All experimental animal procedures were approved by the University of Edinburgh ethical committee and performed in accordance with the Animals Scientific Procedures Act (1986) of the UK Home Office (PPL 70/8754). Female C57BL/6JOlaHsd mice were purchased from Envigo (Hillcrest, UK). Mice were randomised to high fat or control diet (Special Diets Services 826172 – RM 58% AFE Fat or 826171 – RM 11% AFE Fat) for 12 weeks and body weight recorded weekly. Endometrial shedding and repair were simulated in ovariectomised mice as previously described (Brasted et al. 2003, Cousins et al. 2014). In brief, 6- to 9-week-old female mice were ovariectomised on day 1 of the protocol to deplete endogenous steroid production. Mice then received daily subcutaneous injections of oestradiol (E2) in peanut oil (100 ng) on days 7–9. A progesterone implant (P4) was placed subcutaneously on day 13, mice also received daily injections of E2 (5 ng) from days 13 to 15. On day 15, decidualisation of one uterine horn was induced by transcervical injection of 20 μL peanut oil using a non-surgical transfer device (ParaTechsTM, Lexington, KY, USA). Decidualisation is a prerequisite for endometrial breakdown at menses. P4-withdrawal was induced 4 days after decidualisation (day 19) by removal of the P4 implant to trigger endometrial breakdown and repair. Mice received an intra-peritoneal injection of bromodeoxyuridine (BrdU, 0.25 mg) 1.5 h prior to culling. Mice were culled by cervical dislocation 24 h after P4-withdrawal (T24). Uteri were dissected and collected in RNA later (Ambion) and 4% neutral buffered formalin for paraffin embedding. Any animal with failed decidualisation was excluded from the study as there was no endometrial breakdown/repair to grade. This was n  = 2 out of 12 mice fed a normal diet (16.6% failed decidualisation rate) and n  = 9 out of 15 mice on a high fat diet (60% failed decidualisation rate).

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