Bushen Zhuyun Recipe improves endometrial receptivity through kruppel-like factor 4-mediated recruitment of mixed-lineage leukemia 1 to activate homeobox A10 transcription.

[BACKGROUND] The Bushen Zhuyun Recipe (BSZYR) is a traditional Chinese formula for treating infertility that can improve the pregnancy outcome via enhancing endometrial receptivity during in vitro fertilization and embryo transfer in the treatment of infertility patients. However, its exact molecular mechanism remains unclear.

[PURPOSE] Impaired endometrial receptivity is a major cause of embryo implantation failure. Kruppel-like factor 4 (KLF4) and the histone methyltransferase mixed-lineage leukemia 1 (MLL1) are known regulators of endometrial receptivity. This study investigates whether the BSZYR improves endometrial receptivity by modulating the expression and activity of KLF4 and MLL1, and further explores the underlying epigenetic mechanisms.

[METHODS] Ultra high performance liquid chromatography-tandem mass spectrometry was applied to identify the major compounds in BSZYR. Using a controlled ovarian hyperstimulation (COH) mouse model, we evaluated the effects of BSZYR on the number of embryo implantation sites and endometrial morphology. Endometrial epithelial cells were treated with BSZYR-containing serum, adenovirus-mediated KLF4 overexpression or knockdown, or siRNA targeting MLL1. The expression levels of homeobox A10 (HOXA10), MLL1, and KLF4 were assessed by qRT-PCR, immunofluorescence, and Western blot. H3K4me3 modification was detected via immunofluorescence and Western blot. Protein interaction between MLL1 and KLF4 was examined by co-immunoprecipitation, and chromatin immunoprecipitation was used to measure the binding of KLF4, MLL1, and H3K4me3 to the HOXA10 promoter region. Molecular docking was conducted to assess the potential binding interactions of BSZYR compounds with MLL1 and KLF4.

[RESULTS] BSZYR enhanced endometrial receptivity and promoted embryo implantation in a COH mouse model. It upregulates the expression of the receptivity marker HOXA10 both in vivo and in vitro. Notably, BSZYR induced KLF4 expression and promoted its nuclear translocation. Within the nucleus, KLF4 interacts with MLL1 and recruits it to the HOXA10 promoter region, thereby increasing H3K4me3 enrichment and activating HOXA10 transcription. This epigenetic mechanism ultimately enhances endometrial receptivity.

[CONCLUSION] BSZYR improved endometrial receptivity during COH. BSZYR enhances endometrial receptivity by facilitating functional cooperation between KLF4 and MLL1, leading to synergistic activation of HOXA10 transcription through epigenetic mechanisms. These findings not only reveal a novel regulatory axis in endometrial physiology but also provide substantial pharmacological support for BSZYR's clinical application.