SAT-147 - Defining the ATOH8-dependent Transcriptome in Regulating Human Endometrial Stromal Cell Decidualization

Introduction: Decidualization of human endometrial stromal fibroblasts (hESFs) into epithelial-like decidual cells is critical for embryo implantation, placental development, and pregnancy maintenance. Failure of this process contributes to infertility, recurrent pregnancy loss, and conditions such as endometriosis. ATOH8, a basic helix-loop-helix transcription factor, is upregulated during hESF decidualization in vivo and in vitro. We have previously shown that inhibiting ATOH8 expression prevents normal decidualization in vitro. In part, this was due to altered BMP2-WNT/FZD/CTNNB1/FOXO1 signaling axis during decidualization. However, the broader transcriptional program it governs remains unknown.

Methods: hESFs were isolated from the term placenta of three independent patients (N=3, ≤4 passages) and transduced with ATOH8-knockdown (shATOH8) or non-targeting (shNT) lentiviruses (MOI=5), followed by decidualization induction using estradiol, medroxyprogesterone acetate, and 8Br-cAMP for 48h. Transcriptomic analysis was performed by paired-end mRNA-seq (Illumina NextSeq 2000; ~40–45M reads/sample), mapped via RNA-STAR, and the number of reads counted with HT-Seq. Differentially expressed genes (FDR < 0.05, FC>1.5) were identified using edgeR. Validation of differentially expressed genes was assessed by RT-qPCR in cells from six different patients (N=6).

Results: Targeting ATOH8 expression significantly altered expression within the BMP-SMAD1/5, Activin-SMAD2/3, WNT-FZD-CTNNB1, and NOTCH-RBPJ-MAML signaling pathways; all with established roles in decidualization. Cross-referencing with published FOXO1 and PGR RNA-seq and ChIP-seq datasets revealed notable overlaps with ATOH8-regulated genes. Transcription factor binding site enrichment was identified for FOXO1, LEF1, SMAD2/3/4, class I bHLH factors (TCF3/4/12), RBPJ, and PGR, of which three were significantly downregulated upon targeting ATOH8 expression.

Conclusions: These findings expand our understanding of ATOH8 as a central transcriptional regulator of human decidualization, influencing multiple interconnected signaling pathways essential for successful implantation and pregnancy. This work lays the groundwork for future mechanistic studies and positions ATOH8 as a potential target in reproductive disorders, including infertility, recurrent pregnancy loss, and endometriosis.

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