子载体激活的孤雌生殖体。

1.4. Blastocyst Formation, Implantation, and Placental Development
胚胎囊形成、着床和胎盘发育

Initial differentiation of cells in the inner cell mass and trophectoderm is accompanied by differential methylation. Maternal DNA methylation regulates early trophoblast development [33], and miscarriages may be attributed to errors in differential methylation. Methylation that accompanies zygotic genomic imprinting affects further placental development [34]. In the male, DNA methylation has an effect on trophoblastic function in general, and methylation errors will therefore have a negative impact [35]. Defective methylation affects overall trophoblast development and its physiological capacity to sustain implantation and, subsequently, placental development and fetal growth [33,36].
内细胞团和滋养层细胞的初步分化伴随着差异性甲基化。母源 DNA 甲基化调节早期滋养层发育[33]，流产可能归因于差异性甲基化错误。伴随着合子基因组印记的甲基化影响进一步的胎盘发育[34]。在男性中，DNA 甲基化对滋养层功能有影响，因此甲基化错误将产生负面影响[35]。缺陷的甲基化影响整体滋养层发育及其维持着着床和随后的胎盘发育和胎儿生长的生理能力[33, 36]。

Trophoblastic tissue remains hypomethylated relative to embryonic tissues. Alterations in the cellular adhesion profile of trophoblast cells are regulated by methylation of maternal DNA [33]. Imprinting appears to be particularly important both for placental and for embryo development; the development of extraembryonic tissues is under paternal control, whereas embryo growth is directed by maternal genes. For this reason, although cloning by duplication of pronuclear paternal or maternal genomes is possible, it can never progress to a viable outcome, even if implantation takes place. Mouse androgenotes display hypertrophy of the placenta (placentomegaly), and the opposite is observed in gynogenesis. The “sex conflict hypothesis” could be linked to imprinting mechanisms: The maternal goal is to not invest all of her resources in a single offspring and to limit fetal use of maternal resources. The paternal goal is to maximize the size of an individual by maximizing fetal use of maternal resources. However, numerous exceptions to this hypothesis are evident, as demonstrated by IGF2/H19 expression. Paternally expressed IGF2, which is highly expressed in normal mouse and human placenta, regulates placental growth via growth factor activity and nutrient permeability. The gene for maternally expressed H19 lies on the same chromosome as the IGF2 gene, and it allows the synthesis of a 2.3 Kb noncoding RNA (NcRNA). Hypermethylation of the H19 promoter on the paternal allele allows for expression of the paternal allele for IGF2 and decreases (represses) H19 expression. IGF2 drives placental growth as well as the capacity to transfer nutrients to the fetus Maternally expressed H19 genes suppress growth, and expression of H19 is continuously regulated (biallelic to monoallelic and reverse) throughout embryonic development. However, anomalies linked to aberrant placental IGF2 methylation lead to fetal growth restriction [37]. The balance between IGF2 and H19 expression is of major importance for viable fetal gestation, and monoallelic expression can occur in the absence of imprinting.
滋养层组织相对于胚胎组织保持低甲基化状态。滋养层细胞的细胞粘附特性的改变受到母源 DNA 甲基化的调控[33]。印记对胎盘和胚胎发育都特别重要；胚胎外组织的发育受到父源控制，而胚胎生长则由母源基因指导。因此，尽管通过复制父源或母源的原核核体进行克隆是可能的，但即使发生着床，也永远无法进展到可行的结果。小鼠雄性核胚显示胎盘肥大（胎盘肥大症），而雌性核胚则相反。"性冲突假说"可能与印记机制有关：母亲的目标是不将所有资源投入到单个后代，并限制胎儿对母体资源的利用。父亲的目标是通过最大化胎儿对母体资源的利用来最大化个体的大小。然而，IGF2/H19 表达所示，这个假说有许多例外情况。 父源表达的 IGF2 在正常的小鼠和人类胎盘中高度表达，通过生长因子活性和营养渗透调节胎盘生长。母源表达的 H19 基因位于与 IGF2 基因相同的染色体上，它允许合成一个 2.3 Kb 的非编码 RNA（NcRNA）。父源等位基因上 H19 启动子的高甲基化使得父源等位基因表达 IGF2，并降低（抑制）H19 的表达。IGF2 推动胎盘生长以及向胎儿传递营养的能力。母源表达的 H19 基因抑制生长，并且 H19 的表达在胚胎发育过程中持续调控（双等位基因到单等位基因再到反向）。然而，与异常胎盘 IGF2 甲基化相关的异常导致胎儿生长受限[37]。IGF2 和 H19 表达之间的平衡对于胎儿的可行性妊娠非常重要，而单等位基因表达可以在没有印记的情况下发生。

