Comparative Analysis of the Transcriptome and Methylome in Distinct Trophoblast Populations During Embryo Implantation in Mares

Background: During equine implantation, two distinct trophoblast subpopulations are identified: the invasive chorionic girdle (CG) and the non-invasive allantochorion. The invasive CG is believed to modulate the maternal immune response and facilitates embryo attachment to the endometrium, while the allantochorion contributes to placental development without breaching the endometrium. However, the mechanisms underlying differential adhesion and immune modulation by these two lineages remains poorly understood. This study aimed to investigate the regulation of biological processes underlying trophoblast attachment, development and maintenance of immune tolerance by comparing the transcriptomes and methylomes of CG and allantochorion trophoblasts before and after implantation, that is, between gestational days 33 and 42. Results: Irrespective of gestational day, allantochorion (n=4) was enriched in TGFB and its receptors TGFBR1 and TGFBR2. Across the implantation window, expression of regulatory macrophage markers such as IL33, IL4|1, PLG-RKT, and CD163 increased in the allantochorion, along with upregulation of TFEB and SERPINE1. The invasive GC (CG33, n=4) showed higher expression of NLRC5, a transcriptional regulator of MHC I, compared to the non-invasive allantochorion (ALC33, n=4). Notably, NLRC5 expression declined in the allantochorion from day 33 to 42. Furthermore, B2M, which encodes the MHC I light chain, was hypermethylated in the allantochorion. Several genes, including CAVIN1, LHB, and INSR, were both differentially expressed and methylated between the two trophoblast types, while others, such as IFNGR1, CD177, showed differential regulation across the implantation window. Gene Ontology and pathway analyses revealed that differentially expressed and methylated genes were enriched in biological processes and pathways critical for implantation, including cell migration and adhesion, TGFB signaling, fibrosis, wound healing, and leukocyte extravasation. Conclusions: The allantochorion plays an active role in establishing the attachment to the endometrium and sustaining maternal immune tolerance during implantation. The upregulation of IL33, IL4|1, and CD163 suggests the that the allantochorion may promote the recruitment or polarization of macrophages toward a regulatory phenotype, thereby modulating the local immune environment. TFEB may contribute to the steroidogenic activity and functional maturation of the allantochorion. Spatial and temporal differences in gene methylation between CG and allantochorion suggest involvement of epigenetic regulation in trophoblast invasion and attachment to the endometrium. Finally, combined B2M hypermethylation and differential expression of NLRC5 point to epigenetic and transcriptional control of MHC I expression in the equine trophoblasts during implantation. Overall design: Equine allantochorion (ALC) and chorionic girdle (CG) samples were collected with non-surgical uterine lavage at gestational days 33 (N ALC =4; N CG=4) and 42 (N=4) from physiologically progressing pregnancies. RNA was isolated from each sample using PureLink™ RNA Mini Kit (Ambion) with DNAse digestion between washing. Its quality and quantity were measured on a NanoDrop2000 and a TapeStation 2200 system (RNA tapes, Agilent). Acceptable samples had an RIN over 9.5. According to the manufacturer's protocol, cDNA libraries cells were prepared using the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina from 300 ng aliquots of purified RNA samples. The final cDNA library concentration was 10 nM. The quality and quantity of cDNA libraries was assessment using D1000 tapes on TapeStation2200 and on Quibit. The cDNA were sequenced over 150PE cycles on a HiSeq 3000 System (Illumina, San Diego, CA, USA) in outdoor facilities (Admera Health Biopharma Services).

研究背景：在马胚胎着床过程中，可鉴定出两种截然不同的滋养层亚群：侵袭性绒毛膜带（chorionic girdle, CG）与非侵袭性尿囊绒毛膜（allantochorion, ALC）。学界认为侵袭性CG可调控母体免疫应答，并促进胚胎附着于子宫内膜；而ALC则在不突破子宫内膜的前提下，参与胎盘发育。然而，这两种滋养层谱系在差异黏附与免疫调控方面的潜在机制仍有待阐明。本研究旨在通过比较着床前后（即妊娠第33天与第42天）CG与ALC滋养层细胞的转录组（transcriptome）与甲基化组（methylome），探究滋养层附着、发育以及免疫耐受维持相关生物学过程的调控机制。

研究结果：无论妊娠天数如何，ALC（n=4）均富集于转化生长因子β（transforming growth factor β, TGFB）及其受体TGFBR1、TGFBR2相关通路。在着床窗口期内，ALC中调节性巨噬细胞标志物（如IL33、IL4|1、PLG-RKT与CD163）的表达水平显著升高，同时TFEB与SERPINE1的表达也出现上调。与非侵袭性ALC（ALC33，n=4）相比，侵袭性CG（CG33，n=4）的主要组织相容性复合体I类（Major Histocompatibility Complex I, MHC I）转录调控因子NLRC5表达水平更高。值得注意的是，从妊娠第33天至第42天，ALC中NLRC5的表达水平逐渐下降。此外，编码MHC I类轻链的B2M在ALC中呈现高甲基化状态。在两种滋养层细胞之间，CAVIN1、LHB与INSR等多个基因同时存在差异表达与差异甲基化；而IFNGR1、CD177等其他基因则在着床窗口期内呈现差异调控。基因本体（Gene Ontology, GO）富集分析与通路分析显示，差异表达与差异甲基化的基因显著富集于与着床密切相关的生物学过程与通路，包括细胞迁移与黏附、TGFB信号通路、纤维化、伤口愈合以及白细胞渗出。

研究结论：ALC在着床过程中主动参与子宫内膜附着的建立，并维持母体免疫耐受。IL33、IL4|1与CD163的上调表达提示，ALC可能通过促进巨噬细胞向调节表型招募或极化，从而调控局部免疫微环境。TFEB可能参与ALC的类固醇生成活性与功能成熟过程。CG与ALC之间基因甲基化的时空差异表明，表观遗传调控参与了滋养层侵袭与子宫内膜附着过程。最后，B2M高甲基化与NLRC5差异表达的联合作用提示，马着床过程中滋养层细胞的MHC I类表达受到表观遗传与转录水平的双重调控。

实验设计：从生理状态正常的妊娠个体中，通过无创子宫灌洗法分别于妊娠第33天（ALC组n=4，CG组n=4）与第42天（各组n=4）采集马ALC与CG样本。使用PureLink™ RNA迷你提取试剂盒（Ambion）提取总RNA，并在清洗步骤中辅以DNA酶消化以去除基因组DNA；随后采用NanoDrop2000与TapeStation 2200系统（配套Agilent RNA胶带）检测RNA的质量与浓度，合格样本的RNA完整性数（RNA Integrity Number, RIN）需大于9.5。按照试剂盒说明书，以300ng纯化RNA为起始材料，使用VAHTS Universal V8 Illumina RNA测序文库制备试剂盒构建cDNA文库。最终cDNA文库浓度为10nM，采用TapeStation2200的D1000胶带与Quibit荧光定量系统检测文库的质量与浓度。随后在户外设施（Admera Health Biopharma Services）中，使用HiSeq 3000系统（Illumina，美国加利福尼亚州圣地亚哥）进行150bp双端循环测序。