Phase-dependent cytokine dynamics in multicellular in vitro models of the human endometrium

To characterize the dynamic secretion patterns of extracellular cytokines in 3 in vitro human endometrial culture models subjected to a hormonally simulated 28-day menstrual cycle and to assess the influence of cellular composition on paracrine signaling relevant to endometrial receptivity.

Experimental in vitro study.

Three endometrial culture models were developed using combinations of endometrial epithelial cells (RL95-2), stromal cells (T0533), and primary myometrial smooth muscle cells.

The 3 multicellular models were subjected to a sequential hormonal treatment protocol simulating the proliferative, ovulatory, and secretory phases of the human menstrual cycle.

Quantitative profiling of 105 extracellular cytokines across 4 hormonal phases (control, proliferative, ovulatory, and secretory) using a cytokine array platform. We conducted a comparative analysis of cytokine expression patterns, correlation matrices, and hierarchical clustering to identify model-specific and hormone-dependent regulatory networks.

We identified 12 highly expressed cytokines with distinct phase and model-specific expression profiles. Extracellular matrix metalloproteinase inducer, macrophage migration inhibitory factor, and interleukin 8 levels increased consistently across all phases, peaking during the window of implantation. Vascular endothelial growth factor exhibited biphasic expression patterns; epidermal growth factor level was up-regulated by estradiol and down-regulated by progesterone. Dickkopf-related protein 1 was up-regulated in progesterone-dominant phases. Serpin E1 and Dickkopf-related protein 1 showed phase-specific regulation and were also influenced by cellular composition. Insulin-like growth factor-binding protein 3 down-regulated the proliferative and ovulatory phases. Clustering analyses revealed coregulatory modules (e.g., extracellular matrix metalloproteinase inducer/macrophage migration inhibitory factor and growth-regulated oncogen-α /lipocalin-2) and distinct cytokine networks modulated by stromal and myometrial components. Notably, model C (endometrial epithelial cells [EEC] + endometrial stromal cells + myometrial smooth muscle cells) demonstrated profiles more similar to model A (EEC alone) than to model B (EEC + endometrial stromal cells), suggesting myometrial smooth muscle cells may attenuate certain epithelial-stromal paracrine interactions.

The phase-specific and model-dependent cytokine secretion patterns highlight the complexity of endometrial paracrine signaling and underscore the importance of multicellular in vitro models. These findings advance our understanding of cytokine dynamics during the menstrual cycle and provide a platform for future studies on endometrial receptivity and implantation.