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ObjectiveBromocriptine treatment has been shown to reduce menstrual bleeding and pain in women with adenomyosis in a pilot clinical trial. The underlying mechanism contributing to the treatment effect is however unknown. The purpose of this study was to explore the effect of bromocriptine on the proliferation and migration properties of the endometrium in women with adenomyosis, by assessing cellular and molecular changes after six months of vaginal bromocriptine treatment.MethodsEndometrial specimens were collected during the proliferative phase from women with adenomyosis (n=6) before (baseline) and after six months of treatment with vaginal bromocriptine. Immunohistochemistry was used to determine changes in the protein expression of Ki67 in the endometrium of women with adenomyosis. Primary endometrial stromal cells isolated at baseline were expanded in vitro and exposed to different doses of bromocriptine to determine the optimal half-maximum inhibitory concentration (IC50) using CellTiter-Blue® Cell Viability Assay. Cell proliferation was assessed by bromodeoxyuridine ELISA assay and Ki67 gene expression was checked by real-time PCR. The migratory ability of endometrial stromal cells was determined by wound healing and transwell migration assays. Small RNA sequencing was applied on tissues collected from women with adenomyosis before and after bromocriptine treatment to identify differentially expressed microRNAs (miRNAs) after bromocriptine treatment. Bioinformatic methods were used for target gene prediction and the identification of biological pathways by enrichment procedures.ResultsVaginal bromocriptine treatment reduced the Ki67 protein expression in the endometrium of women with adenomyosis and did not change the prolactin mRNA expression and protein concentration of prolactin in endometrial tissues. Bromocriptine significantly inhibited the proliferative and migrative abilities of endometrial stromal cells derived from women with adenomyosis in vitro. Moreover, small RNA sequencing revealed 27 differentially expressed miRNAs between the endometrium of women with adenomyosis before and after six months of vaginal bromocriptine treatment. KEGG pathway analysis on targeted genes of 27 miRNAs showed that several signaling pathways associated with cell proliferation and apoptosis were enriched after bromocriptine treatment.ConclusionBromocriptine treatment exhibits an anti-proliferative effect in the endometrium of women with adenomyosis in vivo and in vitro. Bromocriptine might inhibit the proliferation of endometrial tissue in adenomyosis in part through the regulation of dysregulated microRNAs and proliferation-associated signaling pathways.

Additional file 2. Is the protocol for Clinical Trial 1, ‘Mifepristone treatment prior to insertion of a levonorgestrel releasing intrauterine system for improved bleeding control – a randomized controlled trial’ (EudraCT registration number 2009-009014-40).

Additional file 1: Supplementary Methods. Fig. S1. Elevated levels of estradiol and progesterone in BRCA1 and BRCA2 mutation carriers compared to controls. Fig. S2. Epithelial cell composition in normal tissues adjacent to TNBC compared to cancer-free breasts. Fig. S3. Enrolment and randomisation in Clinical Trial 1. Fig. S4. Enrolment and randomisation in Clinical Trial 2. Fig. S5. Enrolment in Clinical Trial 3. Fig. S6. Effect of mifepristone treatment on breast epithelium cell subtype composition. Fig. S7. Effect of mifepristone treatment on breast epithelium cell subtype composition – RNA-seq data. Fig. S8. The WID-Breast5 index. Fig. S9. The Ki67 index. Fig. S10. Comparing the Ki67 index with the WID-Breast29 index. Fig. S11. Comparing the change (before and after ulipristal acetate) in percentage of immunohistochemistry Ki67-positive cell with change in Ki67-index in DNAme Dataset 4 (Clinical trial 3, ‘A pilot prevention study of the effects of the anti- progestin Ulipristal Acetate (UA) on surrogate markers of breast cancer risk’). Table S1a. Summary statistics about the 20 healthy women (12 BRCA1 mutation carriers and 8 BRCA1 wild type) enrolled in the trial to measure estradiol and progesterone levels throughout the menstrual cycle (see also Figs. 1 and 2). Table S1b. Summary statistics about the 21 healthy BRCA mutation carriers enrolled in Clincal Trial 2 who provided samples for DNAme analysis as part of DNAme Set 3. Table S2. The 37 WID-Breast29 CpGs. Table S3. Summary statistics about the donors of the breast cancer biopsies and matched surrounding normal tissue samples. Table S4. The reference CpGs identified for luminal progenitor, mature luminal, and basal breast epithelial cells. Table S5. Summary statistics about the 15 healthy women who provided samples for RT-PCR and RNA-seq before and after mifepristone treatment.

Additional file 4. Is the protocol for Clinical Trial 3, ‘A pilot prevention study of the effects of the anti- progestin Ulipristal Acetate (UA) on surrogate markers of breast cancer risk’ (EudraCT registration number: 2015-001587-19).

Additional file 3. Is the protocol for Clinical Trial 2, ‘The effect of a progesterone receptor modulator on breast tissue in women with BRCA1 and 2 mutations’ (EudraCT registration number: 2012-003703-35).