Background:

Syncytiotrophoblast (STB) executes critical placental functions and forms via cytotrophoblast fusion. Placenta-specific protease HtrA4 has been shown to be essential for STB differentiation in various models, including human trophoblast stem cells (TSCs) (1), but how it mediates the process remains unknown. This study investigated how HtrA4 is involved in STB differentiation using TSCs and human placental explants.

Methods:

Control and HtrA4 knockdown (HtrA4-KD) TSCs were cultured with and without STB differentiation stimulation. Global transcriptomic changes of these cells were analyzed by RNA-seq to identify molecular pathways blocked in HtrA4-KD cells. Transcriptomic results were further validated by RT-PCR and immunofluorescence in TSCs, and by immunohistochemistry on cultured placental explants. Scanning electron microscopy (SEM) was then employed to track morphological progression of these cells over the time course of STB differentiation.

Results:  

In control cells, STB differentiation triggered a widespread presentation of differentially expressed genes (DEGs). In contrast, HtrA4-KD cells exhibited a severe transcriptional block, displaying only 25% of the control DEGs while the remaining 75% were severely attenuated or entirely blocked. Representative DEGs were validated in TSCs and placental explants. Pathways analysis revealed that early STB differentiation events, such as cell cycle arrest and the initiation of autophagy, occurred independently of HtrA4. However, HtrA4 deficiency severely impaired many subsequent differentiation processes, including hormone activity, extracellular matrix remodeling, and cytoplasm/cytoskeleton restructuring. Altering protein binding emerged as a major functional juncture of HtrA4 influence. Additionally, neural-related pathways were suppressed during STB differentiation, and this suppression was influenced by HtrA4. Morphologically, SEM visually demonstrated that while HtrA4-KD cells could initiate the differentiation process, they failed to progress to cell-cell fusion.

Conclusion:

HtrA4 plays a critical role in STB differentiation, influencing many vital cellular and structural remodeling events. These results provide crucial foundations for understanding how HtrA4 dysregulation may contribute to pregnancy complications.