The main role of HLA-G is to protect the semi-allogeneic embryo from immune rejection by proper interaction with its cognate receptors on the maternal immune cells. Spontaneous abortion is the most common adverse pregnancy outcome, with an incidence rate between 10% and 15%, with immunologic dysregulation being thought to play a role in some of the cases. In this study, we aimed to detect the membrane and soluble HLA-G molecule at the maternal–fetal interface (MFI) and in the serum of women experiencing missed abortion (asymptomatic early pregnancy loss) in comparison to the women experiencing normal early pregnancy. In addition, the proportion of T cells and their cytotoxic profile was evaluated. We observed no difference in the spatial expression of HLA-G at the MFI and in its serum levels between the women with missed abortions and those with normal early pregnancy. In addition, comparable numbers of peripheral blood and decidual total T and γδT cells were found. In addition, as novel data we showed that missed abortion is not associated with altered extravilous invasion into uterine blood vessels and increased cytotoxicity of γδT cells. A strong signal for HLA-G on non-migrating extravilous trophoblast in the full-term normal placental bed was detected. In conclusion, HLA-G production at the MFI or in the blood of the women could not be used as a marker for normal pregnancy or missed abortions.

The role of human leukocyte antigen F (HLA-F) at the maternal-fetal interface (MFI) during viral infection and its regulation by interferon signaling remains poorly understood. Here, we investigated HLA-F expression and regulation in first-trimester trophoblast cells following activation of the type I interferon pathway and viral infection. We demonstrate that HLA-F is significantly upregulated at both mRNA and protein levels in response to Poly(I:C) and IFN-β in a dose- and time-dependent manner, suggesting its regulation as an interferon-stimulated gene (ISG). Zika virus (ZIKV) infection similarly induced HLA-F upregulation over time. In contrast, HSV-2 infection downregulated HLA-F mRNA while maintaining steady protein levels, indicative of virus-specific regulatory mechanisms. Moreover, we identified a soluble HLA-F secreted following Poly(I:C) stimulation. These findings reveal that HLA-F is dynamically regulated in trophoblasts during viral challenge and type I IFN signaling activation, supporting its broader immunomodulatory role in antiviral defense and immune tolerance at the MFI.

The role of human leukocyte antigen F (HLA-F) at the maternal–fetal interface (MFI) during viral infection and its regulation by interferon signaling remains poorly understood. Here, we investigated HLA-F expression and regulation in first-trimester trophoblast cells following activation of the type I interferon pathway and viral infection. We demonstrate that HLA-F is significantly upregulated at both mRNA and protein levels in response to Poly(I:C) and IFN-β in a dose- and time-dependent manner, suggesting its regulation as an interferon-stimulated gene (ISG). Zika virus (ZIKV) infection similarly induced HLA-F upregulation over time. In contrast, HSV-2 infection downregulated HLA-F mRNA while maintaining steady protein levels, indicative of virus-specific regulatory mechanisms. Moreover, we identified a soluble form of HLA-F secreted following Poly(I:C) stimulation. These findings reveal that HLA-F is dynamically regulated in trophoblasts during viral challenge and type I IFN signaling activation, supporting its broader immunomodulatory role in antiviral defense and immune tolerance at the MFI.

Maternal–fetal tolerance mechanisms are crucial during human pregnancy to prevent the immune rejection of the embryo. A well-known mechanism blocking NK-cell cytotoxicity is the interaction of their inhibitory killer-cell immunoglobulin-like receptors (iKIR) with HLA-C molecules on the target cells. In this study, we aimed to investigate the expression of iKIRs (KIR2DL1 and KIR2DL2/3) on the matched decidual and peripheral γδT cells and the localization of HLA-C ligands throughout human pregnancy. The degranulation of γδT cells of pregnant and non-pregnant women in the presence of trophoblast cells was evaluated as well. Our results showed a higher proportion of iKIR-positive γδT cells at the maternal–fetal interface early in human pregnancy compared to the paired blood of pregnant women and full-term pregnancy decidua. In accordance, HLA-C was intensively expressed by the intermediate cytotrophoblasts and decidua-invading extravillous trophoblasts (EVTs) in early but not late pregnancy. Decidual γδT cells during early pregnancy showed higher spontaneous degranulation compared to their blood pairs, but neither decidual nor peripheral γδ T cells increased their degranulation in the presence of Sw71 EVT-like cells. The latter were unable to suppress the higher cytotoxicity of γδT cells, suggesting a complex regulatory landscape beyond NK-like activity inhibition.

Cytotoxic lymphocytes contribute to immune regulation at the maternal–fetal interface, yet their regulating mechanisms during early pregnancy remain elusive. We investigated compartment-specific NK- and γδT-cell degranulation towards trophoblast-derived signals. Blood and decidual mononuclear cells were obtained from non-pregnant women and women in early pregnancy. Spontaneous and trophoblast-mediated degranulation of NK- and γδT-cell subsets was assessed following co-culture with Sw71 extravillous trophoblast-like cells with normal and reduced expression of HLA-G/HLA-C molecules. CD107a-positive frequency and mean fluorescence intensity were quantified by flow cytometry. Statistical analyses included paired testing, multivariate analyses, and false discovery rate correction. Decidual cytotoxic lymphocytes displayed a distinct degranulation profile compared with blood counterparts. At baseline, γδT cells exhibited higher spontaneous degranulation than NK cells. Trophoblast co-culture exerted limited effects on NK-cell degranulation; however, decidual CD56⁺ γδ T cells showed increased degranulation towards trophoblasts with impaired HLA expression. In conclusion, early pregnancy is characterized by a highly compartmentalized regulation of cytotoxic lymphocyte activity. Decidual NK and γδT cells exhibit enhanced degranulation, but only CD56⁺ γδT cells retain substantial responsiveness to trophoblast-derived signals. These findings identify γδT cells as a dynamically regulated cytotoxic population at the maternal–fetal interface, sensitive to local shifts in trophoblast immune signaling.

Successful human implantation depends on precise and adequate invasion of the extravillous trophoblast into maternal decidua. The role of the TWIST1 transcription factor in this process remains incompletely defined. In humans, deficiencies in EVT functionality are associated with great obstetrical syndromes. Understanding the TWIST1-based implantation impairment is limited by the lack of reliable in vitro models. To address this gap, we combined the ex vivo analysis of TWIST1 expression in healthy vs. aborted gestational tissues with in vitro Sw71 TWIST1-CRISPR/Cas9 knocked-out human blastocyst-like surrogate (Sw71TW1-KO BLS). We found strong expression of TWIST1 at the maternal–fetal interface of healthy but not of missed aborted pregnancy. The generated Sw71TW1-KO BLSs preserved blastocyst-like morphology but showed reduced expression of the essential HLA-G and HLA-C molecules. Sw71TW1-KO BLSs’ functionality was assessed by tests for migration and invasion. Sw71TW1-KO BLSs showed markedly impaired migration. Placed on Matrigel-coated endometrial stromal cell scaffold, Sw71TW1-KO BLSs failed to invade the scaffold. The results support that TWIST1 is an important component of the healthy decidua and placenta early in human pregnancy. The 3D model Sw71TW1-KO BLSs with suboptimal HLA expression and impaired function might serve as a tool for investigating TWIST1-associated trophoblast defects.