胎盘外胚层滋养细胞（EVTs）是胎盘形成过程中的关键细胞，主要功能是侵入母体脱落膜，将发育的胎盘固定在子宫上，重构子宫动脉，并调节免疫反应以防止排斥。早期妊娠期间，脱落膜经历低氧和酸性微环境，据显示这种环境参与了肿瘤细胞的迁移、侵袭、生长和血管生成。然而，EVTs如何感知和回应酸性微环境，从而执行其功能，目前尚未完全了解。通过使用JAR团块、小鼠囊胚和HTR-8/SVneo细胞，测试了G蛋白偶联受体65（GPR65）对细胞粘附和其他细胞功能的影响。具体而言，我们利用HTR-8/SVneo细胞进行基因过表达和沉默，以研究GPR65在酸性环境下对滋养细胞功能的作用的潜在机制。此外，利用早期妊娠损失患者获得的绒毛组织样本，探索了GPR65及其相关信号通路分子与该疾病的潜在关联。本研究发现GPR65在滋养细胞中广泛表达，其中在EVTs中含量最高。重要的是，维持正常的粘附、迁移和侵袭需要适度的GPR65水平，而GPR65过表达通过激活cAMP-ERK信号通路、上调肌球蛋白轻链激酶（MYLK）和MYLK3的表达，并随后下调纤维连接蛋白来抑制这些功能。一致地，患有早期妊娠损失的患者中观察到GPR65、MYLK和MYLK3的表达上调。本研究揭示了酸性环境下GPR65对EVT功能的抑制作用，并强调了早期妊娠并发症治疗干预的潜在目标。视频摘要。© 2023，BioMed Central有限公司，Springer Nature部分所有。

Extravillous trophoblasts (EVTs) are essential cells during the formation of the placenta, with the major function of invading the maternal decidua, anchoring the developing placenta to the uterus, remodeling uterine arteries, and regulating immune responses to prevent rejection. During early pregnancy, the decidua undergoes a hypoxic and acidic microenvironment, which has been shown to participate in tumor cell migration, invasion, growth, and angiogenesis. Nevertheless, the mechanisms by which EVTs sense and respond to the acidic microenvironment, thereby executing their functions, remain poorly understood.The effects of G protein-coupled receptor 65 (GPR65) on cell adhesion and other cellular functions were tested using JAR spheroids, mouse blastocysts, and HTR-8/SVneo cells. Specifically, we employed HTR-8/SVneo cells for gene overexpression and silencing to investigate the underlying mechanism of GPR65's impact on trophoblast cell function under acidic conditions. Additionally, villus tissue samples obtained from early pregnancy loss patients were utilized to explore the potential association between GPR65 and its related signaling pathway molecules with the disease.This study identified GPR65 expression widely in trophoblasts, with the highest level in EVTs. Importantly, optimal GPR65 levels are required for maintaining normal adhesion, migration, and invasion, whereas overexpression of GPR65 inhibits these functions by activating the cAMP-ERK signaling pathway, upregulating myosin light chain kinase (MYLK) and MYLK3 expression, and subsequently downregulating fibronectin. Consistently, elevated expression of GPR65, MYLK, and MYLK3 is observed in patients suffering from early pregnancy loss.This work offers insights into the suppressive effects of GPR65 on EVT function under acidic conditions and highlights a putative target for therapeutic intervention in early pregnancy complications. Video Abstract.© 2023. BioMed Central Ltd., part of Springer Nature.