The placenta is the most hypomethylated DNA in human tissue, and DNA methylation increases specifically with gestational age [38], coupled to a high demand for methionine in the third trimester of pregnancy [39]. A striking observation confirms the link between elevated circulating homocysteine, oxidative stress, and methylation [40,41,42]. Homocysteine is both a cause and a consequence of oxidative stress, which can lead to pre-eclampsia [43] A further observation has shown that the oxygen load delivered during parturition in order to stabilize preterm babies affects their methylome. It is not known whether the process is reversible, and the authors [44] suggest that this could affect the response to oxidative stress, DNA repair, and cell progression. Ultimately, placental growth is intricately linked to DNA methylation [40], and correct homeostasis to allow effective methylation avoids adverse pregnancy outcomes such as pre-eclampsia [41,42,43,44,45,46].
胎盘是人体组织中 DNA 甲基化最低的部分，而 DNA 甲基化会随着妊娠年龄的增加而增加[38]，这与妊娠第三期对蛋氨酸的高需求有关[39]。一个引人注目的观察结果证实了循环高同型半胱氨酸、氧化应激和甲基化之间的联系[40, 41, 42]。同型半胱氨酸既是氧化应激的原因，也是其结果，可能导致子痫前期[43]。另外一个观察结果显示，在分娩过程中输送的氧气负荷会影响早产儿的甲基组。目前尚不清楚这个过程是否可逆，作者[44]认为这可能会影响对氧化应激、DNA 修复和细胞进展的反应。最终，胎盘生长与 DNA 甲基化密切相关[40]，通过维持正确的稳态以实现有效的甲基化，可以避免不良的妊娠结局，如子痫前期[41, 42, 43, 44, 45, 46]。

