van 't Hof Liseanne J, Kapsenberg Johanna M, Drabbels Jos J M, van der Meeren Lotte E, Roelen Dave L, Eikmans Michael, van der Hoorn Marie-Louise P
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands.
Department of Pathology, Leiden University Medical Center, Leiden, Netherlands.
Front Immunol. 2025 May 9;16:1575086. doi: 10.3389/fimmu.2025.1575086. eCollection 2025.
Maternal-fetal HLA compatibility influences pregnancy outcome, including preeclampsia risk. Cervical extravillous trophoblasts (EVT) in early pregnancy provide a non-invasive source for fetal genome acquisition, potentially enabling fetal HLA typing for obstetric risk assessment. This study aimed to achieve fetal HLA typing through EVT isolation using HLA-G-coupled nanoparticle immunomagnetic separation (TRIC) and fluorescence-activated cell sorting (FACS).
Cervical samples from 32 pregnant women were collected by cytobrush. Saliva and umbilical cord blood (n=13) served as maternal and fetal HLA genotype controls, respectively. Cervical samples from non-pregnant women, primary cultured EVT, and cryo-sectioned term placentas served as controls for cell phenotype, protein expression, and effect of fixation. FACS and TRIC were applied to isolate EVT from maternal cells, followed by RSSO-PCR for HLA typing. EVT presence pre- and post-isolation was determined through HLA-G, β-hCG, and Cytokeratin-7 (CK-7) expression. TRIC was optimized by improving antibody-binding-efficiency, and comparing three (nano)beads types and two magnets.
Purity and yield of HLA-Gβ-hCGCK-7 cells after TRIC failed to match pre-isolation HLA-G cell counts, despite protocol optimization. FACS revealed a fetal HLA genotype. In contrast, only the maternal HLA genotype was detected in TRIC-isolated cells.
EVT counts and maternal cell contamination limit reliable fetal HLA typing from cervical samples. Refining non-invasive EVT isolation techniques may enable fetal HLA typing to be included in risk assessment of pregnancy complications.
母胎人类白细胞抗原（HLA）相容性会影响妊娠结局，包括子痫前期风险。妊娠早期的宫颈外绒毛滋养层细胞（EVT）为获取胎儿基因组提供了一种非侵入性来源，有可能实现用于产科风险评估的胎儿HLA分型。本研究旨在通过使用HLA - G偶联纳米颗粒免疫磁珠分离法（TRIC）和荧光激活细胞分选法（FACS）分离EVT来实现胎儿HLA分型。
通过细胞刷收集32名孕妇的宫颈样本。唾液和脐带血（n = 13）分别作为母体和胎儿HLA基因型对照。来自非孕妇的宫颈样本、原代培养的EVT以及足月胎盘的冰冻切片用作细胞表型、蛋白质表达和固定效果的对照。应用FACS和TRIC从母体细胞中分离EVT，随后进行基于连接序列特异性寡核苷酸的聚合酶链反应（RSSO - PCR）进行HLA分型。通过HLA - G、β - 人绒毛膜促性腺激素（β - hCG）和细胞角蛋白 - 7（CK - 7）表达来确定分离前后EVT的存在情况。通过提高抗体结合效率、比较三种（纳米）磁珠类型和两种磁体对TRIC进行了优化。
尽管对方案进行了优化，但TRIC后HLA - Gβ - hCGCK - 7细胞的纯度和产量未能与分离前的HLA - G细胞计数相匹配。FACS揭示了胎儿HLA基因型。相比之下，在TRIC分离的细胞中仅检测到母体HLA基因型。
EVT计数和母体细胞污染限制了从宫颈样本中进行可靠的胎儿HLA分型。完善非侵入性EVT分离技术可能使胎儿HLA分型能够纳入妊娠并发症的风险评估中。
