van Vliet Marjolein M, Boers Ruben G, Boers Joachim B, Schäffers Olivier J M, van der Meeren Lotte E, Gribnau Joost, Schoenmakers Sam, Steegers-Theunissen Régine P M
Department of Obstetrics and Gynecology, Erasmus MC, Rotterdam, The Netherlands.
Department of Developmental Biology, Erasmus MC, Rotterdam, The Netherlands.
Mol Hum Reprod. 2025 Apr 3;31(2). doi: 10.1093/molehr/gaaf011.
Placental DNA methylation varies across gestation and is associated with obstetrical complications. Cell-free DNA (cfDNA) from maternal plasma could provide a noninvasive approach to study placental DNA methylation in ongoing pregnancies. However, research on maternal cfDNA methylation is limited and technologically challenging. Therefore, we aimed to investigate DNA methylation in maternal cfDNA and placental tissues across gestation using the innovative methylation DNA sequencing (MeD-seq) technology. Secondly, we explored the origins of methylation differences in maternal cfDNA across gestation, and aimed to identify gestational age-associated placental DNA methylation markers directly in cfDNA. We longitudinally collected maternal cfDNA in all three trimesters and at birth (n = 10), alongside placental tissues from first trimester, second trimester, and term pregnancies (all n = 10), and used previously collected maternal blood buffy coat samples (n = 20). Different placental cell types, including syncytiotrophoblasts/cytotrophoblasts (SCTs/CTBs) (n = 10), extravillous trophoblasts (n = 7), and syncytial knotting (n = 3), and maternal cell types including spiral arteries (n = 3) and endometrial epithelium (n = 3), were isolated using laser capture microdissection. Differentially methylated regions (DMRs) identified in cfDNA from pregnant compared to non-pregnant women (n = 6) ranged from 798 to 2163 in first and third trimesters, respectively. Gradual DNA methylation changes were observed across gestation in cfDNA, placental tissues, and trophoblasts. We showed an increase in DMRs in cfDNA, that overlap with DNA methylation in placental tissues and especially trophoblasts, and in DNA methylation of placenta-specific markers across gestation, reflecting an increased placental-originated cfDNA fraction. Among 110 DMRs between first trimester and term placental tissues, those related to NXPH4, EPS8L2, AMOTL1, and IRX2 had the strongest association with gestational age in cfDNA, for which comparable associations were found in SCTs/CTBs. These DMRs were all hypomethylated in maternal buffy coat samples. This study indicates the feasibility of identifying gestational age-dependent placental DNA methylation marks in maternal cfDNA and can serve as a reference for future studies.
胎盘DNA甲基化在整个孕期会有所不同，并且与产科并发症相关。来自母体血浆的游离DNA（cfDNA）可为研究正在进行的妊娠中的胎盘DNA甲基化提供一种非侵入性方法。然而，关于母体cfDNA甲基化的研究有限且在技术上具有挑战性。因此，我们旨在使用创新的甲基化DNA测序（MeD-seq）技术研究整个孕期母体cfDNA和胎盘组织中的DNA甲基化。其次，我们探讨了整个孕期母体cfDNA甲基化差异的来源，并旨在直接在cfDNA中鉴定与孕周相关的胎盘DNA甲基化标记物。我们在所有三个孕期以及分娩时纵向收集了母体cfDNA（n = 10），同时收集了孕早期、孕中期和足月妊娠的胎盘组织（均n = 10），并使用了之前收集的母体血液白膜层样本（n = 20）。使用激光捕获显微切割技术分离了不同的胎盘细胞类型，包括合体滋养层细胞/细胞滋养层细胞（SCTs/CTBs）（n = 10）、绒毛外滋养层细胞（n = 7）和合体结（n = 3），以及母体细胞类型，包括螺旋动脉（n = 3）和子宫内膜上皮（n = 3）。与未怀孕女性（n = 6）相比，在孕早期和孕晚期从孕妇cfDNA中鉴定出的差异甲基化区域（DMRs）分别为798至2163个。在cfDNA、胎盘组织和滋养层细胞中观察到整个孕期DNA甲基化的逐渐变化。我们发现cfDNA中的DMRs增加，这些DMRs与胎盘组织尤其是滋养层细胞中的DNA甲基化以及整个孕期胎盘特异性标记物的DNA甲基化重叠，这反映了源自胎盘的cfDNA比例增加。在孕早期和足月胎盘组织之间的110个DMRs中，与NXPH4、EPS8L2、AMOTL1和IRX2相关的DMRs在cfDNA中与孕周的关联最强，在SCTs/CTBs中也发现了类似的关联。这些DMRs在母体白膜层样本中均为低甲基化。这项研究表明在母体cfDNA中鉴定与孕周相关依赖的胎盘DNA甲基化标记物是可行的，并可为未来的研究提供参考。