1.5. Embryo Growth, DNA Methylation, and Fetal Testis and Ovary
胚胎生长、DNA 甲基化和胎儿睾丸和卵巢

DNA methylation plays a major role throughout embryo growth; however, tracking the sequence of variations is very complex due to the fact that epigenetic alterations will modify both gene and promoter methylation, leading to epipolymorphism [41]. All of the methylation/epigenetic processes can be modified by the environment, and modifications can be transferred to the next generation via gametogenesis. This feature has now been clearly established [47,48,49]. The progeny of female rats injected with plastic endocrine disruptors during pregnancy shows severe pathologies for at least 3 generations, with altered germline epigenome transmission observed between generations. Experiments carried out with the yellow agouti mouse model demonstrated that the results involved methylation. The deleterious impact of endocrine disruptors and other xenogenic chemical compounds has been clearly demonstrated [42,47,48,49,50,51]. Supporting the one-carbon cycle with appropriate exogenous compounds can rescue normal gametogenesis, and thus, the impact appears to be clearly due to erratic methylation [51,52,53]. Fetal ovaries and testes are very sensitive to environmental perturbation, including food, air pollution, and nanoparticles. DNA methylation and gametogenesis are intricately linked due to the fact that primordial germ cells are profoundly demethylated and subsequently re-methylated during a later developmental period: prenatal life in males and postnatal development in females. For this reason, the environmental impact on the testis is more significant [54,55], as resetting of methylation may be impaired via toxicants carried by maternal blood and amniotic fluid. The fetal testis is a major target for endocrine disruptors such as herbicides, pesticides, and PolychloroBisphenyls PCBs, and determining whether the anomalies have occurred during fetal life or during the prepuberal period can be complicated. The effects can include inducing low sperm count, testicular cancer, cryptorchidism, undescended testis, ambiguous genitalia, and “testis dysgenesis syndrome (TDS)”: environmental exposure is the primary factor involved in the phenotypes associated with this syndrome. The adult endocrine system may be affected either directly or indirectly via epigenetic mechanisms. The genetic possibility of errors cannot be totally excluded, but the consensus is in favor of an environmental problem affecting epigenesis and methylation [2,47,48,49,54,55,56].
DNA 甲基化在胚胎发育过程中起着重要作用；然而，由于表观遗传改变会同时修改基因和启动子的甲基化，导致表观多态性的序列变化追踪非常复杂[41]。所有的甲基化/表观遗传过程都可以被环境改变，并且这些改变可以通过配子发生传递给下一代。这一特征现在已经得到明确确认[47, 48, 49]。在怀孕期间注射塑料内分泌干扰物的母鼠的后代至少连续 3 代出现严重病理学问题，且观察到代际间生殖细胞系表观基因组的改变。使用黄色斑点小鼠模型进行的实验表明，结果与甲基化有关。内分泌干扰物和其他异源化学物质的有害影响已经得到明确证明[42, 47, 48, 49, 50, 51]。通过适当的外源化合物支持一碳循环可以挽救正常的配子发生，因此，影响似乎明显是由于不规则的甲基化[51, 52, 53]。 胎儿的卵巢和睾丸对环境干扰非常敏感，包括食物、空气污染和纳米颗粒。由于原始生殖细胞在较晚的发育阶段（雄性为产前期，雌性为产后期）被深度去甲基化和重新甲基化，DNA 甲基化和配子发生密切相关。因此，对睾丸的环境影响更为显著[54, 55]，因为母血和羊水中携带的毒物可能会影响甲基化的重置。胎儿睾丸是内分泌干扰物（如除草剂、杀虫剂和多氯联苯 PCBs）的主要靶点，确定异常是在胎儿期还是在青春期前期发生的可能会很复杂。影响可能包括导致精子计数减少、睾丸癌、隐睾、睾丸下降不全、性别不明确和“睾丸发育异常综合征（TDS）”：与该综合征相关的表型主要受环境暴露影响。成年内分泌系统可能会直接或间接受到表观遗传机制的影响。 遗传错误的可能性不能完全排除，但共识是环境问题影响了表观遗传和甲基化[2, 47, 48, 49, 54, 55, 56]。

1.6. Methylation Errors in Gametogenesis, Postnatal Life, and Adults
1.6. 生殖细胞发生、出生后期和成年期的甲基化错误

Numerous aspects of modern lifestyle have contributed to an increase in life expectancy since World War II. However, we are now faced with a downturn that seems to be related to environmental issues. This paradigm parallels an increased prevalence of problems associated with fertility: decreased sperm quality, an increase in premature ovarian failure, and diminished ovarian reserve syndromes. The decline in male fertility is no longer a matter for debate and has been linked to the effect of environmental oxidative stress on methylation. As mentioned earlier, resetting of DNA methylation in germinal cells is initiated during fetal life and the fetal testis is more sensitive than the fetal ovary to exogenous (chemical) toxins carried in maternal blood. In women, the scarcity of material for study makes the situation difficult to analyze; damage arises over a short period of time during oocyte maturation, but the problems may start earlier, during “quiescent periods”, when the oocytes have no protection against exogenous hazards [57].
自二战以来，现代生活方式的许多方面都导致了预期寿命的增加。然而，我们现在面临的衰退似乎与环境问题有关。这种模式与与生育相关的问题的普遍增加相似：精子质量下降，早期卵巢功能衰竭增加，卵巢储备综合征减少。男性生育能力的下降已不再是一个争论的问题，并且已与环境氧化应激对甲基化的影响相关联。如前所述，胚胎期间始发的生殖细胞 DNA 甲基化重置在胎儿生活期间开始，胎儿睾丸对母体血液中的外源（化学）毒素更为敏感，而胎儿卵巢则较不敏感。在女性中，研究材料的稀缺使得情况难以分析；损害在卵母细胞成熟期间的短时间内产生，但问题可能在更早的“静止期”开始，此时卵母细胞对外源危害没有保护[57]。

1.7. Methylation Errors in the Male
男性中的甲基化错误

During spermatogenesis, DNA methylation is active during spermatogonial mitosis and meiosis and less active during spermiogenesis [58]. Histone methylation has an immediate effect during fertilization; it exerts a positive effect on the embryonic S-phase and on subsequent preimplantation development [7,8]. It is now recognized that pathologies can be transmitted across generations via epimutations that occur in adult gametogenesis [59,60,61,62,63]. Oxidative stress plays a pivotal role in this process, with glucose, lipids, and homocysteine as well as chemicals at the center of the problem. Diabetic patients have a high level of oxidative stress due to excess ROS generated by glucose metabolism at the level of the Krebs cycle (succinyl dehydrogenase) and inhibition of hypoxanthine-guanine phosophoribosyltransferase (HPRT). Although epigenetic disorders can be transmitted via sperm [6,60,63], ROS may also affect the primary sperm DNA structure directly via the formation of DNA adducts such as 8-oxo deoxyguanosine: all of the nuclear bases may be affected by oxidation, but guanine is the most sensitive. The oocyte has a redundant but finite capacity for DNA repair, which decreases with maternal age [57,64]. Interestingly, it has been shown that oxidized sperm also has an effect on epigenetic reprogramming of the preimplantation embryo [65]. Zygotic DNA repair machinery (base excision repair) is activated at the expense of active demethylation of paternal DNA, another unexpected observation that links oxidative stress to errors in methylation [55,56]. In obese patients, peroxidized lipids may oxidize sperm DNA and membranes. In the case of ART and intracytoplasmic sperm injection (ICSI) in particular, injected oxidized membrane lipids and DNA can induce oxidative stress (OS) in the oocyte. Since spermatogenesis is a continuous process, it is difficult to determine at what point the problems might begin [66]; fortunately, therapeutic treatments can easily cover a complete cycle of Tspermatogenesis [15,53,67] The genetic background of MTHFR SNPs (A1298C and C677T) significantly perturbs the sperm methylome [15,68]: patients who carry these polymorphisms are not able to metabolize synthetic folic acid, with accumulation of unmetabolized folic acid (UMFA). This must be taken into consideration to avoid further damage to the sperm methylome [68].
在精子发生过程中，DNA 甲基化在精原细胞有丝分裂和减数分裂期间活跃，而在精子发生期间活跃较少[58]。组蛋白甲基化在受精过程中具有即时效应；它对胚胎 S 期和随后的植入前发育产生积极影响[7, 8]。现在已经认识到，病理可以通过在成年配子发生过程中发生的表观突变代际传递[59, 60, 61, 62, 63]。氧化应激在这个过程中起着关键作用，葡萄糖、脂质和同型半胱氨酸以及化学物质是问题的核心。糖尿病患者由于在 Krebs 循环（琥珀酸脱氢酶）水平上由葡萄糖代谢产生的过量 ROS 和次黄嘌呤鸟苷酸磷酸核糖转移酶（HPRT）的抑制而具有高水平的氧化应激。尽管表观遗传紊乱可以通过精子传递[6, 60, 63]，但 ROS 也可能通过形成 DNA 加合物（如 8-氧脱氧鸟苷酸）直接影响原始精子 DNA 结构：所有核碱基都可能受到氧化的影响，但鸟嘌呤最为敏感。 卵母细胞具有冗余但有限的 DNA 修复能力，随着母亲年龄的增长而减少[57, 64]。有趣的是，研究表明氧化的精子也会影响着床前胚胎的表观遗传重编程[65]。配子的 DNA 修复机制（碱基切除修复）在代价是父系 DNA 的主动去甲基化的情况下被激活，这是另一个将氧化应激与甲基化错误联系起来的意外观察[55, 56]。在肥胖患者中，过氧化脂质可能会氧化精子的 DNA 和膜。在辅助生殖技术和特别是细胞内单精子注射（ICSI）的情况下，注射的氧化膜脂和 DNA 可以在卵母细胞中引起氧化应激（OS）。由于精子生成是一个连续的过程，很难确定问题可能从何时开始[66]；幸运的是，治疗方法可以轻松覆盖一个完整的精子生成周期[15, 53, 67]。MTHFR 基因的 SNP（A1298C 和 C677T）显著扰乱了精子的甲基组[15, 68]：携带这些多态性的患者无法代谢合成叶酸，导致未代谢叶酸（UMFA）的积累。 这必须考虑到，以避免对精子甲基组造成进一步的损害[68]。

Plastic endocrine disruptors (EDCs) exert a transgenerational effect: they induce oxidative stress (Figure 3) via the estrogen receptor (ER), proliferation of activated peroxisome receptors and constitutive androstane receptor (CAR), pregnan X receptor (PXR), and aryl carbon (Ah) receptors.
塑料内分泌干扰物（EDCs）具有代际效应：它们通过雌激素受体（ER）、活化过氧化物酶体受体和构成性雄烷受体（CAR）、孕烷 X 受体（PXR）和芳基碳（Ah）受体等诱导氧化应激（图 3）的作用。

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Figure 3 图 3

The link between oxidative stress and methylation disturbance: Xenobiotics (endocrine disruptors (EDCs)) interactions with some receptors.
氧化应激与甲基化紊乱之间的联系：外源物质（内分泌干扰物（EDCs））与某些受体的相互作用。

The underlying process has been elucidated and demonstrates an immediate impact on methylation [55]. When cytosine is oxidized at the level of differentially methylated regions (DMRs) in DNA, CpG islands, and/or imprinting control regions (ICRs), there is a risk of interference with regulatory processes by demethylation. Oxidative stress affects histone acetylation and de-acetylation [69], with indirect perturbation of “the epigenetic landscape”—methylation tags are transmitted to the offspring. In general, these posttranslational modifications (PTMs) act as good markers of sperm quality. Methylation occurs during spermatid elongation, and this is the most important PTM. The biochemical process is driven by methyltransferases that are specific to Arginine (R) or Lysine. (K), with S-AdenosylMethionine (SAM) as a co-effector. Decreased methylation on H3K will lead to disorganized spatiotemporal embryo development. These positioned methyl tags can regulate transcription by either repressing or increasing gene expression. The spermatozoon allows ancestral history to be transmitted via methylation, as demonstrated by the clear example of the 1944–1945 Dutch Famine. Individuals exposed prenatally to food shortage were found to have reduced methylation status of the imprinted IGF2 gene several decades later. Together with methylated DNA, methylated histones carry epigenetic and imprinting marks which will modulate phenotypic and biochemical variations. Sperm histones/DNA methylation should be considered as a means of transmitting acquired heredity, and this finely tuned system can be impaired by environmental factors.
潜在的过程已经被阐明，并且显示出对甲基化的直接影响[55]。当胞嘧啶在 DNA 的差异甲基化区域（DMRs）、CpG 岛和/或印记控制区域（ICRs）水平被氧化时，脱甲基化可能会干扰调控过程。氧化应激影响组蛋白乙酰化和去乙酰化[69]，间接干扰“表观遗传景观”，甲基化标记会传递给后代。总的来说，这些翻译后修饰（PTMs）作为精子质量的良好标志。甲基化发生在精子成熟过程中，这是最重要的 PTM。生化过程由特异于精氨酸（R）或赖氨酸（K）的甲基转移酶驱动，S-腺苷甲硫氨酸（SAM）作为辅助因子。H3K 上的甲基化减少将导致胚胎发育的时空混乱。这些定位的甲基标记可以通过抑制或增加基因表达来调节转录。精子通过甲基化传递祖先历史，这在 1944-1945 年荷兰饥荒的明显例子中得到了证明。 曝露于孕期食物短缺的个体在几十年后发现其印记 IGF2 基因的甲基化状态降低。甲基化的 DNA 与甲基化的组蛋白一起携带表观遗传和印记标记，这些标记将调节表型和生化变异。精子组蛋白/DNA 甲基化应被视为传递后天遗传的一种方式，而这个精细调节的系统可能会受到环境因素的损害。

Methylation anomalies, either hypomethylation or hypermethylation depending upon the target (genes or their promoters), may lead to testicular cancer, initiated from the period of gonadal development. However, as with other genital tract cancers, the true impact of methylation anomalies is not proven.
甲基化异常，无论是低甲基化还是高甲基化，取决于目标（基因或其启动子），可能导致睾丸癌，起源于性腺发育期。然而，与其他生殖道癌症一样，甲基化异常的真正影响尚未得到证实。